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The H1P1 family of cytoskeletal-associated proteins Chopra, Vikramjit 2003-12-31

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THE HIP 1 FAMILY OF CYTOSKELETAL-ASSOCIATED PROTEINS by Vikramjit Chopra B.Sc, McGill University, 1992 M.Sc., McGill University, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES Department of Medical Genetics We accept this thesis as conforming tojthe required standards THE UNIVERSITY OF BRITISH COLUMBIA August 25, 2003 © Vikramjit Chopra Abstract Huntingtin-interacting protein 1 (HJPT) is a membrane associated protein which interacts with huntingtin, the protein altered in Huntington Disease (HD). It was determined that the H1P1 gene consists of 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIPl-1 and HIP1- 2. Additionally, a novel homolog of HIP1, termed HIP12, was identified. HIP1 and HIP12 share significant amino acid identity and both proteins contain an epsin N-terminal homology (ENTH), coiled coil and talin-Iike domains. While overexpression of HTP1 is toxic in cell culture, HIP12 did not confer toxicity in the same assay systems. Interestingly, HIP12 did not interact with huntingtin but was able to interact with HJPT suggesting a potential interaction in vivo which may influence the function of each respective protein. Tissue distribution studies indicated that HIP1 was expressed predominantly within the brain whereas HIP12 expression was more ubiquitous. Within cells, HIP1 and HJPT2 displayed a similar punctate distribution at the plasma membrane and underlying cortical actin cytoskeleton. HIP1 was shown to interact with the endocytic proteins clathrin heavy chain and AP2. However, HIP12 displayed weak binding to clathrin heavy chain and no interaction with AP2. HIP1 and HIP12 were also shown to interact with the clathrin light chain. In addition, it was also revealed that HJPT and HIP12 were capable of inducing the assembly of the clathrin coat in vitro. Finally, it was determined that HIP12 interacts with F-actin suggesting that HIP12 may interact with the cortical actin ii cytoskeleton underlying clathrin-coated pits and vesicles. HIP1 may be indirectly associated with the actin cytoskeleton by forming heterodimers with HIP12. Thus, HTP1 and HTP12 may act as a molecular "link" between the endocytic machinery and the cortical actin cytoskeleton. Taken together, these results demonstrate that HIP1 and HIP12 are components of the endocytic machinery that participate in the recruitment, assembly and stabilization of the clathrin lattice structure. The observation that huntingtin associates with HIP1 suggests that it may function in endocytosis and transport. Since mutant huntingtin displays an altered interaction with HIP1, it raises the possibility that HD may be the result of defects in endocytosis and transport. The determination of the cellular functions of the HJP1 family and huntingtin in endocytosis and transport may provide valuable insights into the pathogenic mechanism(s) underlying HD. iii Table of Contents Abstract II Table of Contents V List of Tables XList of Figures XII List of Abbreviations V Dedication XVAcknowledgements XVII Chapter 1: Introduction 1 1.1 Huntington Disease: Clinical Presentation, Epidemiology and Natural History 2 1.2 Neuropathology of Huntington Disease 3 1.3 Huntington Disease Gene 4 1.4 CAG Repeat Length is Inversely Correlated with the Onset of HD 4 1.5 Do CAG Expansions lead to a Gain of Function Mutation? 6 1.6 Inclusions and Aggregates in Huntington Disease 7 1.7 Proteolysis of Huntingtin 9 1.8 Cellular Functions of Huntington 10 1.8.1 Cellular Survival1.8.2 Transcriptional Regulation 11 iv 1.8.3 Intracellular Signaling 12 1.8.4 Cellular Transport and Endocytosis 13 1.9 Interacting Proteins involved in Endocytosis and Cellular trafficking 16 1.9.1 Huntingtin Associated Protein 11.9.2 a-Adaptin-C 18 1.9.3 Endophillin A3 9 1.9.4 Pacsin 1 20 1.9.5 Huntingtin Interacting Protein 14 21 1.9.6 CDC42-Interacting Protein 4 2 1.9.7 Huntingtin Interacting Protein 1 23 1.9.7.1 The Pro-Apoptotic Function of H1P1 24 1.9.7.2 The Role of HIP1 in Cellular Transformation 26 1.9.7.3 The HIP1 Family of Proteins 30 1.9.7.4 Saccharomyces cerevisiae Sla2p 32 1.9.7.5 The Caenorhabtidis elegans HJPT (CeHIPl) 34 1.9.7.6 Huntingtin Interacting Protein 12 (HIP12) 35 1.10 Clathrin-Mediated Endocytosis and the Cortical Actin Cytoskeleton 36 1.11 Hypothesis 41 1.12 Thesis ObjectivesChapter 2: Materials and Methods 43 2.1 Materials and Methods for Chapter 3 4 v 2.1.1 Isolation of HTP12 44 2.1.2 DNA and Amino Acid Sequence Analyses 46 2.1.3 Determination of Additional 5' HTP1 Sequences 46 2.1.4 Genome mapping of HIP12: FISH Analysis 48 2.1.5 Northern Blot Analyses 42.1.6 Generation of Antibodies 9 2.1.7 Protein Preparation and Western Blot Analyses 49 2.1.8 Construction of Yeast Two-Hybrid Vectors 50 2.1.9 Immunocytochemical Analyses of Neuronal NT2 cells and ES Cell Derived Neurons 51 2.1.10 Cell Viability Assays 2 2.1.11 Caspase 3 Activation Assays 53 2.2 Materials and Methods for Chapter 4 4 2.2.1 Antibodies 52.2.2 DNA constructs 4 2.2.3 Immunofluorescence 55 2.2.4 Transferrin uptake assays2.2.5 Purification of CCVs 56 2.2.6 Pull-down and binding assays 52.2.7 Immunoprecipitation analyses 7 2.3 Materials and Methods for Chapter 5 8 2.3.1 Antibodies 52.3.2 DNA Constructs and Recombinant Proteins 58 vi 2.3.3 Pull-down Assays 59 2.3.4 Immunoprecipitation Assays 60 2.3.5 Immunofluorescence2.3.6 Actin-binding Assay 61 2.3.7 Clathrin Purification and Clathrin Assembly Assay 62 Chapter 3: HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin 63 3.1 Introduction 64 3.2 Results 6 3.2.1 Identification of HIP12, a paralog of HIPl 66 3.2.2 Alternative splicing at the human HIP1 locus 68 3.2.3 Genomic organization of human HIP1 70 3.2.4 Sequence comparisons of HIP12 with HJP1, Sla2p and ZK370.3 70 3.2.5 mRNA Expression of Human HIP1 and H1P12 78 3.2.6 Protein Expression of Human HIP1 and HIP12 79 3.2.7 Immunofluorescence in NT2 cells and ES Cell-Derived Neurons 82 3.2.8 Yeast 2-Hybrid Interactions of HIPl, HTP12 and Huntingtin 82 3.2.9 Overexpression of HIPl and HIP12 in Human Embryonic Kidney (293T) Cells 85 vii 3.3 Discussion 88 Chapter 4: HIP1 Interacts with Clathrin and AP2 91 4.1 Introduction 92 4.2 Results 4 4.2.1 Co-localization of HTPl with components of the endocytic machinery 94 4.2.2 Identification of HIP1 domains necessary for endocytic targeting 96 4.2.3 HTPl interaction with AP2 and clathrin terminal domain 98 4.2.4 HTPl plays a role in clathrin-mediated endocytosis 101 4.3 Discussion 103 Chapter 5: Differential Binding of HIP1 and HIP12 to Components of the Endocytic Machinery 106 5.1 Introduction 107 5.2 Results 9 5.2.1 Comparison of HTPl andHIP12 protein expression 109 5.2.1.1 FfJPl and HIP12 protein expression in adult and embryonic rat brain 109 5.2.1.2 Subcellular fractionation studies 109 viii 5.2.2 Interaction of HIPl with HTP12 110 5.2.2.1 Co-immunoprecipitation 115.2.2.2 Confocal microscopy 114 5.2.2.3 Affinity purifications using deletion constructs 114 5.2.3 Differential binding of HIPl and HIP12 to Clathrin 116 5.2.4 Differential binding of HIPl and HTP12 to F-actin 117 5.2.4.1 Co-sedimentation studies 115.2.4.2 Confocal microscopy 119 5.2.5 The Coiled-Coil Domains of HIPl and HIP12 Bind to Clathrin 119 5.2.6 Clathrin Coat Assembly using the Coiled-Coil Domains of HIPl andHIP12 120 5.2.7 HIPl and HTP12 Binding to the Clathrin Light Chain 123 5.3 Discussion 125 Chapter 6: General Discussion 129 6.1 Summary 130 6.2 HJPl and HIP 12 are components of the endocytic machinery 130 6.3 HIPl and HIP 12 Interact with the Cortical Actin Cytoskelton 136 6.4 In vivo Evidence of HIPl Function in Endocytosis 137 6.5 The Role of Huntingtin in Endocytosis and Transport 138 6.5.1 Endocytic and Transport Proteins that interact with Huntingtin 139 ix 6.5.2 Huntingtin-Interacting Proteins that Influence Endocytosis and Transport 140 6.6 The Potential Role of Endocytosis Defects in the Pathogenesis of HD 146 6.7 Future Directions 147 6.8 Conclusion 150 Chapter 7: References 151 x List of Tables Table 1.1 The CAG trinucleotide repeat diseases 5 Table 1.2 Huntingtin interacting proteins and their functions 14 Table 3.2.3. Splice-acceptor and splice-donor sequences at the HIPl locus 72 Table 3.2.4. Pairwise comparisons between the nucleotide and amino acid sequences of HIPl-1, HIP1-2, HIP12, Sla2p and ZK370.3 73 xi List of Figures Figure 1.1. Two potential mechanisms how HIP1 induces apoptotic cell death 28 Figure 1.2. The different stages of clathrin mediated endocytosis 38 Figure 2.1 Mouse ESTs Identified by BLAST Search Using the Human HTPl Nucleotide Sequence 45 Figure 3.2.1. Genomic mapping of the HTP12 locus 67 Figure 3.2.2. Alternative splicing at the HTPl locus 9 Figure 3.2.3. Genomic organization of HIP1 71 Figure 3.2.4. Amino acid alignment of HIP1-1, HIP1-2, HTP12, Sla2p andZK370.3 75 Figure 3.2.5. Northern blot analysis of the human HTPl and HIP12 80 Figure 3.2.6. HIP1 and HIP12 protein expression in normal human CNS and peripheral tissues 81 Figure 3.2.7. Immunolocalization of HIP1 and HIP12 in NT2 and ES cell-derived neurons 83 Figure 3.2.8. Yeast 2-Hybrid interactions of HTPl and HIP12 with various GAL4 DNA-binding domain constructs 86 xii Figure 3.2.9. MTT and caspase-3 activation assays as a measures of cell viability and apoptosis 87 Figure 4.2.1. HIPl colocalizes with markers of endocytosis and is enriched in CCVs 95 Figure 4.2.2. HIPl targeting to CCVs 7 Figure 4.2.3. HIPl binds clathrin and AP2 100 Figure 4.2.4. Transferrin uptake in COS7 cells 102 Figure 5.2.1. HIPl and HIP12 are coat proteins of CCVs and display distinct developmental expression profiles 111 Fig. 5.2.2. HIPl and HIP12 interact via their helical domains 113 Fig. 5.2.3. HIPl and HIP12 display differential binding to clathrin and AP2 115 Fig. 5.2.4. HIPl does not bind actin in contrast to HTP12 118 Fig. 5.2.5. The helical domains of HIPl and HIP12 bind to clathrin but not AP2 121 Fig. 5.2.6. HIPl and HIP12 promote clathrin assembly through their helical domains 122 Fig. 5.2.7. HIPl and HIP12 bind directly to the clathrin light chain through their helical domains 124 Fig. 6.1. HIPl and HIP12 domains 131 Fig. 6.2 Domain structures of HIPl and HIP12 135 Fig. 6.3 Potential roles for huntingtin interacting proteins in endocytosis 145 xiii List of Abbreviations Adapter protein 1 (AP2) Adaptor protein 180 (AP180) a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) Analysis of variance (ANOVA) Base pair (bp) Brain-derived neurotrophic factor (BDNF) Calmodulin (CAM) Cdc42-interacting protein 4 (CIP4) Clathrin assembly lymphoid myeloid leukemia protein (CALM) Clathrin-coated vesicle (CCV) Clathrin heavy chain (CHC) Clathrinlight chain (CLC) Clathrin-mediated endocytosis (CME) Chronic myelomonocytic leukemia (CMML) Death effector domain (DED) Dentatorubropalliduysian atrphy (DRPLA) Epsin N-terminal Homology (ENTH) Y-amino butyric acid (GABA) Glutathione S-transferase (GST) Hemagglutinin (HA) Huntingtin associated protein 1 (HAP1) Huntingtin interacting protein 1 (HIP1) Huntingtin interacting protein 12 (HTPl2) Huntingtin interacting protein 14 (HIP 14) HTPl protein interactor (HTPPI) Huntington Disease (HD) Huntington Disease Collaborative Research Group (HDCRG) xiv Huntingtin, elongation factor 3, protein phosphatase 2A subunit 65, and the lipid kinase TOR (HEAT) type 1 inositol (l,4,5)-triphosphate receptor (InsPsRl) Kilo-base pair (kb) Neuronal intranuclear inclusion (Nils) N-methyl-D-aspartate (NMDA) Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] Platelet-derived growth factor P receptor (PDGF(3R) Postsynaptic density 95 (PSD95) Spinal and Bulbar muscular atrophy (SBMA) Spinocerebellar ataxia type 1 (SCA1) Spinocerebellar ataxia type 2 (SCA2) Spinocerebellar ataxia type 3 (SCA3) Spinocerebellar ataxia type 6 (SCA6) Spinocerebellar ataxia type 1 (SCA7) Src homology 2-containing inositol 5-phosphatase (SHIP1) Synthetic Lethal with Actin binding protein-1 #2 (Sla2) Transgenic mouse model for prostate cancer (TRAMP) Ubiquitin-conjugating enzyme (hE2-25K) Wiskott-Aldrich syndrome protein (WASP) Yeast artificial chromosome (YAC) xv Dedication To my wife, mother, father, and brother for their love, patience, and support. xvi Acknowledgements I would very much like to thank Dr. M. R. Hayden for giving me the opportunity to work in his laboratory and providing me with guidance throughout my graduate training. I would also like to thank members of my thesis committee, Dr. F. Jirik, Dr. D. Mager, and Dr. R. McMaster for keeping me on the right track through their thoughtful comments throughout the years. I am also very grateful to all the past and present members of Dr. Hayden's laboratory and my fellow colleagues at the Centre for Molecular Medicine who generously answered my many questions and provided me valuable advice. 1 would especially like to thank Roshni, Brett, Susie, Keith, Ryan, Simon, Ed and Rebecca. I would like to thank my collaborators Dr. M. Metzler, members of Dr. P. McPherson's Lab, and members of Dr. D. Nicholson's team at Merck Frosst Canada. I would like to give thanks and love to my mom and dad for their incredible support and love. All my achievements are because of you two. The mistakes are mine. Finally, I would like to give love and thanks to my wife, Manjit for her love, patience and faith in me. I cannot thank you enough for everything that you have given me. But I will try. This thesis was supported in part by scholarships from the Huntington Disease Society of Canada, the University of British Columbia Graduate Fellowship, and the British Columbia Children and Women's Research Institute. xvii Chapter 1: Introduction 1 1.1 Clinical Presentation, Epidemiology and Natural History Huntington Disease (HD) is a severe autosomal dominant neurodegenerative disorder that occurs with equal frequency in both sexes and has no known cure (Hayden, 1981). The incidence of HD in North American and European populations is --1/10000 with other populations showing lower frequencies. HD is characterized by chorea, dementia and psychiatric disturbances (Hayden, 1981). The initial symptoms can include behavioral disturbances and minor motor disturbances. As the disease progresses, the classical hallmark of HD, chorea, becomes apparent (Martin and Gusella, 1986). During the later stages of the disease the motor disturbances worsen resulting in rigidity, dystonia, dysphagia and dysarthria. HD patients often develop marked weight loss. Furthermore there is significant cognitive deficits including intellectual decline, memory loss and dementia. Although the age of onset in HD is variable, in most instances symptoms first appear at approximately 40 years of age. The mean life expectancy after first presentation of the symptoms is 18 years. HD displays the phenomenon of genetic anticipation where, in this case, earlier age of onset and more rapid progression of the disorder correlate with inheritance of the mutant allele from the father. Individuals with onset under 20 years of age are defined as juvenile HD and comprise 5 to 10% of total cases (Hayden, 1981). Juvenile HD displays somewhat different clinical features including rigidity, spasticity, and severe intellectual decline. Furthermore, the progression of the disorder is much more rapid in juvenile patients (Hayden, 1981). 2 1.2 Neuropathology Neuropathological findings in HD brains indicate that there is a selective loss of neurons in the neostriatum (caudate nucleus and putamen) and neurons within the deeper layers of the cerebral cortex (Vonsattel et al., 1985). Medium spiny neurons of the striatum produce the neurotransmitters y-amino butyric acid (GABA) which upon release exerts inhibitory effects (via the direct and indirect pathways) on neurons within the globus pallidus. In HD patients the loss of the GABAergic medium spiny neurons results in the loss of inhibition of neurons in the globus pallidus, which in turn results in choreic movements (Albin et al., 1992; Kremer et al., 1992; Vonsattel and DiFiglia, 1998). Loss of regulation of neurons in the internal segments of the globus pallidus also results in the rigid state seen in the advance stages of the disease (Kremer et al., 1992). Glutamate receptor hyperactivity, referred to as excitotoxic cell death, may also play a role in the selective neuronal loss seen in HD patients and mouse models of HD (Albin et al., 1992; Hodgson et al., 1999; Zeron et al., 2002). Overactivation of glutamate receptors results in high calcium influx that activates calcium-dependent degradative proteases and endonucleases and creation of reactive oxygen species that can lead to cellular dysfunction and death (Albin et al., 1992; Zeron et al., 2002). In addition to the loss of medium spiny neurons, the striatum also exhibits signs of inflammation such as astrogliosis, microgliosis and complement activation (Singrao et al., 1999). Examination of the degenerating neurons has demonstrated that these cells contain swollen mitochondria, nuclear and cytoplasmic inclusions, condensed cytoplasm, and condensed and marginalized heterochromatin (Difiglia et 1997). 3 1.3 HD Gene The HD gene was first identified by the HD Collaborative Research Group in 1993 (HDCRG, 1993). The HD gene is located on the short arm of chromosome 4 (4pl6.3), spans ~210kb and consists of 67 exons. The HD gene encodes 2 differentially polyadenylated transcripts that are 10.3 and 13.6kb in size that are ubiquitously expressed (Lin et al., 1994). Furthermore, no differences are observed between normal and affected individuals, with respect to expression levels or patterns (Li et al, 1993). These transcripts encode a 348kDa protein (called huntingtin) which, like the mRNA, is expressed in all tissues of normal and affected individuals (Sharp et al., 1995; Trottier et al., 1995). Within exon 1 of the HD gene is a tract of multiple [CAG]n, which code for glutamine residues. The mutation underlying HD stems from an expansion of this CAG tract beyond 35 repeats. The nature of the mutations has placed HD into a growing list of neurodegenerative disorders associated with repeat expansions within the coding portion of their respective genes (table 1.1). 1.4 CAG Repeat Length is Inversely Correlated with the Onset of HD The normal range of CAG repeats in huntingtin is 9-35 with a mean of ~18. The range in HD patients is 36-150, with a mean of 46 (Duyao et al., 1993; Snell et al., 1993; Andrew et al., 1993; Read et al., 1993; Nance et al., 1999). There is a significant inverse correlation between the age of onset and the repeat size length (Trottier et al., 1994; Brinkman et al., 1997). For example, the range of repeats observed in juvenile HD is 44-121 (Telenius et al., 1993). Although the sex incidence of juvenile HD is equal, ~80% of 4 w u z Oi W u. w oi co" so o ? 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Taken together, these findings show that the CAG repeat is more unstable upon transmission through the male germline. It has been suggested that the higher instability of CAG tracts in the paternal germline and the inverse correlation between the age of onset and the repeat length represent the molecular basis of genetic anticipation in HD. 1.5 Do CAG Expansions lead to a Gain of Function Mutation? The development of HD knockout mice has indicated that the polyglutamine expansion in the huntingtin protein results in a gain of function mutation. Nasir et al. (1995), Duyao et al. (1995) and Zeitlin et al. (1995) generated targeted disruption in exon 5, in exon 4 and the promoter of the HD gene, respectively. Mice heterozygous for the disruption did not develop a phenotype reminiscent of HD. Instead, mice homozygous for the disruption died before embryonic day 8.5 demonstrating that the HD gene plays a critical role in development. Examination of the embryos indicated that they were able to initiate gastrulation, but do not proceed to form somites or undergo organogenesis. For example, using embryonic stem cells lacking any HD gene expression Metzler et al. (2000) demonstrated that huntingtin is also required for the normal generation and expansion of hematopoietic cells. The development of conditional HD gene knockout mice, however, has provided evidence that the disease may in part be due to a loss of huntingtin function. Dragatsis et 6 al. (2000) selectively inactivated wild-type huntingtin in the brains and testes of adult mice. These mice developed progressive neurodegeneration within the striatum and cortex and displayed abnormal limb-clasping behavior, slight tremor and hypoactivity (Dragatsis et al., 2000). 1.6 Inclusions and Aggregates in HD The hypothesis that polyglutamine expansion results in a gain of function mutation may explain the development of insoluble aggregates or inclusions within the cytoplasm and nucleus of neurons in HD mouse models as well as affected individuals. These aggregates contain ubiquinated amino-teiminal fragments of huntingtin (Davies et al.1997; DiFiglia et al., 1997). In addition, the aggregates contain a number of other proteins including chaperones (Jana et al., 2000), and transcription factors (Boutell et al, 1999; Steffan et al., 2000; Suhr et al, 2001; Nucifora et al, 2001). The aggregates were initially observed in transgenic mice that over-expressed exon 1 of the HD gene containing an expanded CAG repeat length (Davies et al., 1997). The mice contained neuronal intranuclear inclusions (Nils) in several regions of their brains including the striatum, cortex, cerebellum and spinal cord. Nils have been observed in post mortem brain material from HD patients (DiFiglia et al., 1997; Becher et al., 1997). In patients affected with HD, these aggregates are found solely in the dystrophic neurites and neuropil but never in glial cells (DiFiglia et al., 1997). In addition, larger numbers of aggregates have been found in the striatum and cortex in the more severe juvenile-onset HD (DifFiglia et al., 1997). Interestingly, aggregate accumulation seems to be common feature in a number of different trinucleotide repeat 7 disorders including SCA-1 (Skinner et al, 1997), SCA-3 (Paulson et al., 1997), SCA-7 (Holmberg et al., 1998), SBMA (Li et al., 1998) and DRPLA (Becher et al., 1997). It has been demonstrated that the accumulation of aggregates in neurons does not necessarily play a causal role in cell death. Saudou et al. (1998) demonstrated that nuclear import of mutant huntingtin is sufficient to induce cell death and does not require the formation of Nils. Furthermore, neurodegeneration without the appearance of aggregates has been observed in a YAC transgenic mouse model for HD (Hodgson et al., 1999; Slow et al., 2003). It has been hypothesized that aggregates are not the cause of cell death but are rather a marker for cellular dysfunction. At this time it is unclear what are the underlying mechanisms underlying the formation of aggregates. Recently, it has been demonstrated that the phosphorylation occurs of mutant ataxin-1 at discrete sites (Emamian et al., 2003). The phosphorylated ataxin-1 interacts with the regulatory protein 14-3-3, which in turn, leads to the formation of aggregates (Chen et al., 2003). Two additional biochemical mechanisms have also been proposed to explain why mutant huntingtin tends to form aggregates. One possibility is that the expanded polyglutamine tract in huntingtin can form stable hairpin structures consisting of anti-parallel (3-sheets (Perutz, 1999, Thakur et al, 2002). Another possibility is that huntingtin and other polyglutamine containing proteins may be the target of transglutaminases. Transglutamination would result in the formation of protein complexes that are cross-linked via isopeptide bonds (Green, 1993). Interestingly, transglutaminase activity has been shown to be increased in HD patients (Karpuj et al., 1999). 8 1.7 Proteolysis of Huntingtin The presence of amino terminal fragments of huntingtin has been demonstrated in brain material from HD patients (DiFiglia et al., 1997; Becher et al., 1997). The generation of these fragments may in part be due to cleavage of huntingtin by proteases. In vitro studies have shown that huntingtin is a target for proteolysis by caspase-3 and 6 in a polyglutamine length-independent manner (Wellington et al., 2000; Wellington et al., 1998; Goldberg et al., 1996). Caspases are a family of cysteine aspartic acid proteases involved in the execution phase of apoptosis (Thornberry et al., 1997). The presence of activated caspase 3 and 6 has been confirmed in the brains of a YAC mouse model of HD, as well as post-mortem brain material from HD patients (Wellington et al., 2002; Kim et al., 2001). In addition, huntingtin cleavage can occur in the absence of an apoptotic stimulus suggesting that caspase cleavage of this protein may be a normal physiological mechanism to regulate huntingtin levels within the cell (Wellington et al., 2000; Wellington et al., 2002). It has been hypothesized that caspase cleavage of huntingtin having an expanded polyglutamine tract may lead to amino terminal fragments which may be toxic to the cell, and also lead to further activation of caspases (and other proteases) that ultimately lead to cell death. Indeed, it has been established that amino terminal fragments of expanded huntingtin can induce neuronal cell death (Hackam et al, 1998; Martindale et al., 1998; Saudou et al., 1998, Cooper et al, 1998). Moreover, inhibition of cleavage of expanded huntingtin by caspases inhibitors has been shown to greatly reduce both cellular toxicity and the accumulation of cytoplasmic and nuclear inclusions (Kim et al., 1999; Wellington 9 et al., 2000). These studies suggest that cleavage of mutant huntingtin may play an important role in the pathogenesis of HD. Huntingtin can also undergo proteolysis by calpains, a family of Ca2+-dependent cysteine proteases (Kim et al., 2001; Gafhi and Ellerby, 2002). Calpains cleave huntingtin in a polyglutamine-length dependent manner, where larger tracts confer greater cleavage (Gafni and Ellerby, 2002). These studies may be linked to previous findings indicating that intracellular Ca2+ levels are altered in both in vitro and in vivo models for HD (Li et al., 2000, Hodgson et al., 1999; Chen et al, 1999). Recently, Lunkes et al (2002) have demonstrated that an undefined aspartic endopeptidase can cleave huntingtin to generate two amino-terminal fragments termed cp-A and cp-B. These two fragments appear to play an important role in the biogenesis of inclusion within the nucleus and cytoplasm. The cp-A fragments are found exclusively in nuclear aggregates whereas the cp-B is found in the cytoplasmic inclusion in association with cytosolic proteins (Lunkes et al., 2002). 1.8 Cellular Functions of Huntington Although the exact functions of huntingtin are unknown, a growing body of literature has indicated that this protein may play an important role in a number of different cellular processes including cellular survival, transcriptional regulation, intracellular signaling, and cellular transport and endocytosis. 1.8.1 Cellular Survival Several groups have demonstrated that neuronal cell death in HD may involve apoptosis (Nasir et al., 1995; Thomas et al., 1995; Portera-Cailliau et al., 1995; Dragunow 10 et al, 1995). Similarly, Zeitlin et al. (1995) reported homozygote HD knockout mice that displayed higher levels of apoptosis. They postulated that wild-type huntingtin is involved in processes to counterbalance the apoptotic pathway. In vivo studies have shown that wild-type huntingtin can also mitigate the toxic effects of mutant huntingtin in a YAC mouse model of HD (Leavitt et al., 2001). In vitro studies suggest that expression of wild-type huntingtin can protect both neural and non-neuronal cells from toxic stressors such as serum deprivation, 3-nitropropionic acid (a mitochondrial toxin), and the expression of either full-length or amino-terminal fragments of mutant huntingtin (Rigamonti et al., 2000; Ho et al., 2001; Cattaneo et al., 2001). Huntingtin is thought to mediate its protective effect by down-regulating apoptotic signals upstream of caspase-3 activation and downstream of pro-apoptotic Bcl-2 family members, Bik and Bak (Rigamonti et al., 2000). Interestingly, Zuccato et al. (2001) have demonstrated that normal huntingtin can upregulate transcription of brain-derived neurotrophic factor (BDNF) whereas mutant huntingtin decreases BDNF expression. The loss of BDNF in cells expressing mutant huntingtin may ultimately lead to cell death (Zuccato et al., 2001; Zuccato et al., 2003). Finally, wild-type huntingtin can interact with the pro-apoptotic protein huntingtin interacting protein 1 (HIP-1) and thereby prevent it from inducing apoptosis (Hackam et al., 2000). HIP-1 will be discussed in greater detail in later sections. 1.8.2 Transcriptional Regulation It has been hypothesized that huntingtin may play a role in transcriptional regulation (Perutz, 1999). This theory is based, in part, on the observation that glutamine-rich regions are present in a number of transcription factors including the TATA binding protein (Kao et al, 1990) and Spl protein (Courey and Tjian, 1988). The glutamine 11 repeats within the HD protein may allow it to interact with proteins in the transcriptional machinery and the DNA itself (Perutz 1999; Perutz, 1994; Stot et al., 1995). The glutamine-rich region in the androgen receptor, which is expanded in SBMA, is believed to be important for interaction with other transcription factors. In addition, huntingtin can be found within the nucleus where it may interact with a number of different transcription factors and influence their activity. For a complete list of huntingtin-interacting transcription factors and their known functions please refer to table 1.2. Finally, examination of mouse models of HD expressing either full-length or an amino terminal fragment of mutant huntingtin exhibit marked alterations in the expression of various genes (Luthi-Carter et al, 2002; Chan et al., 2002). 1.8.3 Intracellular Signaling Huntingtin has been shown to be play a role of intracellular signaling based on its interaction with a number of different signaling proteins such as epidermal growth factor receptor signaling complex (Liu et al., 1997) and the JNK activator, mixed lineage kinase 2 (MLK2; Liu et al., 2000). For a complete list of huntingtin-interacting signaling proteins and their known functions please refer to table 1.2. Recently, huntingtin has also been shown to be phosphorylated by the serine / threonine kinase, Akt (Humbert et al, 2002). Activation of Akt has been shown to have a neuroprotective effect in models of neurodegenerative diseases (For reviews see Dore et al., 2000; Brunet et al., 2001). Interestingly, in vitro studies indicated that the phosphorylation abrogates the ability of mutant huntingtin to induce apoptosis (Humbert et al., 2002). 12 1.8.4 Cellular Transport and Endocytosis Immunohistochemical studies indicate that huntingtin is localized to the intracellular membranes and the cytoskeleton. Huntingtin is present in the axon and nerve terminals where it appears to be associated with microtubules and synaptic vesicles (DiFiglia et al, 1995; Gutekunst et al., 1995; Tukamoto et al., 1997). In addition, Velier et al. (1998) have demonstrated that huntingtin may play a role in vesicle transport in the endocytic pathways through associations with clathrin-coated vesicles. Consistent with these findings is the observation that huntingtin contains HEAT repeat domains (Andrade and Bork, 1995; for a review see Andrade et al., 2001). The HEAT domains mediate protein-protein interactions and are present in proteins that perform multiple cellular functions. Of particular interest is importin, a HEAT repeat containing protein, which is involved in the transport of proteins from the cytoplasm to the nucleus (Andrade and Bork, 1995). By analogy, huntingtin may also play a role in transport. Huntingtin has also been shown to interact with a number of proteins that are proposed to have a role in transport and endocytosis (see table 1.2). These interacting proteins will be discussed in greater detail below. 13 CU u a c #© "-C u e c GO O v-CL c/l cd Pi CD Cu CD CO cd C 15 _c '5 o L. CL —1 o D. 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Since HD results in the selective loss of neurons, it has been suggested that the mutant HD protein mediates its effects by interacting with other brain-specific proteins. Several studies have determined that huntingtin interacts with components of the endocytic machinery and cellular transport. These proteins include HAP1, (Li et al., 1995), HIPl (Wanker et al., 1997; Kalchman et al., 1997); SH3GL3 (Sittler et al, 1998), a-adaptin-C (Faber et al., 1998), HIP 14 (Singaraja et al., 2002) and Cdc42-interacting protein 4 (Holbert et al., 2003). Each of these proteins have been implicated in the process of clathrin-mediated endocytosis (CME) and vesicle transport. CME will be discussed in greater detail in section 1.10. Please refer to figure 1.2 for a diagram of the different stages of clathrin mediated endocytosis. The nature of these interactions and their relevance to the pathogenesis of HD are discussed below. 1.9.1 Huntingtin Associated Protein 1 The first known huntingtin-interacting protein HAP1 (Huntingtin associated protein) was identified using the yeast 2-hybrid system (Li et al., 1995). The interaction between HAP1 and huntingtin has been shown to be enhanced by an expanded polyglutamine tract (Li et al., 1995). HAP1 is highly expressed in the neurons of the cortex and striatum, regions involved in HD pathology. However, it is also expressed in the cerebellum and brainstem, regions unaffected in HD (Gutekunst et al., 1998). Interestingly, the expression of HAP 1 in the striatum was more widespread than huntingtin suggesting that the interaction between these two proteins only occurs in a 16 subset of the striatal neurons (Gutekunst et al., 1998). HAP1 shows no similarity with other known proteins that could provide clues as to its normal cellular function. Within the neuron, HAP1 and huntingtin displayed a similar cellular distribution, including several different membranous organelles such as mitochondria, endoplasmic reticulum, lysosomal, endocytic and synaptic vesicles (Gutekunst et al., 1998; Martin et al., 1999). In addition, huntingtin and HAP1 were also associated with microtubules (Gutekunst et al., 1998). Taken together these findings suggest that HAP1 and huntingtin may play a role in vesicle trafficking and organelle transport. Consistent with this idea is the observation that HAP1 interacts with pl508'ued, a subunit of the microtubule-based transporter complex dynein (Englender et al, 1997, Li et al., 1998). In addition, HAP1 has been shown to interact with duo, a guanine nucleotide exchange factor that is a regulator of actin cytoskeletal dynamics (Colomer et al., 1997). HAP1 has also been shown to interact with hepatocyte growth factor-regulated tyrosine kinase (Hrs), a protein involved in the regulation of vesicle transport from the early endosomes to the late endocytic compartments (Li et al., 2002). At this time it is unclear how the enhanced binding of HAP1 to mutant huntingtin may lead to some of the pathological changes seen in HD. It may be possible that the increased interaction may somehow perturb HAP1 's ability to transport vesicles which may ultimately lead to the cellular dysfunction or death seen in HD. Recently, it has been demonstrated that HAP1 interacts with type 1 inositol (l,4,5)-triphosphate receptor, InsP3Rl (Tang et al., 2003). I11SP3RI is an intracellular calcium channel which functions in calcium signaling within neurons. In addition, Tang et al. (2003) have identified a ternary complex consisting of InsPsRl, HAP1 and 17 huntingtin. Interestingly, activation of InsPsRl by its ligand, InsP3, is sensitized by the presence of mutant huntingtin, resulting in an increase in intracellular calcium. It is postulated that the increased calcium levels may activate calcium-dependent degradative enzymes and generate reactive oxygen radicals, both of which result in cellular dysfunction and death (Albin et al., 1992). Indeed, several studies have reported that intracellular calcium levels are elevated in HD patients and mouse models of HD (Albin and Greenamyre et al, 1992, Hodgson et al., 1999; Zeron et al., 2002). Chan et al. (2002) have generated mice with a targeted disruption in the HAP1 gene (HAPV1'). The HAP1~'~ mice displayed a depressed feeding behavior which resulted in postnatal death. The role of HAP 1 in normal feeding activity is unclear. 1.9.2 a-Adaptin-C A yeast two hybrid screen of a human fetal brain library demonstrated that huntingtin with an expanded polyglutamine tract (58 and 62 repeats) interacted with the endocytic machinery component a-adaptin-C (Faber et al., 1998). At this time it has not been tested whether a-Adaptin-C also interacts with wild-type huntingtin. a-adaptin-C is a subunit of the adapter protein 2 complex (AP2). AP2 consists of four subunits: a-adaptin, p2-adaptin, u2-adaptin and o-adaptin (Slepnev and DeCamilli, 2000). This heterotetrameric complex has been shown to interact with membrane lipids and various components of the endocytic machinery (Slepnev and DeCamilli, 2000). Indeed, the AP2 complex has been shown to play a role in the cellular process referred to as clathrin mediated endocytosis (CME) at the cytoplasmic membrane (Brodsky et al., 2001). During CME, AP2 is implicated in the recruitment and polymerization of clathrin 18 subunits which ultimately form clathrin-coated vesicles (Brodsky et al., 2001). Furthermore, AP2 is also thought to help synchronize the process of endocytosis with intracellular signaling pathways (Slepnev and DeCamilli, 2000; Brodsky et al., 2001). The process of CME will be discussed in greater detail in section 1.12. Since it is unknown whether wild-type and mutant huntingtin differ in their binding affinity to a-adaptin-C, it is difficult to speculate as to role these interactions play in the pathogenesis of HD. 1.9.3 Endophillin A3 Sittler et al. (1998) have demonstrated that huntingtin can interact with the endocytic protein endophillin A3 (also known as SH3GL3). Endophillin A3 contains a SH3 domain which has been shown to mediate its interaction with the polyproline-rich domain in huntingtin. Moreover, an increased strength of interaction was shown to correspond to larger lengths in the polyglutamine tract of huntingtin (Sittler et al., 1998). Although the exact function of endophillin A3 is unclear, it has been postulated that it plays a role in the induction of membrane curvature during clathrin-dependent endocytosis (Slepnev and De Camilli, 2000). Similar to huntingtin, the highest levels of endophillin A3 expression are seen in the brain and testes (Sittler et al., 1998). In vitro studies using cultured cells have indicated that endophillin A3 co-localizes with mutant huntingtin in both nuclear and cytoplasmic inclusions (Sittler et al., 1998). In addition, over-expression of endophillin A3 was shown to enhance the accumulation of aggregates in cultured cells expressing mutant huntingtin (Sittler et al., 1998). These findings suggest that endophillin A3 plays a role in aggregate formation that is seen in the brains 19 of HD patients. Furthermore, endophillin A3's functions in endocytosis may be compromised due to sequestration within aggregates. Thus, disruption of clathrin-mediated endocytosis might play a role in the pathogenesis of HD. 1.9.4 Pacsin 1 Recently, huntingtin has been shown to interact with the neurospecific protein Pacsin 1 (also known as syndapin; Modregger et al., 2002). The interaction was mediated by the SH3 domain of Pacsin 1 and the polyproline region of huntingtin. In addition, the strength of this interaction was enhanced by increased length of the polyglutamine tract in huntingtin (Modregger et al., 2002). Pacsin 1 is thought to play an important role in the recruitment and stimulation of the actin polymerization machinery at the site of newly formed endocytic vesicles (Modregger et al., 2002; Qualmann and Kessels, 2002). Within the neuron, Pacsin 1 is normally located throughout the cytoplasm and along neurites and presynaptic boutons (Modregger et al., 2002). Immunohistochemical studies by Modregger et al. (2002) have demonstrated that huntingtin shows partial co-localization with Pacsin 1 in the cytoplasm of neurons. However, examination of brain material from early-stage HD patients indicated a conspicuous loss of Pacsin 1 in the neuronal processes (Modregger et al., 2002). The altered distribution of Pacsin 1 may be due to its enhanced interaction with mutant huntingtin. Taken together, the loss of Pacsin 1 at the synaptic boutons may cause defects in endocytosis which may play a role in the pathogenesis of HD. 20 1.9.5 Huntingtin Interacting Protein 14 Singaraja et al. (2002) have recently demonstrated that huntingtin interacts with a novel trafficking and endocytosis protein termed huntingtin interacting protein 14 (HIP 14). The strength of the interaction was found to be inversely correlated with the length of the polyglutamine tract in huntingtin (Singaraja et al., 2002). HIP 14 was found to be predominately expressed in neurons within the brain. Interestingly, HIP 14 was found to co-localize with huntingtin in the striatal medium spiny neurons; the cells that are selectively lost in HD (Singaraja et al., 2002). Within these neurons, HIP 14 was localized to the Golgi apparatus and cytoplasmic vesicles. Similarly, previous studies by Velier et al. (1998) have indicated that huntingtin is also enriched on Golgi membranes and cytoplasmic vesicles. HIP 14 has been shown to have 41% amino acid conservation to the Saccharomyces cerevisiae endocytic protein Akrlp. Akrlp has been shown to be essential for the endocytosis and transport of the yeast pheromone receptor Ste3p to degradatory vesicles (Kao et al., 1996). Thus, by analogy, HIP14 may also play a role in endocytosis and vesicle trafficking within neurons. Consistent with this hypothesis is the observation that HIP 14 can partially restore endocytosis of Ste3p when expressed in S. cerevisiae cells lacking Akrlp (Singaraja et al., 2002). Thus, it may be postulated that the decreased interaction between HIP 14 and mutant huntingtin may disturb endocytosis and intracellular transport and that these dysfunctions may play a role in the pathogenesis of HD. 21 1.9.6 Cdc42-Interacting Protein 4 Using the yeast two-hybrid system to screen a Caenorhabtidis elegans cDNA library, Holbert et al. (2003) demonstrated that huntingtin can interact with Cdc42-interacting protein 4 (CIP4). The two-hybrid and biochemical analyses indicated that CIP4 exhibits greater affinity for mutant huntingtin than wild-type. Furthermore, Holbert et al. (2003) showed that the SH3 domain present in CIP4 mediates binding to the proline-rich domain in huntingtin. Presumably, the enlarged polyglutamine tract in mutant huntingtin causes a conformational change in the protein that enhances CIP4 interaction. Immunohistochemical examination of the brain material from HD patients and age-matched controls indicated that CIP4 protein levels positively correlated with the severity of neuropathology in the striatum of HD patients (Holbert et al., 2003). Within the neurons CIP4 was found to partially colocalize with ubiquitin-positive aggregates (Holbert et al., 2003). Interestingly, overexpression of CIP4 in cultured rat striatal neurons resulted in apoptotic cell death (Holbert et al., 2003). As the name implies, CIP4 interacts with the activated form of the small GTPase, Cdc42 (Aspenstrom et al., 1997). In addition, CIP4 also independently interact with Wiskott-Aldrich syndrome protein (WASP; Tian et al., 2000). Both Cdc42 and WASP have been shown to be important regulators of cytoskeletal dynamics (Ramesh et al., 1999). In addition, CIP4 interaction with WASP has been shown to play an essential role in the transport and recycling of the glucose transporter 4 (Glut4; Jiang et al., 2002). Taken together, these findings suggest that CIP4 is involved in several cellular functions including endocytosis, transport and regulation of actin cytoskeleton. It is possible that 22 the enhanced interaction of mutant huntingtin with CIP4 may result in disruptions of these cellular functions. 1.9.7 Huntingtin Interacting Protein 1 Two research groups independently isolated the huntingtin interacting protein, HIP1, using the yeast 2-hybrid system (Kalchman et al., 1997; Wanker et al., 1997). HIP1 has 2 features that are suggestive of its role in HD: 1) HIP1 is highly expressed in the brain, although the mRNA can be found throughout the body (Kalchman et al., 1997). 2) Increased length of the huntingtin polyglutamine tract is associated with decreased interaction with HIP1 (Kalchman et al., 1997). The HIP1 gene has been shown to map to human chromosome 7ql 1.2 and produce a 120kDa protein. Subcellular localization of HIP 1 in the brain revealed that the protein is associated with the intracellular membranes in a manner similar to many cytoskeletal and cytoskeletal-associated membrane proteins including actin (Kalchman et al., 1997). As mentioned previously, huntingtin is membrane-associated, co-localizes with microtubules and may be involved in vesicle trafficking (Difiglia et al., 1995; Gutekunst et al., 1995). The fact that HIP1 is seen exclusively bound to the cytoskeleton, a site where huntingtin can also be found, suggests that the huntingtin-HIPl interaction occurs at this site (Kalchman et al., 1997). Interestingly, HIP1 also shares significant sequence similarity and biochemical properties with the known cortical cytoskeletal protein Sla2p (Synthetic Lethal with Actin binding protein-1 #2) in Saccharomyces cerevisiae and CeHIPl (or ZK370.3) in Caenorhabtidis elegans (Kalchman et al, 1997; Wanker et al., 1997). Furthermore, HIP1 has been shown to have a homolog termed HIP 12 (or HIPIr) which 23 will be discussed in depth throughout this thesis (Chopra et al., 2000, Engqvist-Goldstein et al., 1999; Seki et al., 1999). Taken together, these findings suggest that huntingtin-HIP1 interaction may play an important role in the normal membrane dynamics and vesicular trafficking and that the loss of normal interaction may play a role in HD. Recently, Metzler et al. (2003) have generated mice with a targeted disruption in the HIPl gene (HIPl'1') to determine the cellular function of HIPl in vivo. The HIPl'1' mice exhibited gait ataxia, tremor and the development of a rigid thoracolumbar kyphosis which was the result of neurological dysfunction. Furthermore, these mice exhibited a profound dose-dependent defect in the internalization of glutamate subtype, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor. These findings suggest that HIPl regulates AMP A receptor internalization in the CNS. It may be postulated that by association huntingtin may also play a role in AMPA receptor trafficking. HIPl expression has been shown to correlate with neoplastic changes resulting in uncontrolled cellular growth (Ross et al., 1998; Ross and Gilliland, 1999; Saint-Die et al., 2001; Rao et al., 2002, Rao et al., 2003). Paradoxically, it has also been suggested that HIPl is a pro-apoptotic protein in light of evidence demonstrating that it can induce both the intrinsic and extrinsic cellular death pathways (Hackam et al., 2000; Gervais et al., 2002). In order to gain greater understanding of the role of HIPl in HD pathogenesis, the following sections will review evidence implicating HIPl in apoptosis, neoplasia, cytoskeletal dynamics, and endocytosis. 1.9.7.1 The Pro-Apoptotic Function of HIPl As mentioned previously, several lines of evidence suggest that activation of cell death pathways may play a role in the pathogenesis of HD. Interestingly, Hackam et al. 24 (2000) have demonstrated that overexpression of HIP 1 in NT2 cell lines results in the morphological changes consistent with apoptosis including retraction of cytoplasmic extensions, cell rounding, blebbing and fragmentation of nuclear DNA. In addition, overexpression of HIP 1 in vitro induced caspase 3 activation in a caspase 8 and usurpin-independent manner. These findings suggest that HIP1 can activate the intrinsic cell death pathway. The intrinsic pathway is normally activated by cytochrome c release from the mitochondria. This release leads to the activation of caspase 9 that in turn, activates caspase 3 (for review see Ashkenazi and Dixit, 1999). Sequence analyses have demonstrated that HIP1 contains an 82 amino acid death effector domain (DED) starting at amino acid position 376 (Hackam et al., 2000). Furthermore, site-directed mutagenesis of a phenylalanine residue at amino acid residue 398 completely inhibited the ability of HIP 1 to induce apoptosis. Several activators of apoptosis such as caspases 8 and 10, also contain DEDs that are thought to mediate protein-protein interactions that play a role in the induction of apoptosis (for review see Rasper et al., 1998). Since HIP1 binds mutant huntingtin with reduced affinity, it is possible that the unbound HIP 1 may induce cell death via the intrinsic pathway. Thus, the "free" HIP1 may play a role in the apoptosis seen in brain tissue of individuals affected with HD. More recently, in vitro studies have reported that HIP 1 can also induce the activation of the extrinsic cell-death pathway (Gervais et al., 2002). The extrinsic pathway is usually initiated by the activation of either the fas or the tumor necrosis factor receptor. The stimulated receptor interacts with DED-containing proteins that recruit pro-caspase 8 to the plasma membrane and activate it. The activated caspase 8 induces 25 caspase 3 activity which results in the initiation of apoptosis (for review see Ashkenazi and Dixit, 1999). Gervais et al. (2002) established that HIPl interacts with a novel DED-containing protein termed HIPPI (HIPl protein interactor). Immunohistochemical studies indicated that HIP 1 and HIPPI proteins have similar patterns of expression in the brain and peripheral tissues (Gervais et al., 2002). Interestingly, the HIPl - HIPPI heterodimers were shown to interact with and activate caspase 8 (Gervais et al., 2002). The HIP1-HIPPI mediated activation of caspase 8 may represent the signal inducing apoptosis. In HD, mutant huntingtin would be expected to bind HIPl with decreased efficiency. The unbound HIPl may then interact with HIPPI and induce activation of the extrinsic cell death pathway. HIPl activation of the intrinsic and extrinsic cell death pathways are illustrated in figure 1.1. 1.9.7.2 The Role of HIPl in Cellular Transformation There is accumulating evidence to suggest that HIPl may play a role in the development of neoplastic changes in cells. Ross et al (1998) identified a t(5;7)(q33;ql 1.2) chromosomal translocation in a patient with chronic myelomonocytic leukemia (CMML). The translocation resulted in the generation of a chimeric protein containing amino acids 1 to 950 of HIPl fused with the transmembrane and tyrosine kinase domain of the platelet-derived growth factor (3 receptor (PDGFPR). Ross et al. (1998) demonstrated that expression of the HIPl - PDGFPR chimeric protein (HIPl/ PDGFpR) can induce cellular transformation of the murine hematopoietic cell line, Ba/F3. Within the transformed Ba/F3 cells, HIPl/ PDGFpR was shown to form oligomers and contain a constitutively active PDGFPR tyrosine kinase domain (Ross and 26 Gilliland, 1999). It has been demonstrated that HIP1/ PDGFPR expression correlates with tyrosine phosphorylation of cell signaling proteins STAT5 and Src homology 2-containing inositol 5-phosphatase (SHIP1) (Ross and Gilliland, 1999; Saint-Die et al., 2001). The tyrosine phosphorylated form of STAT5 has been shown to induce DNA synthesis and cell division (deGroot et al., 1998; Saint-Die et al., 2001). However, phosphorylation of SHIP1 has been shown to inhibit its ability to down-regulate hemapoietic proliferation (Saint-Die et al., 2001). Thus, activation of STAT5 and inhibition of SHIP 1 may result in unregulated cell growth resulting in transformation. Primary tissue microarray studies have indicated that HIP 1 protein is highly expressed in aggressive forms of prostate and colon cancer (Rao et al., 2002). Rao et al. (2002) examined cancer tissue microarrays using samples generated from small biopsies from patients with various types of cancer including prostate, colon, central nervous system, lung, ovary breast, kidney, and various leukemia and melanomas. These studies found that HIP1 was overexpressed in prostate and colon cancers relative to normal epithelium of prostate and colon. Furthermore, analysis of tissue microarrays from patients with various stages of prostate or colon cancer progression indicated that the level of HIP 1 protein was directly correlated with the severity of these cancers. Immunohistochemical examination in the transgenic mouse model for prostate cancer (TRAMP) demonstrated that HIP1 was highly expressed in the prostate tumors as compared to adjacent benign tissue (Rao et al., 2002). Interestingly, HIP1 was not highly expressed in all cells of the TRAMP mouse prostate tumors suggesting that HIP1 overexpression is not a prerequisite to tumor formation. 27 Figure 1.1. Two potential mechanisms how HIP1 induces apoptotic cell death. HIP1 displays reduced interaction to mutant huntingtin. The "unbound" HIP1 may then stimulate apoptosis through a pathway that involves activation of caspase-9, which then activates the downstream effector caspase-3. Caspase-9 is normally a component of the intrinsic cell death pathway that is activated by cytochrome c release from the mitochondria. HIP1, when combined with the HIP1 interactor HIPPI, has also been shown to elicit cell death via activation of caspase-8, which then activates caspase-3. Caspase-8 is part of the "extrinsic" cell death pathway that is normally stimulated by activation of cell-surface death receptors such as tumor necrosis factor. 28 Recently, Rao et al., (2003) have reported that overexpression of HIPl in vitro promotes transformation of NIH/3T3 fibroblasts and is also associated with alterations in the trafficking of several receptors including epidermal growth factor receptor (EGFR). Interestingly, overexpression of HIPl within the brains of HIPl transgenic mice does not appear to result in tumor formation (Dr. Metzler, personal communication). Thus, it is possible that the in vitro studies by Rao et al (2003) do not necessarily reflect physiological events underlying tumor formation in vivo. Alternatively, HIPl may simply perform different cellular functions in the brain as compared to peripheral tissues. 1.9.7.3 The HIPl Family of Proteins HIPl is a member of a growing family of membrane cytoskeletal-associated proteins. HIPl has been shown to share significant sequence identity and biochemical characteristics with the known SLA2p in S. Cerevisiae and CeHIPl in C. elegans and the hypothetical protein CGI0971-PA in D. melanogaster. Furthermore, humans and mice express a HIPl homolog termed HIP 12 (Chopra et al., 2000; Engqvist-Goldstein et al., 1999; Seki et al., 1999). All of the HIPl family of proteins are similar in molecular weight and contain an amino-terrninal ENTH (Epsin N-terminal Homology), a central coiled coil and a carboxy terminal talin-like domain. The ENTH domain is postulated to induce clathrin-mediated endocytosis through binding to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]-containing membranes (Ford et al., 2001; Itoh et al., 2001; DeCamilli et al., 2002). Furthermore, ENTH domains are also found in several other endocytic proteins including epsin 1 and 2, adaptor protein 180 (API80) and its homologue, clathrin assembly lymphoid myeloid leukemia protein 30 (CALM; Ford et al, 2002; Ford et al, 2001; Itoh et al., 2001). Interestingly, each of these proteins also binds to clathrin. The ENTH domains of epsin 1 and 2, contain a type-I clathrin-binding sequence, referred to as a "clathrin box" which interacts with the amino-terminal domain of the clathrin heavy chain (Ford et al, 2002; Ford et al., 2001; Itoh et al., 2001). Proteins that bind both PtdIns(4,5)P2 and clathrin are thought to promote the accumulation and assembly of clathrin coat proteins to sites of receptor-mediated endocytosis (DeCamilli et al., 2002 Kay et al., 1999; Rosenthal et al., 1999). The central region of HIP 1, HIP 12, Sla2p, CeHIPl and CGI 0971 contains a predicted coiled coil domain that is postulated to mediate protein-protein interactions (Engqvist-Goldstein et al, 1999; Kalchman et al., 1997; Lupas, 1996; Parker, 2001; Wanker et al., 1997). In the case of Sla2p and HIP 12, the coiled coil domains are critical for the localization of these proteins to the plasma membrane (Engqvist-Goldstein et al., 1999; Wesp et al., 1997; Yang et al., 1999). It may be speculated that the coiled coil domains may tether Sla2p (or HIP 12) to proteins that are present at the plasma membrane (Yang et al., 1999; Barr, 2000). The carboxy-terminal domains of the HIP1 family of proteins share significant homology with the mammalian membrane cytoskeletal-associated protein, talin (Kalchman et al., 1997). Talin is found at sites where the cytoskeletal element actin is linked to the extracellular membrane via integral membrane proteins (Critchley, 2000; Moulder et al., 1996). The talin-like domain present in the HIP1 family members consists of four sub-domains collectively referred to as the I/LWEQ module. The I/LWEQ modules of Sla2p and HIP 12 have been shown to interact with F-actin (McCann and Craig, 1997; Engqvist-Goldstein et al., 1999). Thus, it is likely that the talin-like domain 31 in the HIPl family of proteins plays an essential role in targeting these proteins to the cortical cytoskeleton. Taken together, the ENTH, coiled coil and talin-like domains suggest that the HIPl family of proteins may be involved in the functioning of the actin cytoskeleton and endocytosis. The following sections will discuss what is known about the cellular functions of Sla2p and CeHIPl in order to suggest possible functions for HIPl. 1.9.7.4 Saccharomyces cerevisiae Sla2p The yeast actin-binding protein Sla2p has been shown to play an essential role in the assembly and function of the actin cytoskeleton in Saccharomyces cerevisiae (Holtzman et al., 1993). In addition, Sla2p has also been implicated in endocytosis, vesicle transport and cAMP signaling (Mulholland et la., 1997; Wesp et al., 1997; Blader et al., 1999). Sla2p was originally characterized as a null mutation (Sla2~/~) causing temperature sensitive growth defects related to a general disorganization of cell surface and actin cytoskeleton (Holtzman et al., 1993). For example, the Sla2~'~ cells possess abnormally thick cell walls and display a spherical shape as opposed to the characteristic ellipsoid appearance seen in wild-type cells (Holtzman et al., 1993). Moreover, during cellular division the Sla2~'~ cells do not undergo polarized cell surface growth. These cells have actin patches distributed evenly across the surface of the mother and bud, whereas in wild-type cells the cortical actin patches are spatially restricted to growing domains within the cell cortex (Holtzman et al, 1993; Yang et al, 1999). Interestingly, these defects in bipolar budding are also been reported in yeast with mutations in various components of the cortical actin cytoskeleton (Holtzman et al, 1993; Raths et al 1993, Na et al, 1995). 32 The two other known alleles at the SLA2 gene locus are referred to as End4 and Mop2 (Na et al 1995, Raths et al 1993). The mutants described as end4 were originally described as having temperature-sensitive growth phenotype and defects in receptor-mediated endocytosis (Raths et al., 1993). The mutants referred to as mop2 were shown to have temperature-sensitive growth defects that resulted an inability to regulate the levels of the yeast H+-ATPase (Pmalp) in the plasma membrane (Na et al., 1995). Taken together these findings suggest that Sla2p is required for endocytosis and the maintenance and distribution of integral membrane proteins. The generation of yeast cells with various SLA2 deletion mutants has helped to delineate the role of the talin-like, ENTH, and coiled-coil domains in Sla2p function. Biochemical studies by McCann and Craig (1997) have demonstrated that Sla2p can bind to F-actin through its talin-like domain. In addition, it has been shown that the ENTH domain contains a localization sequence that allows Sla2p to be associated with the cortical actin cytoskeleton. Thus, it would seem that the ENTH and talin-like domain both play a role in the localization of Sla2p to the cortical actin cytoskeleton (Wesp et al., 1997; Ayscough et al., 1997; Yang et al., 1999). At this time it is unclear what subtle differences in distribution can be attributed to these two domains. The central coiled-coil domain has been shown to play an essential role for the cellular activity of Sla2p. Yeast cells expressing Sla2p that lack the central coiled-coil domain display normal cellular distribution, however, they also contain defects in endocytosis, cellular growth and cytoskeletal organization (Yang et al., 1999). It has been postulated that the coiled-coil domain mediates its effects by interacting with other proteins involved in cytoskeletal dynamics and endocytosis (Lupas et al, 1991; Yang et al, 1999). 33 1.9.7.5 The Caenorhabtidis elegans HIPl (CeHIPl) The CeHIPl gene spans 4808 bp, contains 10 exons and codes for a 2798 bp message. The CeHIPl is 927aa in length and has a molecular weight of 104kDa. Within the adult hermaphrodites, CeHIPl is present in the pharynx, spermatheca, vulvar muscles and the gonads. In the adult male C. elegans, CeHIPl was found in the pharynx, vas deferens and cloacae (Parker, 2001). Through chemical mutagenesis (using trimethylpsoralen and ultraviolet light) and RNA interference (RNAi) studies it was determined that the CeHipl protein was found to be essential for normal development since no animals with a homozygous or heterozygous deletion of CeHIPl could be detected. Transgenic C. elegans containing an inducible CeHIPl construct were also developed to determine the effect of CeHIPl over-expression in these worms. Upon induction of the transgene the worms failed to thrive and developed intracellular vacuoles suggesting that excess CeHIPl may in some be toxic to the worm (Parker, 2001). RNAi studies were performed to silence the CeHIP 1 gene in adult C. elegans (Fire 1998; Parker 2001). The silencing of CeHIPl expression resulted in altered morphology and functioning of the pharynx as well as reduced production of fertilized oocytes (Parker, 2001). The exact function of CeHIPl in pharynx and gonads is unclear at this time. It has been postulated that since CeHIPl contains a talin domain, the protein most likely interacts with the actin cytoskeleton and thereby helps to maintain the structural integrity of the cells within the pharynx and the gonads (Parker, 2001). Furthermore, since CeHIPl contains an ENTH and coiled coil domains, it may also perform a critical role in the endocytic and transport pathways within the pharynx and gonads of C. elegans (For a review see Riddle et al 1997; Parker, 2001). 34 1.9.7.6 Huntingtin Interacting Protein 12 (HIP12) The role of the HIP1 in membrane dynamics and vesicle transport is supported by the observation that it is structurally similar to its homolog HIP 12 (Chopra et al., 2000; Engqvist-Goldstein et al., 1999). The murine homolog of HIP 12, mHIP12 (or mHIPlr) has been shown to play an essential role in receptor-mediated endocytosis and vesicular transport (Engqvist-Goldstein et al., 1999). mHIP12 was initially isolated by our laboratory by screening a mouse brain cDNA library with mouse expressed sequence tags (ESTs) showing significant nucleotide identity (60%) with human HIP1 (Engqvist-Goldstein et al., 1999). The mHIP12 protein is 1068 amino acids in length and has a molecular mass of approximately 120 kDa. Like HIP1, Sla2p, and CeHIPl, the mHIP12 protein contains an amino-terminal ENTH domain, a central coiled-coil domain and a carboxy-terminal talin domain. Furthermore, mHIP12 is expressed in mouse embryos 7 days post-fertilization suggesting that this protein may play an important role during development. In the adult mouse, mHIP12 is expressed in both the central nervous system and peripheral tissues including kidney, liver, lung, spleen, testes (Engqvist-Goldstein et al., 1999). Within the cell, mHIP12 was shown to exhibit a distinctive punctate staining throughout the cytoplasm but not in the nucleus. Several lines of experimental evidence suggest that mHIP12 is involved in clathrin mediated endocytosis and actin cytoskeleton dynamics. In vitro binding assays indicated that the mHIP12 talin-like domain interacts with F-actin (Engqvist-Goldstein et al., 1999). Similarly, immunohistochemical studies demonstrated that mHIP12 colocalized with F-actin at the cortical cytoskeleton and in the perinuclear region of the cultured cell lines (Engqvist-Goldstein et al., 1999). At the plasma membrane, mHIP12 35 was found to colocalize with components of the receptor-mediated endocytic machinery. It was determined that mHIP12 co-localizes with the coat protein, clathrin, which is essential for vesicle formation at the plasma membrane and the trans-Golgi network (Engqvist-Goldstein et al., 1999; Brodsky et al., 2001). Moreover, mHIP12 was also found to colocalize with the adapter protein, AP2, which is known to play a role in the recruitment of clathrin at sites undergoing receptor-mediated endocytosis (Brodsky et al., 2001). In addition, mHIP12 was also found to colocalize with newly endocytosed vesicles containing labeled transferrin. However, as the transferrin-containing vesicles traveled further into the cytoplasm, their co-localization with mHIPlr decreased (Engqvist-Goldstein et al., 1999). Taken together, these findings suggest that mHIPlr may play an important role in the initial stages of receptor-mediated endocytosis. The generation and expression of various mHIP12 deletions constructs in culture have indicated that the ENTH, coiled-coil and talin-like domains play an essential role in cellular localization and receptor mediated endocytosis. Engqvist-Goldstein et al. (1999) demonstrated that both the ENTH and coiled-coil domains are required for normal cellular distribution and association with the endocytic machinery (Engqvist-Goldstein et al., 1999). The mHIP12 talin-like domain has been shown to be necessary for localization to F-actin. These findings suggest that mHIP12 may act a link between the endocytic machinery and the underlying actin cytoskeleton. 1.10 Clathrin-Mediated Endocytosis and the Cortical Actin Cytoskeleton As previously mentioned, HIP 12 (and potentially HIPl) have been shown to play a role in clathrin-mediated endocytosis (CME; Engqvist-Goldstein et al., 2000; Waelter et 36 al., 2001). CME is the major mechanism for the internalization and transport of proteins and lipids from the plasma membrane to the cytoplasm (Hirst and Robinson, 1998). For example, at the neuronal synapse, CME has been shown to be critical in the recycling of synaptic vesicles following neurotransmitter release (Slepnev and DeCamilli, 2000). CME in neurons can be divided into 4 different stages: 1) nucleation, 2) budding, 3) scission and 4) uncoating of the clathrin-coated vesicle (please refer to figure 1.2). During nucleation, the adapter proteins, AP2 and API80, are recruited to discrete sites on the plasma membrane. The adapter proteins aid in the recruitment of the clathrin units to the cell membrane. The clathrin units are composed of three heavy and light chains that form a three-legged triskelion. These clathrin triskelia self-assemble into the curved polygonal lattice structure which is referred to as the clathrin-coated pit (Brodsky et al., 2001). During the budding phase, the clathrin-coated pit develops a pronounced curvature until a narrow neck forms at the plasma membrane. The highly complex budding process is regulated by the coordinated activity of several proteins that are associated with the clathrin-coat. These proteins include the adapter proteins amphiphysin, intersectin, EPS 15, and epsin. In addition, the phospholipid metabolism enzymes endophillin and synaptojanin have also been shown to regulate the budding process (Brodsky et al., 2001; Slepnev and DeCamilli, 2000). During fission, ring-like structures form around the neck of the clathrin-coated pits and "pinch" off clathrin coated vesicles. The ring-like structure is composed of the GTPase, dynamin, in association with amphiphysin and endophillin (Slepnev and DeCamilli, 2000). Finally, the clathrin-coated vesicle is rapidly uncoated through the activity of the chaperone HSP70 and its neuron-specific cofactor auxilin (Slepnev and DeCamilli, 2000). 37 Figure 1.2. The different stages of clathrin mediated endocytosis (CME; see section 1.10). The light blue line represents the cytoplasmic membrane. The green hexagons represent the clathrin subunits. The red "Y" structure represent AP2. The dark blue ovals represent other coat proteins such as AP180 (and potentially HIPl and HIP12). The plum circles represent other accessory proteins including amphiphysin, endophillin and intersectin. The grey ovals represent dynamin. Finally, the yellow rods represent actin filaments. The huntingtin interacting proteins HAP1, AP2, endophillin, pacsin, HIPl, HIP 14, and CIP4 are all thought to play important during four stages of CME. 38 The cortical actin cytoskeleton has been speculated to perform a number of different functions associated with CME. The actin cytoskeleton may help to anchor the endocytic machinery to specific sites on the plasma membrane by either binding to the endocytic components or restraining their mobility (Qualmann et al., 2000). Consistent with this idea are the observations that exposure of cultured cells to the actin depolymerizing drug latrunculin B, causes a diffuse localization of the endocytic machinery across the cell surface and an inability to perform CME (Santini et al., 2002). The cortical actin cytoskeleton is also hypothesized to undergo deformation or invagination at the site of endocytosis, and thereby assist in the generation of the clathrin-coated pits (Qualmann et al., 2000). Alternatively, there may be a selective depolymerization of the actin cytoskeleton at the site of endocytosis to allow the expansion and growth of the clathrin-coated pit (Fujimoto et al., 2000; Qualmann et al., 2000). Finally, rapid polymerization of actin at the site of endocytosis may generate the force necessary to release the clathrin-coated vesicle from the plasma membrane and transport it further into the cytoplasm (Qualmann et al., 2000; Merrifield et al., 1999). The propulsive force of the actin cytoskeleton has been visualized as a "comet tail" where long chains of actin extend from the plasma membrane to the newly formed clathrin-coated vesicle (Merrifield et al., 1999).These findings suggest that clathrin-mediated endocytosis requires the complex interplay between endocytic machinery and the underlying cortical actin cytoskeleton. Interestingly, HIP1 and HIP 12 contain structural domains that allow these proteins to interact with both the endocytic machinery and the 40 cortical actin cytoskeleton. Thus, HIP1 and HIP 12 may represent a molecular link between the endocytic machinery and the underlying cortical actin cytoskeleton. 1.11 Hypotheses The following hypotheses will be addressed in this thesis 1) HIP1 is a member of a family of cytoskeletal-associated proteins that exhibit differences in their ability to both interact with huntingtin and to induce apoptosis. 2) The HIP1 family of proteins are components of the clathrin-mediated endocytic machinery and the actin cytoskeleton. 1.12 Thesis Objectives To address the above hypotheses, these are specific objectives for this thesis: 1) To identify members of the huntingtin interacting protein 1 family by using ESTs to screen both mouse and human cDNA libraries. 2) To sequence and assess the similarities or identities of the isolated HIP1 family members with HIP1, Sla2p and CeHIPl. 3) To identify the chromosomal location of the isolated HIP1 family members. 4) To characterize the mRNA and protein expression pattern of the isolated HIP1 family members. 5) To assess whether the novel HIP1 family members interact with huntingtin or HIP1 using the yeast two-hybrid system. 6) To address whether HIP1 family members activate apoptosis. 41 7) To determine whether the human HIPl family members are associated with components of clathrin-mediated endocytic machinery. 8) To determine whether the human HIPl family members interact with actin filaments. 9) To identify domains present in the human HIPl family members that are required for normal function and localization. 42 Chapter 2: Materials and Methods 43 2.1 Materials and Methods for Chapter 3 2.1.1 Isolation ofHIP12 Human HIP 12 was initially identified as a result of our efforts to obtain a murine homolog of HIPl. The published nucleotide sequence of human HIPl cDNA (accession # U79734) was used to screen the GenBank EST database (dbEST) using BLASTN. We obtained 2 different murine ESTs which showed homology to HIPl: aal 10840 (clone 520282) and w82687 (404331). Please see figure 2.1. A 1.3kb Eco RI & Nco I fragment of clone 404331 was used to screen a mouse brain lambda ZAPII cDNA library (Stratagene) using manufacturer's suggested protocols. We isolated a 3.9 Kb cDNA clone, which we termed mHIP12, that showed nucleotide and amino acid identity with HIPl. The entire mHIP12 cDNA sequence was used to screen the non-redundant database of GenBank EST Division. We identified a human EST, T08283 (clone HIBBB80), which showed homology to mHIP12 but not HIPl. A 1.6Kb Hind IIII & Not I fragment of clone 404331 (containing EST T08283) was used to screen a human frontal cortex lambda ZAPII cDNA library (Gift from M. Montal, University of California, San Diego). We isolated a 3.2 Kb cDNA clone, termed HIP12, that showed nucleotide and amino acid identity with mHIP12 and HIPl. The remaining 5' portion of the HIPl2 cDNA was isolated using the 5' Rapid Amplification of cDNA Ends (RACE) Kit (Canadian Life Technologies). The first strand cDNA was synthesised using the oligo HIP 12-950 (5' ACC GCC 44 CO c g CD z CD sz O o h- o CO 00 CD oo h- <o H < ™ CN 00 CN CN CO o to 00 T" CD M\ CD CN 00 CO 00 CN CO . —1 o> I CM 1 m / 1 t CO CM 00 m CN 55 co If) If) 00 22 CM 00 CD CO CN ] oo 2-CD -Q CM O 00 CN 00 CM O 00 CL o n T~ O T- CD 5" ^ 9r oo -Q oo ^ co g CD CO CN CO co -Q CO O) T- O 5a CL CL X E CD CD CO h-CO CO >> XI "D 0 if= c CD £ 0 •5 +J CD SZ CM 2 CD O c CD CT 0 CO CD T3 '-4—' O 0) O C CL X c CD E Zi co .E CO LU CO O) CD -+—* CD O c 0 cr CD co T3 0 CO CO S> Q. X 0 0 CO Zl o CO c CO 0 CO CD jQ CD +J CD T3 !— CO LU c o CD E 75 O 'oi o o o 0 L— •+-» c 0 O ATT GAT GAT GGT AT). After dCTP tailing the first strand cDNA with terminal deoxynucleotide transferase, 2 rounds of 30 cycles (94 °C for 1 min; 58 °C for 1 min; 72 °C for 2 min) of PCR using HIP 12300 (5' CAC ATG GAC GAG CTC ACT GT) and the anchor primer (5'(CAU)4 GGC CAC GCG TCG ACT AGT ACG GGIIGG Gil GGG IIG) (Canadian Life Technologies) were performed. The subsequent 1.5Kb PCR product was cloned using the TA cloning system (Invitrogen) and sequenced. 2.1.2 DNA and Amino Acid Sequence Analyses The DNA sequences were analysed using the BLAST server at NIH (http://www.ncbi.nlm.nih.gov) to search for homology with nucleotide (BLASTN) and protein (BLASTP) sequences. Amino acid alignments were performed using CLUSTALW, converted to MSF format and transferred to GeneDoc. Coiled-coil prediction was performed using COIL at BCM search launcher (http://kiwi.imgen.bcm.tmc.edu:8088/search-launcher /launcher, html). 2.1.3 Determination of Additional 5' HIPl sequences A theoretical translation of the published HIPl 5' UTR showed significant sequence similarity to the amino terminal sequences of Sla2p, ZK370.3 and HIP12. 5' RACE and RT-PCR analyses were performed to determine whether HIPl contained additional 5' coding sequence. 5' RACE Analysis: 0.5 pg of Human Brain Marathon-ready cDNA library (Clontech) was used to isolate the 5' portion of HIPl by Rapid Amplification of 46 cDNA ends (RACE), following the manufacturer's recommendations. The primers used in the first round of PCR were a gene-specific primer-1 (5' CTT TCA ACT TTG TTA ACT GCT CC) and the supplied adaptor primer 1. The primers used for the second round of PCR were a nested gene-specific primer-2 (5' GGT TGC TGG AGC GGT AGA ACA G) and a supplied nested adaptor primer 2. The resulting PCR product was subcloned using the TA cloning system (Invitrogen) and sequenced. RT-PCR Analysis: 5 pg of total human brain RNA (Clontech) was used for first strand cDNA synthesis using the SUPERSCRIPT™ preamplification system (Canadian Life Technologies). The synthesized cDNA was diluted 1:10 and 1 pi of cDNA was used for PCR amplification (30 cycles: 92°C for 10 seconds; 60°C for 30 seconds and 72°C for 1 minute). The following primers were used: an exon 1-specific primer (PI) encompassing the upstream translation initiation site (5' GGA TGG CCA GCT CCA TGA AG), an exon 2-specific primer (P2; 5' GAG AGC TTC GAG CGG ACT CAG) and two exon 3-specific primers (P3 and P4) encompassing the downstream translation initiation site (5' GCC TCG GTC ATG GAT GTG AGC or 5' GGT CAT GGA TGT GAG CAA GA, respectively) were used in combination with an exon 10-specific primer (5' GAC ATG TCC AGG GAG TTG AA). The PCR products were separated on a 1% agarose gel and visualized following ethidium bromide staining. 47 2.1.4 Genome mapping of HIP12: FISH detection system and image analysis The full-length HIP 12 cDNA obtained by screening the human brain cDNA library and 5' RACE amplification was mapped by fluorescent in situ hybridization (FISH) to normal human lymphocyte chromosomes counter-stained with propidium iodide and DAPI. Biotinylated probe was detected with avidin-fluorescein isothiocyanate (FITC). Images of metaphase preparations were captured by a thermoelectrically cooled charge coupled camera (Photometries). Separate images of DAPI banded chromosomes and FITC targeted chromosomes were obtained. Hybridization signals were acquired and merged using image analysis software and pseudo coloured blue (DAPI) and yellow (FITC) as described and overlaid electronically. 2.1.5 Northern Blot Analyses A commercially prepared blot with mRNA from various human tissues and different brain regions (Clontech) was hybridized with a 1.2Kb HIPl fragment labelled using random-priming and incorporation of [a32 P]-dCTP. After washing the blot according to manufacturer's recommendations, the filter was exposed to X-ray film (Hyperfilm, Amersham). The filters were then stripped using 0.5% sodium dodecyl sulfate, reprobed using a [a32 P]-dCTP-labelled 675bp Pvu II HIP 12 -jo fragment and washed as before. The blots were then hybridized with randomly [a P]-dCTP-labelled (3-actin as an internal control to ensure that the mRNA on the blots was intact and to ensure equal loading. 48 2.1.6 Generation of Antibodies The HIPl and HIP 12 antibodies were generated using GST-tagged HIPl and HIP 12. The HIPl-GST expression vector was constructed by subcloning a 1.2 Kb HIPl Sal I fragment into pGEX-5X-l (Pharmacia). The HIP12-GST expression vector was generated by subcloning a 675bp HIP 12 Pvu II fragment into pGEX-4T-2 (Pharmacia). Each of the vectors was transformed into E. coli DH5-alpha (Canadian Life Technologies). The HIPl-GST and HIP12-GST proteins were isolated using methods previously described (Wanker et al., 1997). New Zealand white rabbits were injected with either HIPl-GST or HIP12-GST protein in Freund's adjuvant (Pierce, Rockford, Illinois). Antibodies against HIPl fusion protein (HIPlFP)or HIP 12 fusion protein (HIP12FP) were purified from rabbit sera using affinity column with low pH elution. The affinity columns were made by incubation of either HIPl-GST or HIP12-GST with activated thiol-sepharose (Pharmacia). 2.1.7 Protein Preparation and Western Blot Analyses Frozen human tissues were homogenized using a Polytron homogenizer in a buffer containing 0.25 M sucrose, 20 mM Tris-HCl (pH 7.5), 10 mM EGTA, 2 mM EDTA supplemented with a protease-inhibitor cocktail (Roche Diagnostics). The homogenate was centrifuged at 4000 rpm for 10 min at 4 °C to remove cellular debris. 100 pg / lane of protein was separated on a SDS-PAGE (7% acrylamide) gel and transferred to Immobilon-P membranes (Millipore) using standard protocols (Towbin et al., 1979). Western blot analyses were performed using 49 purified antibodies HIP1FP (1:100), HIP12FP (1:100) or a control monoclonal antibody for glyceraldehyde phosphodehydrogenase (1:500; Chemicon) and then detected using enhanced chemiluminescence (Amersham). 2.1.8 Construction of Yeast Two-Hybrid Vectors Huntingtin cDNA constructs with either 15 or 128 CAG repeats were generated encompassing amino acids 1-548 of the published huntingtin sequence. The yeast expression vectors pGBT9-1955-15 and pGBT9-1955-128 were constructed by subcloning a EcoR\-Sal\ fragment from pCI1955-15 or pCI1955-128 into pGBT9 (Clontech; Kalchman et al., 1997). Similarly, the EcoRl-Sal 1 fragment from pCI1955-15 or pCI 1955-128 into pGBT9 (Clontech)were fused in-frame adjacent to the GAL4-activating domain (AD) of the yeast two-hybrid vector pGAD (Clontech) generating pGAD-1955-15 and pGAD-1955-128. A 1513bp PCR product comprising nucleotide 642 to 2155 of HIP 12 was subcloned into the pCR2.9 TA cloning vector (HIP12pCR2.9). The fragment was sequenced to ensure that it was intact, excised from the TA vector using EcoRX, and blunt-end ligated into the Sma I site of both pGAD424 (HIP12pGAD) and pGBT9 (HIP12pGBT9). Yeast transformations were performed using a modified lithium acetate transformation protocol and grown at 30 °C using the appropriate synthetic complete (SC) dropout media (Geitz et al., 1996). P-galactosidase chromogenic assays were performed using procedures previously described (Kalchman et al., 1997). 50 2.1.9 Immunocytochemical Analyses of Neuronal NT2 cells and ES Cell Derived Neurons The neuronal precursor cell line NT2 (gift from Dr. L. Ellerby, Buck Institute, San Francisco, California) were washed three times with PBS and fixed with 4% (w/v) paraformaldehyde at room temperature (RT) for 15 minutes. Cells were permeabilized in PBS containing 1% paraformaldehyde and 0.3% Triton X-100 for 5 minutes, washed with PBS three times, and incubated with either H1P1FP or HIP12FP antibodies in PBS, 2%o normal goat serum overnight at 4°C. After extensive washing in PBS, slides were incubated with goat anti rabbit Alexa 488 (Molecular Probes, Eugene, Oregon) at a 1:800 dilution in PBS containing 2% normal goat serum for 2 hours at RT. Cells were washed 3 times with PBS, counterstained with TO-PRO (Molecular Probes) mounted in Mowiol (Aldrich) and analyzed by confocal laserscanning microscopy (BioRad Radiance Plus Confocal Microscope). Images were analyzed using NIH Image software. Double immunofluorescence was performed on embryonic stem (ES) cell-derived neurons 12 days after plating as previously described (Metzler et al., 1999). After fixation and permeabilization, the cells were incubated with either the HIP1FP or HIP12FP antibodies overnight at 4°C. Subsequently, cells were washed with PBS and incubated with a mouse monoclonal antibody directed against GM130 (Transduction Laboratories, Lexington, Kentucky) in PBS containing 2% normal goat serum. After extensive washing in PBS, slides were incubated with goat anti-rabbit Alexa 488 and goat anti-mouse Alexa 594 antibodies (Molecular Probes) at a 1:800 dilution in PBS containing 2% normal goat serum for 2 hours at 51 RT. Cells were washed and mounted in Mowiol (Aldrich). Immunofluorescence was detected using an Axioscope microscope (Carl Zeiss Inc.) Images were captured digitally with a CCD camera (Princeton Instruments Inc.). 2.1.10 Cell Viability Assays The entire coding sequence of HIP 1-2 and HIP 12 were each fused into the mammalian expression vector pCI (Promega). Human embryonic kidney 293T cells were plated in 96 and 6 well dishes (Becton-Dickinson) for the MTT and Caspase-3 activation assays, respectively in Dulbecco's modified eagle medium, 10% fetal bovine serum, 50 units/ml penicillin-streptomycin, and 2 mM L-glutamine (Canadian Life Technologies) and cultured at 37°C in 6% CO2. Cells were transfected with either the HIP 1-2 or HIP 12 constructs using the CaP04 method (Jordan et al., 1996), and the media was changed 24 hours post transfection. Transfection efficiency was 80-95%> as measured by fj-galactosidase staining. The HIPl and HIP 12 constructs produced similar levels of protein as determined by immunoblot assays. These transfected cells were used for either MTT or Caspase 3 activation assays. MTT Assays: At 48 hours post transfection, 10 pi of the cell proliferation reagent WST-1 (Roche Diagnostics) was added to each well, and the plates were incubated for an additional hour. The assay was quantified using a test filter of 450 nm and a reference filter of 630 nm. Statistical analyses were performed using one way ANOVA with a Neuman-Keuls post-test. 52 2.1.11 Caspase 3 Activation Assays At 24 hours post transfection, 2 ml of media, containing 35 LlM tamoxifen (Sigma) was added to each well for 4 hours. Cells were scraped on ice, washed with cold PBS, and DEVD-ase assays were performed as specified using the ApoAlert fluorometric kit (Clontech). The fluorescent substrate N-acetyl-Asp-Glu-Val-Asp-AFC (7-amino-4-trifluoromethyl coumarin) (BIOMOL) was used at a final concentration of 50|lM instead of the Clontech substrate. The plate was incubated in a Labsystems Fluoroscan Ascent fluorescent plate reader at 37°C for 75 minutes, with a reading obtained every 5 minutes, using excitiation at 390nm and emission at 510nm and Ascent Research edition 2.24 software. The DEVD-ase activity was normalized to protein concentrations. Statistical analyses were performed using one way ANOVA with a Neuman-Keuls post-test. 53 2.2 Materials and Methods for Chapter 4 2.2.1 Antibodies The mouse monoclonal anti-HIPl antibody (mAb HIPl#9) was generated by immunization of Balb/C mice with a GST-fusion protein encoding the huntingtin-interacting domain of human HIPl (1). Hybridomas were generated and cloned and HIPl mAbs were selected by ELISA with purified HIPl. Positive hybridomas were further characterized by Western blot and mAb HIPl#9 was found to specifically interact with human and mouse HIPl. Mouse mAbs recognizing a-adaptin, CHC or Rab5 were from Transduction Laboratories; mouse mAb directed against CHC was from Affinity Bioreagents Inc.; rabbit polyclonal anti a-adaptin was from Santa Cruz. Mouse mAbs directed against the Flag- and tetra-His epitope were from Sigma and Qiagen, respectively. 2.2.2 DNA constructs HIPl mammalian expression constructs were generated with HIPl splice variant 2 (4) that encodes the 1003 amino acid isoform of HIPl. HIPl cDNA sequences were cloned into the mammalian expression vector pCI (Promega) and tagged at their respective C-termini with the following cDNA sequence 5'-GGAGGTGGAGACTACAAGGACGACGATGACAAGTAG. This cDNA encodes a linker of 3 glycines, the Flag-tag, and a stop codon. The following HIPl-GST-fusion proteins were created by PCR amplification from full-length HIPl cDNA with subsequent cloning into the appropriate pGEX vectors (Pharmacia Biotech): GST-HIP 1-219-616, GST-HIP1-276-335. For the His6-tagged terminal domain (TD) of clathrin, a 54 GST-construct encoding the 579 amino acids of the CHC in pGEX-2T (generous gift of James Keen, Thomas Jefferson University, Philadelphia) was digested with Bamlil and EcoRl and the resulting insert was subcloned into the pTrcHisA (Invitrogen) at the same sites. All expression constructs were verified by DNA sequencing. 2.2.3 Immunofluorescence Cells were washed in phosphate-buffered saline (PBS) (20 mM NaH2P04, 0.9% NaCl, pH7.4), fixed in 4% paraformaldehyde in PBS, and permeabilized in 0.3% Triton X-100, P/o paraformaldehyde. Non-specific binding sites were blocked by incubation with 3% normal goat serum (NGS) in PBS. For double immunofluorescence, HIP1 polyclonal antibody was incubated in 2% NGS in PBS at 4°C overnight followed by incubation with mAbs directed against endocytic markers for 2 h at room temperature. Cells were extensively washed in PBS between each incubation. Cells were then incubated with secondary antibodies, washed, mounted and observed by confocal microscopy. 2.2.4 Transferrin uptake assays COS-7 cells plated on poly-L-lysine-coated coverslips were transfected, cultured 24 h and serum-starved overnight. The cells were then incubated with Cy3-conjugated human transferrin (25 Lig/ml) for 20 min at 37°C, washed three times with PBS, and fixed with 3.5%> paraformaldehyde in PBS. Following fixation, coverslips were processed for immunostaining with a monoclonal anti-Flag antibody as above. 55 2.2.5 Purification of CCVs CCVs from rat brain were purified as described (Maycox et al., 1992). A short version of the purification was adapted for transfected cells. Specifically, cells were homogenized in buffer A [0.1 M 2-(N-morpholino)ethanesulfonic acid pH 6.5, 1 mM EGTA, 0.5 mM MgCl2, 0.83 mM benzamidine, 0.23 mM PMSF, 0.5 pg/ml aprotinin and 0.5 pg/ml leupeptin] using a glass-Teflon homogenizer (10 strokes, 1500 rpm). The homogenate (H) was centrifuged at 17,800 X gmax for 20 min and the supernatant (SI) was collected and centrifuged at 56,100 X gmax for 1 h. The pellet (P2) was resuspended in buffer A using a glass-Teflon homogenizer (3 strokes, 1500 rpm) followed by dispersion through a 25-gauge needle. The resuspended pellet was loaded on top of a solution containing 8% sucrose in buffer A made in D20 and centrifuged for 2 h at 115,800 gmax. The pellet containing the CCVs was collected. 2.2.6 Pull-down and binding assays Adult rat brains were homogenized in buffer B (10 mM HEPES pH 7.4, 0.83 mM benzamidine, 0.23 mM PMSF, 0.5 pg/ml aprotinin and 0.5 pg/ml leupeptin) and a cytosolic fraction was generated by centrifugation at 205,000 gmax for 30 min at 4°C. Triton X-100 was added to the cytosolic fraction (2 mg) to a 1% final concentration and the samples were incubated overnight at 4°C with GST-fusion proteins pre-coupled to glutathione-Sepharose beads. After incubation, samples were washed three times in buffer B containing 1% Triton X-100. In other cases, a His6-tagged fusion protein encoding the clathrin terminal domain (TD; 25 pg) was incubated overnight at 4°C in 56 buffer B with the various GST-fusion proteins pre-coupled to glutathione-Sepharose beads. After incubation, samples were washed three times in buffer B. For all assays, specifically bound proteins were eluted in SDS-PAGE sample buffer and processed for Western blot analysis. 2.2.7 Immunoprecipitation analyses Forty-eight hours following transfection, 293T cells were washed in PBS, scraped in buffer B, sonicated and Triton X-100 was added to a 1% final concentration. The lysates were rocked for 30 min at 4°C and then centrifuged at 245,000gmax for 15 min. Supernatants were incubated overnight at 4°C with anti-Flag antibody pre-coupled to protein G-Sepharose. After incubation, samples were washed three times in buffer B containing 1% Triton X-100. Bound proteins were eluted in SDS-PAGE sample buffer and processed for Western blot analysis. 57 2.3 Materials and Methods for Chapter 5 2.3.1 Antibodies Rabbit polyclonal antibodies against HIP1 (HIP1FP) and HIP 12 (HIP12FP) and a mouse monoclonal antibody against HIP1 (mAb HIP no. 9) were previously described (section 2,1.5 and 2.2.1). Monoclonal antibodies against the clathrin heavy chain and x-adaptin were purchased from Transduction Laboratories. Monoclonal antibody against the clathrin light chain was purchased from Santa Cruz. Monoclonal antibodies against the FLAG, HA, and Hise epitopes were purchased from Sigma, Roche Molecular Biochemicals, and Qiagen, respectively. 2.3.2 DNA Constructs and Recombinant Proteins Mammalian expression constructs encoding full-length HIP1 and HIP 12 and the talin homology domain of HIP 1, each with a FLAG epitope tag at the C terminus, were previously described (Metzler et al., 2001). A construct encoding human full-length HIP 12 with a C-terminal HA epitope tag was generated by insertion of a cDNA sequence, 5'-GGAGGTGGATATCCCTATGATGTCCCCGATTATGCC, encoding a linker of 3 glycines followed by the HA tag. The integrity of the HA-tagged HIP 12 construct was verified by DNA sequencing. Bacterial fusion proteins encoding His6-tagged terminal domain of the clathrinheavy chain and the amino acids 276-335 of HIP1 fused to GST (GST-HIP1 -(276-335)) were previously described (Metzler et al., 2001). A His6-tagged construct encoding full-length clathrin light chain b was generated by subcloning the GFP-clathrin light chain b cDNA (kindly provided by Dr. Juan Bonifacino) into the 58 pTrcHisB bacterial expression vector (Qiagen). The following HIPl and HIP 12 GST fusion proteins were created by PCR amplification from either full-length HIPl or HIP 12 cDNAs with subsequent cloning into pGEX-2T or pGEX-4T vectors (Amersham Biosciences): GST-HIP 12-(302-348), GST-HIP 1-(336-610), GST-HIP 12-(349-644), GST-HIP 1 -(731 -1003) (GST-HIP 1 -talin), and GST-HIP 12-(765-1068) (GST-HIP 12-talin). 2.3.3 Pull-down Assays Rat brains were homogenized in buffer A (10 mM HEPES-OH, pH 7.4, 0.83 mM benzamidine, 0.23 mM phenylmethylsulfonyl fluoride, 0.5 pg/ml aprotinin, and 0.5 pg/ml leupeptin) and the homogenates were centrifuged at 205,000 x gmax for 30 min at 4 °C. The supernatant (cytosolic extract) was collected and Triton X-100 was added to 1% final concentration. Aliquots of the extract (2 mg) were incubated overnight at 4 °C with GST fusion proteins pre-coupled to glutathione-Sepharose beads. After incubation, samples were washed three times in buffer A containing 1% Triton X-100. For binding experiments, purified His6-tagged clathrin terminal domain or clathrin light chain b fusion proteins (15 pg) in buffer A were incubated overnight at 4 °C with various GST fusion proteins pre-coupled to glutathione-Sepharose beads. After incubation, samples were washed three times in buffer A. In all cases, proteins specifically bound to the beads were eluted with gel sample buffer and analyzed by SDS-PAGE and Western blot. For pull-downs from transfected cells, HeLa cells were grown on 10-cm2 dishes and transfected with 6 pg of HIPl or HIP 12 cDNA using FuGENE (Roche Molecular Biochemicals). Twenty-four h post-transfection, cells were washed twice with ice-cold 59 phosphate-buffered saline (PBS) and lysed in buffer A (containing 1% Triton X-100 and 10 u.M Pefabloc ( asulfonyl-type serine protease inhibitor; Roche Molecular Biochemicals). Samples were incubated overnight at 4 °C with GST fusion proteins pre-coupled to glutathione-Sepharose beads. After incubation, samples were washed three times in buffer A containing 1% Triton X-100 and 10 pM Pefabloc. Bound proteins were analyzed by SDS-PAGE and transferred to nitrocellulose for Western blot analysis. 2.3.4 Immunoprecipitation Assays HEK-293T cells were grown on 10-cm2 plates and transfected with 4 pg of HIP 12 cDNA by CaPOl precipitation. Transfected and nontransfected cells were washed 24 h post-transfection with ice-cold PBS and lysed in ABL buffer (20 mM Tris-Cl, pH 7.5,150 mM NaCl, 10% glycerol, 1% Triton X-100) supplemented with a 1:100 dilution of aprotinin (Sigma), 20 ng/ml leupeptin (Roche Molecular Biochemicals), and 10 pM Pefabloc. The cell lysates were sonicated and centrifuged at 245,000 x gmax for 15 min. The supernatants were then incubated for 6 h at 4 °C with mouse monoclonal antibody HIPl no. 9 pre-bound to protein G-Sepharose (Amersham Biosciences). Subsequently, samples were washed 6 times in ABL buffer and specifically bound proteins were analyzed by SDS-PAGE and Western blot. 2.3.5 Immunofluorescence Cells were plated onto gelatinized glass slides in 6-well tissue culture plates and transfected with 1 pg of HIPl or HIP 12 cDNA. Twenty-four h post-transfection, the cells were washed in PBS, fixed in PBS containing 4% paraformaldehyde for 15 min at room 60 temperature, and permeabilized in PBS containing 0.3% Triton X-100 and 1% paraformaldehyde for 5 min. The cells were then incubated in PBS containing 3% normal goat serum for 30 min to block nonspecific binding. Depending on the experiment, the cells were then incubated with monoclonal antibodies against the FLAG or HA epitopes for 1 h at room temperature or overnight at 4 °C with a polyclonal antibody against HIP1, followed by a 1 -h incubation at room temperature with appropriate secondary antibodies in PBS containing 2% normal goat serum. Staining of the actin cytoskeleton was achieved by incubation with Texas Red-phalloidin. Cells were then extensively washed in PBS, mounted, and observed on a BioRad Radiance Plus confocal microscope (BioRad, Hercules, CA) using Laser Sharp software (BioRad). 2.3.6 Actin-binding Assay Purified human non-muscle monomeric actin (Cytoskeleton Inc.) was polymerized as described previously (Engqvist-Goldstein et al., 1999) and incubated with purified GST-HIP 1-talin and GST-HIP 12-talin (20 jig each) for 1 h at room temperature in a final volume of 50 ul. The samples were then centrifuged for 30 min at 362,000 x gmax. Protein components of the pellets and supernatants were analyzed by SDS-PAGE followed by Coomassie Blue staining. Gels were scanned on a ScanJet 6300 (Hewlett Packard) and protein amounts were determined using Quantity One (Bio Rad) software. Statistical analysis was done by the Student's t test. 61 2.3.7 Clathrin Purification and Clathrin Assembly Assay CCVs were purified from adult rat brains as described (Maycox et al., 1992). To purify clathrin, coats were stripped from the vesicles by incubation in buffer B (0.5 M Tris, pH 7.0, 2 mM EDTA, 0.83 mM benzamidine, 0.23 mM phenylmethylsulfonyl fluoride, 0.5 pg/ml aprotinin, and 0.5 pg/ml leupeptin) for 15 min on ice. The samples were then centrifuged for 15 min at 245,000 x gm3X with the purified coat fraction remaining in the supernatant. The stripping and centrifugation steps were repeated a second time and the supernatants were pooled. The pooled supernatants were loaded onto a continuous 5-20% sucrose gradient made in buffer B and then centrifuged for 3.5 h at 195,000 x gmax in a Sorvall Step Saver vertical rotor. Fractions of 2 ml were collected and analyzed by SDS-PAGE andCoomassie Blue staining. Peak clathrin fractions were pooled anddialyzed overnight in clathrin assembly buffer (10 mM Tris-Cl,pH 8.5) and subsequently used for clathrin assembly assays. Clathrin assembly assays were performed with 0.5 pM purified clathrin and different concentrations (0.12-2 pM) of fusion proteins in a final volume of 90 pi of clathrin assembly buffer. Assembly was initiated at 4 °C by addition of 10 pi of 1 M 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.7. The mixture was kept on ice for 45 min and then centrifuged at 400,000 x gmax for 6 min. The supernatant (80 pi) was loaded on a SDS-PAGE and the clathrin assembly was quantified by Coomassie Blue staining. 62 Chapter 3: HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin The data presented in this chapter contributed to the manuscript: Chopra, V. S., Metzler, M., Rasper, D. M., Engqvist-Goldstein, A. E., Singaraja, R., Gan, L., Fichter, K. M., McCutcheon, K., Drubin, D., Nicholson, D. W., and Hayden, M. R. (2000) HIP 12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin. Mamm. Genome 11, 1006-1015. Unless otherwise stated the data presented in this chapter was obtained by myself. All contributions by other individuals will be discussed in the results section 3.2. 3.1 Introduction Huntingtin-interacting protein 1 (HIPl) is a 120 kDa membrane associated protein that was first identified through its interaction with huntingtin, a polyglutamine-containing protein associated with Huntington Disease (HD) (Kalchman et al., 1997; Wanker et al., 1997). The interaction between HIPl and huntingtin is polyglutamine-length dependent; the longer the polyglutamine repeat the less the interaction with huntingtin. Although huntingtin is expressed in all cell types, HD results from a selective loss of medium spiny neurons in the neostriatum and neurons within the deeper layers of the cerebral cortex (Hayden, 1981; Vonsattel et al., 1985; Sharp et al., 1995; for a review see Wellington et al., 1997). This selectivity of neuronal degeneration may be influenced by altered interactions with proteins such as HIPl, which is highly expressed in the brain. HIPl shows homology to Sla2p in Saccharomyces cerevisiae and to ZK370.3 in Caenorhabditis elegans (Wanker et al., 1997; Kalchman et al., 1997). Sla2p is essential for endocytosis and the assembly and function of the cytoskeleton (Holtzman et al., 1993, Raths et al., 1993 and Na et al., 1995) and for vesicle transport from the Golgi apparatus in yeast (Mulholland et al, 1997; Blader et al, 1999). A potential role in vesicle transport has also been suggested for huntingtin which is a cytosolic protein and often found associated with intracellular membranes, microtubules, synaptic and clathrin-coated vesicles (DiFiglia et al. 1995; Gutekunst et al., 1995; Velier et al., 1998). These observations suggest that the interaction between HIPl and huntingtin is necessary for vesicle trafficking and maintenance of the membrane cytoskeleton. As a consequence, the 64 altered interaction of HIP 1 with huntingtin could result in a disruption of cellular transport leading to neuronal cell death. Interestingly, huntingtin has been shown to be a substrate for the proapoptotic protease caspase 3 (Goldberg et al, 1996; Wellington et al., 1998). Furthermore, the caspase 3 cleavage sites present in huntingtin are adjacent to the domain which interacts with HIP1 (Wellington et al., 2000). Thus, it is possible that the reduced interaction between HIP1 and huntingtin containing an expanded polyglutamine tract may render huntingtin more susceptible to cleavage by caspases. In this chapter we determined the genomic organization of HIP1 and demonstrated that alternative splicing occurs at the HIP1 locus. We also identified a novel protein termed HIP 12 that shows profound sequence and biochemical similarities to HIP1 and also shows high sequence similarity to Sla2p and ZK370.3. However, HIP1 and HIP 12 differed in their abilities both to interact with huntingtin and to activate proapoptotic pathways. 65 3.2 Results 3.2.1 Identification of HIP12, a paralog of HIP1 In an attempt to identify the murine homolog of HIP I, a mouse brain lambda ZAPII cDNA library was screened with the mouse EST w82687 (clone 404331). EST w82687 was 420bp in length and showed 66% nucleotide identity with HIP1 cDNA. The EST was initially isolated from the non-redundant database of GenBank EST Division by performing a BLASTN search using a fragment of the human HIP I cDNA as the query. Subsequently a cDNA clone was isolated, termed mHIP12, that exhibited 47% nucleotide identity and 65% amino acid conservation with HIP I. The characterization of mHIP12 is published elsewhere (Engqvist-Goldstein et al., 1999). The entire mHIP12 cDNA sequence was used to screen the non-redundant database of GenBank EST Division. We identified a human EST, T08283, which showed 87% nucleotide identity with mHIP12 cDNA but not HIP1, indicating that HIP1 is a member of a gene family. T08283 was used to isolate a 3.2 Kb cDNA clone from a human frontal cortex cDNA library. Subsequently, 5' RACE was used in order to isolate 1.2 Kb of additional 5'sequence resulting in a 4.4 Kb cDNA sequence which contained an open reading frame of 3204 nucleotides encoding a protein of 1068 amino acids in length, which was termed hHIP12. Flourescence in situ hybridization demonstrated that hHIP12 was localized to chromosome 12q24.32 (figure 3.2.1). Recently, Seki et al. (1998) have also identified an 888 amino acid protein which is identical to HIP 12 but lacks the first 180 amino acid residues. 66 Propidium Iodide DAPI \ 12 Figure 3.2.1. Genomic mapping of the HIP12 locus. A single genomic locus for HIP12 was identified at 12q24.32 using the original cDNA probe isolated from the two-hybrid screen as the probe. 67 3.2.2 Alternative splicing at the human HIPl locus Protein sequence comparisons between HIP 12, HIPl, ZK370.3 and Sla2p suggested that the published sequence of HIPl (Genbank, accession number U79734) may be missing additional amino terminal sequences. Translation of the published HIPl 5' UTR showed significant sequence similarity to the amino terminal sequences of Sla2p and ZK370.3. Moreover, antibodies generated against a HIPl fusion protein detected an approximately 120 kDa protein which is larger than the expected molecular weight of approximately 100 kDa predicted from the previously published HIPl encoded by 914 amino acids. Subsequent analysis of human HIPl genomic sequences deposited in GenBank (accession number AC004491), and RT-PCR and 5' RACE analyses performed on total human brain RNA confirmed that human HIPl contained additional 5' sequences. Moreover, these studies led to the identification of 2 alternative splice variants, HIP1-1 and HIP1-2, which differ in their 5' sequences (figure 3.2.2A and B). The identification of the HIPl isoforms was a collaborative effort by Dr. M. Metzler, D. Rasper and myself. Both HIPl variants contain in frame translation initiation sites with a strong Kozak consensus sequence present in HIP 1-2. HIP 1-1 contains 2 in frame translation initiation sites that are 12 nucleotides apart. The more upstream ATG is surrounded by sequences which conform better to the Kozak consensus sequence (figure 3.2.2A). Therefore, this site was chosen as the translation initiation site and produced a protein of 1034 amino acids. The HIPl-2 open reading frame is 3009 nucleotides long and encodes a smaller protein isoform of 1003 amino acids. Both splice forms share a high degree of homology with Sla2p and ZK370.3 among their respective N-termini. 68 A HIP1-1 PI GAATTCGGCTTACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGTCGGHIGCCAGCT CC^AAGCAGGTGCCCAACCCACTGCCCAAGGTGCTGAGCCGGCGCGGGGTCGGCGCT P2 GGGCTGGAGGCGGCGGAGCGCGAGAGTTCGAGCGGACTCAGACTGT HIP1-2 P4 P3 GCAGCCTCGGJCATGGATGTGAGCAAGATGACTGT Figure 3.2.2. Alternative splicing at the HIP1 locus. A: Sequences of splice variant 1 (HIP1-1) and 2 (HIP1-2) are shown. The translation initiation sites are shaded in grey and the Kozak consensus sequence preceeding the translation initiation site in HIP1-2 is shown as the underlined sequence. Arrows P1 and P2 represent forward primers that recognize 5' sequences in HIP1-1 whereas arrows P3 and P4 denote primers that recognize 5' sequences sequences in HIP1-2. B: RT-PCR amplification of alternative transcripts at the HIP1 locus using total RNA isolated from human brain. The forward primers P1, P2, P3 and P4 were used in combination with the same reverse primer (lanes 1, 2, 3, and 4 respectively). This results in the amplification of a 619 bp fragment in lane 1, a 527 bp fragment in lane 2, a 536 bp fragment in lane 3 and a 531 bp fragment in lane 4. In lane 5 no RNA was present for reverse transcription. Lanes 1 and 2 confirm the presence of HIP1-1 sequences whereas lanes 3 and 4 confirm the presence of HIP1-2 sequences. 69 Moreover, RT-PCR analyses indicate that both splice variants are widely expressed since they are present in all tissues analyzed (data not shown). Thus far, 5' and 3' RACE analyses of HIP 12 sequences have lead to the identification of a single transcript indicating that no alternative splicing occurs at the HIP 12 locus (data not shown). 3.2.3 Genomic organization of human HIP1 The genomic organization of human HIP1 was determined using HIP1 cDNA specific primers followed by PCR amplification across intronic sequences. In addition, exon-intron boundaries were identified comparing HIP1 genomic sequences with HIP1 cDNA sequences deposited in GenBank (accession # AC004491). The HIP1 gene consists of 32 exons spanning approximately 215 Kb of genomic sequence (figure 3.2.3 and table 3.2.3). Moreover, translation initiation sites are present in exon 1 and exon 3 and alternative splicing occurs either from exon 2 to exon 4 or from exon 3 to exon 4. At present it is unclear whether additional untranslated 5' exons exist at the HIP1 locus. Interestingly, exons 1, 2, 4, 5 did not conform to the GT-AG rule for splice-donor and splice-acceptor sites. 3.2.4 Sequence comparisons of HIP12 with HIP1, Sla2p and ZK370.3 Protein sequence alignments of HIP 12, HIP1, ZK370.3 and Sla2p displayed significant sequence similarities. HIP 12 showed 47% identity and 64% amino acid conservation with HIP1. Moreover, HIP 12 and HIP1 showed virtually identical levels of amino acid identity and amino acid conservation with ZK370.3 and Sla2p (Table 3.2.4). 70 ATG ATG TAG Figure 3.2.3. Genomic organization of HIP1. The relative distance between exons is shown by horizontal bars; exons are shown by vertical grey bars. Alternative splicing occurs from exon 2 or exon 3 to exon 4. Exon 32 contains the stop codon and the 3' UTR is shown as a grey box. 71 Table 3.2.3. Splice-acceptor and splice -donor sequences at the human HIP1 gene. Exonic sequences are indicated by capital letters. Intronic sequences are shown by small letters. Exon 5' Splice junction cDNA position 3' splice junction Exon size (bp) Intron size (Kb) 1* ATGGCCAGCT 1-62 GGCGCGGGGTtcctgctaagcatcacac 62** 1 2 tggggatcccggggcagccCGGCGCTGGG 63-111 GCGGACTCAGgtgaggacgggggaccc 49 101 3* ATGGATGTGA 112-129 GAGCAAGATGgtaagtgtgagcaggggc 18** 40 4 ccccccttcttgtttttcagACTGTCAGCATCA 130-193 CACGCCAGAAatatccttttggatgttgctt 64 5.6 5 ttttccataaccccccctcacCGTGCATACT 194-337 ACACCCGAACgtgagt t c ctggggct atgggg 144 0.35 6 gtgttcttttgcccctgcagGTCCTGAAGGAC 338-395 AGCAGGATGTgtgtgagtttggagatgtact 58 5.2 7 ttcccctcctcttcctaaaagGGCCACCTGA 396-476 CCACACCAAAgtgagtctctgcggacagtt 81 4.6 8 ttcctctgcctcccttccagAATCCCAGGT 477-553 TGAACAACTTgtaagtggctcctgccctgag 77 0.8 9 ctctttcctcttgggtccagTTTCCAGTTA 554-615 TTCCAAACAGgtgagtctcttccctcccgt 62 10 10 ccctcacttttatttcctagTATTCAACTC 616-756 CTCCACTCCTgtgagtaccgcgggccagat 141 5.8 11 ctctttccaatttcttccagGCCTCCCAGC 757-814 AGTTTACAAAgt aagtggt tcaagtaacag 58 5.5 12 aggtccttctccacccctagGTTGAAAGAT 815-890 GCTGCCTGAGgtaagcatgcccaaccaca 76 0.1 13 ggatgtgtctccgtcttcagACCCACCCAA 891-1031 CTCTCAGCAGgtgaggaccacttgggagaga 141 1.5 14 tcactcctttttggtcaaccacagAATTTATTT 1032-1129 AGGATGAGAAgtgagtccaagctgggttcaa 98 1.1 15 cattccccttctctttcccagGGACCACTTAAT 1130-1202 GAAGACTGAGgtataactttggatctgctctgcc 73 1.6 16 tgaccggagtccccccacagAGCCAGCGGG 1203-1386 GAGATAGAAAgtgagcggtgggtgggggcg 184 1.4 17 cttggtcctttacaatacagGGAAAGCTCAAG 1387-1475 GCTGCGGAAGgtaagaccctcagcccctgtca 89 0.8 18 acactgttcttcttttgcagAATGCAGAGGT 1476-1592 GGCCAGCGGAAGgtgagtgggacgaggagc 117 1 19 ctgtttttcttctctttcagACTCAAGAACA 1593-1694 CTTCTGCCCAGgtaaatacctcctttttttt 102 1 20 ccttgtggtcacctttgcagTCAGAAGCAAA 1695-1839 AGCACAGAGGCAGgtcacggacatggac 145 0.8 21 acctccctggaccatttttagAATCTATGTG 1840-1974 GGGTCTGCAGgtacacttgcaattgcccagct 135 1.4 22 ccctgtgttccctctcacagATCACCTCCT 1975-2061 TGCCCAGAAGgtaagaatggccaaggacagt 87 0.3 23 cttctctttccaatcctggcagACATCAGTGG 2062-2168 CCTGCCGACTgtgagtactggggcatgagg 107 0.5 24 ctattgccttgtatctccagCACTTGACCGA 2169-2305 CATCGGCGAGgtacttggagtagtatcattg 137 5 25 cctttgtgatgtgttcacagGAGCTCCTGCCC 2306-2416 CCAGAATAGAGgtaggaggttcctgcaggatc 111 1.7 26 accatgcttctttttcacagGAGATGCTCAG 2417-2475 TGAATGAAAGgtcggtctgagcggcatggtgg 59 1.3 27 tcgacttgtttgtttggcagGATCCTTGGTTG 2476-2569 GAGCGGCAGGgtgagcgtgggtgtgggccct 94 2.2 28 actattttttatttccctagGGTACAGCATCCC 2570-2670 CCACTGTCATGgtaagtatctattggtaccaa 101 3.8 29 cccaggggacac agggc aagGTGGATGCAG 2671-2776 TGCATCCAAGgtaggacctggctggacctcc 106 1.8 30 aatctgctttgtcctggtagGTGAAAGCTGAA 2777-2899 GAAGAGACAGgt age c 111 ccaaagggacc c t 123 3.6 31 ttgtttccatccttgcagACAACATGGAC 2900-3070 TGGGAAGAAGgtaagctgactcaaaggat 171 1.4 32 aacataaattatcattgtcttttagGAACAGAGG 3071-3123 3' UTR 53*** * First 10 nucleotides within respective exon starting at the translation initiation site. :* Length of nucleotide sequence in between translation initiation site and 3' end of exonic sequences. *** Length of nucleotide sequence in between 5' end of exon 32 and translation stop codon. 72 Table 3.2.4. Pairwise comparisons between the nucleotide and amino acid sequences of HIP1-1, HIPl-2, HIPl2, Sla2p and ZK370.3. Alignment Percentage Nucleotide Identity Percentage Amino Acid Identity Percentage Amino Acid Conservation HIP1-1 vs HIP1-2 98 96 96 HIP1-1 vs HIP12 64 47 65 HIP1-1 vs ZK370.3 34 30 49 HIP 1-1 vs Sla2p 31 17 39 HIP1-2 vs HIP12 63 46 64 HIP 1-2 vs Zk370.3 34 30 50 HIP 1-2 vs Sla2p 32 18 40 HIP 12 vs ZK370.3 33 30 47 HIP 12 vs Sla2p 32 18 36 HIP 12 shows high nucleotide and amino acid identity with HIPl. HIPl and HIP 12 show similar levels of nucleotide and amino acid identity with ZK370.3 and Sla2p. 73 The carboxy-terminal region of HIP12, like Sla2p, ZK370.3, and HIPl shows considerable homology to the cytoskeletal protein talin. In Sla2p, the talin domain has been shown to contain 4 sub-domains referred to as the I/LWEQ module (McCann and Craig, 1997) that play a critical role in the binding of Sla2p to F-actin (figure 3.2.4). Amino acid residues in subdomain Q in HIPl and HIP 12 are likely to be essential in mediating actin binding for these proteins. Using the COILs program, HIP 12 similar to HIPl, Sla2p and ZK370.3 contains predicted coiled-coil domains (figure 3.2.4B and C). The coiled-coil domain in Sla2p has been shown to play an important role in actin polarization and protein dimerization in yeast (Wesp et al., 1997; Yang et al., 1999; Turunen et al., 1998). These observations suggest that HIPl and/or HIP 12 may play a role in actin polarization in mammalian cells. Further analyses of the HIP 12 protein sequence revealed that, similar to HIPl and Sla2p, HIP 12 contains a leucine zipper at amino acid residues 514 to 535 (LEKLKPvELEAICAGELARAQEAL). Leucine zippers are known to mediate protein-protein interactions observed among cytoskeletal proteins and transcriptional activators (Luscher and Larsson, 1999). 74 Figure 3.2.4. A: Amino acid alignment of HIPi-1, HIP1-2, H1P12, Sla2p and ZK370.3. The alignment between the 5 proteins is shown with the dark shading representing identical or conserved amino acids between all 5 proteins. The lighter shading representing identical or conserved amino acids in 3 or 4 of the proteins. T Refers to the amino and carboxy terminal sequences present in the HIPl yeast 2-hybrid vectors. A Refers to amino and carboxy terminal sequences present in the HIP 12 yeast 2-hybrid vectors. Boxed sequences represent the I/LWEQ sub-domains. B) The COILS program was used to predict the presence of coiled-coil domains in HIP 1-2 and HIP 12. The probability of the coiled-coil domain is plotted as a function of the amino acid position in the protein. C) Schematic representation of the various domains within HIP 1-2 and HIP 12, where the striped regions represent predicted coiled coil domains, solid portions represent the leucine zipper and hatched regions represent the talin domain. 75 A HIPl-1 HIPl-2 1 HI PI 2 1 ZK370.3 1 Sla2 1 consensus 1 HIPl-1 101 HIPl-2 70 HIP12 95 ZK370.3 79 Sla2 72 consensus 101 HIPl-1 200 HIPl-2 169 HIP12 194 ZK370.3 178 Sla2 170 consensus 201 HIPl-1 296 HIPl-2 265 HIP12 290 ZK370.3 274 Sla2 26b consensus 301 1 MASSMKQVPNPLPKVLSRRGVGAGLEJAEH: --MNSIKNVPARVLSRR-PGHSL: MDH; nf| rat % BRKIP E3|YBV|RF| SCONLFDNKF! SQQNLFDNKF: STGPPAMPWV, llJMl.l.NI . PARTP--ARTPTPTPPWMPAISPRPV HIPl-1 HIPl-2 HIP12 ZK370.3 Sla2 consensus HIPl-1 HIPl-2 HIP12 ZK370.3 Sla2 consensus jESEL-EJG|ALI§AEGDAG, !LYSQLRQEHLNL|PRFKK|QI,: HIPl-1 585 El HIPl-2 554 EJ HIP12 570 LLAAQSLVFf, ZK370.3 535 |AE!E Sla2 542 L|LEI ' consensus 601 . . HJ 33 1 ml 'pfl mmSk - —1 Hs.i'MBjB wrs ITYI I ifljlEl Ll:s|lS'l§[ &Y|fcs|LSWSG HIPl-1 HIPl-2 HIP12 ZK370.3 Sla2 consensus I/L HIPl-1 HIPl-2 HIP12 ZK370.3 Sla2 consensus HIPl-1 HIPl-2 HIP12 ZK370.3 Sla2 consensus HIPl-1 HIPl-2 HI PI 2 ZK370.3 Sla2 consensus HIPl-1 HIPl-2 HIP12 ZK370.3 Sla2 consensus 1055 DKKDGIYPAQLVNY 76 3.2.5 mRNA Expression of Human HIPl and HIP12 In order to determine the expression pattern of HIPl and HIP12, Northern blot analyses were performed on various human tissues. Using a 1.2Kb fragment of the HIPl cDNA as a probe, we observed a 9.0 Kb transcript, which was present in all tissues with significant levels of expression found in the brain (figure 3.2.5A). Within the brain, HIPl mRNA expression was observed in all brain regions with highest expression levels present in the frontal cortex and putamen (figure 3.2.5B). RT-PCR analyses indicated that both splice variants, HIP 1-1 and HIP 1-2, are expressed in various human brain regions and peripheral tissues (data not shown) however, the difference in transcript size is too small to be detectable by Northern blot analysis. Northern blot analyses of HIP 12 indicated a different expression pattern in comparison with HIPl. Using a 675 bp cDNA of HIP 12, a 5.0 Kb transcript was present in the brain, heart, kidney, pancreas, and liver but not in the lung or placenta (figure 3.2.5C). In addition, a 7.0 Kb transcript was also present in the heart and skeletal muscle. Furthermore, a 3.5 Kb transcript was observed in the skeletal muscle and occipital lobe. RT-PCR was performed in order to determine the source of the 7.0 and 3.5 KB transcripts. Primer pairs within the coding region of HIP 12 amplified identical size products in all tissues examined which indicated that the source of the larger transcript is not due to alternative splicing within the coding region (data not shown). We performed 3' RACE to determine whether these transcripts arose due to differential polyadenylation and did not detect any differences in RT-PCR products that could account for the 3.5 and 7.0 Kb bands (data not shown). Therefore, it is likely that the 3.5 and 7.0 kb transcripts result from cross reactivity of the HIP12-specific cDNA probe. 78 3.2.6 Protein Expression of Human HIPl and HIP12 Western blots were performed using novel polyclonal antibodies generated against either a HIPl or HIP 12 fusion protein. The HIPl and HIP 12 fusion protein antibodies recognized 120 and 130 kDa proteins, respectively. The specificity of each anti-fusion protein antibody was confirmed by competition studies using either excess HIPl or HIP 12 fusion proteins (data not shown). Within the brain, both HIPl and HIP 12 were found at similar levels in all brain regions examined (figure 3.2.6B and D). In various tissues examined, HIPl was found to be expressed at highest levels in the frontal cortex and the spleen. Significant levels of HIPl were also observed in the liver, and kidney (figure 3.2.6A). Similarly, HIP 12 was expressed predominantly in the brain, liver, and to a lesser extent in the kidney (figure 3.2.6C). HIPl Western blots exhibited faint lower molecular weight proteins in the spleen, kidney, cerebellum, caudate and putamen. And, in the case of HIP 12, faint lower molecular weight proteins were detected in the heart, liver, lung and cerebellum. These bands most likely represent proteolytic degradation products. 79 Figure 3.2.5. Northern blot analysis of the human HIP1 and HIP12. Northern blots containing 2ug of poly A+ mRNA from various adult human tissues were hybridized with either a 1.2 Kb fragment of HIP1 (A and B) or a 675 bp fragment of HIP12 (C and D). The lower panel represents the same blot hybridized with the human actin cDNA to confirm RNA quality and relative loading (C and D). RNA size markers are shown on the right and left. 80 Figure 3.2.6. HIP1 and HIP12 protein expression in normal human CNS and peripheral tissues. HIP1 protein expression in normal human CNS and peripheral tissues (A). HIP1 protein expression in normal human brain regions (B). HIP12 protein expression in normal human CNS and peripheral tissues (C). HIP12 protein expression in normal human brain regions (D). The lower panels represent the same blots reprobed with the antibody against human glyceraldehyde phosphodehydrogenase to confirm protein quality and relative loading. 81 3.2.7 Immunofluorescence in NT2 cells and ES Cell-Derived Neurons. In order to determine the subcellular localization of HIPl and HIP 12, immunofluorescence studies were undertaken in the neuronal precursor cell line NT2 and functional ES cell-derived neurons (Metzler et al, 1999; figure 3.2.7). These studies were performed in collaboration with Dr. M. Metzler and myself and revealed that HIPl and HIP 12 display a very similar intracellular distribution. In NT2 cells, HIPl and HIP 12 exhibited a non-uniform punctate staining pattern throughout the cell body except the nucleus (figure 3.2.7A-F). Omission of the primary antibodies resulted in no detectable staining within the cells (data not shown). In ES cell-derived neurons, HIPl and HIP 12 were highly both expressed and distributed throughout the cytoplasm and cell processes with enrichment within the cw-Golgi (figure 3.2.7 G and L). However, only a few strong punctae were visible (indicated by arrows) at high levels within these cells (figure 3.2.7 H and J). 3.2.8 Yeast 2-Hybrid Interactions of HIPl, HIP12 and Huntingtin In order to determine if HIP 12, like HIPl, can interact with huntingtin, we performed yeast 2-hybrid assays. A 1.5 Kb fragment of HIP 12, corresponding to amino acids 514 to 738 was fused in frame, adjacent to both the GAL4-DNA binding domain (BD) of the yeast two-hybrid vector pGBT9 and to the GAL4-activating domain (AD) of the yeast two-hybrid vector pGAD. This 1.5 Kb fragment of HIP 12 corresponds to the region in HIPl that interacts with huntingtin. We determined that unlike HIPl, HIP12 does not interact with huntingtin containing 15 polyglutamine repeats. Furthermore, 82 Figure 3.2.7. Immunolocalization of HIPl and HIP 12 in NT2 (A-F) and ES cell-derived neurons (G-L). Expression of HIPl (E, green) and HIP 12 (B, green) in NT2 cells was visualized by confocal immunofluorescence microscopy using the DNA stain TO-PRO to visualize the nuclei (A, D, red). In ES cell-derived neurons HIPl (H, green) and HIP 12 (K, green) expression was visualized by conventional immunofluorescence microscopy and compared with the cis-golgi marker GM130 (G, J, red). Images in the left and middle panel were overlaid and merged electronically and are shown in the right panel (C, F, I, L). 83 K 84 HIP 12 did not show any interaction with huntingtin containing 128 polyglutamine repeats (data not shown). However, HIP 12 can interact with HIPl and may therefore compete with huntingtin in the binding to HIPl. Moreover, HIPl can interact with itself indicating that HIPl may form dimers in vivo (figure 3.2.8). 3.2.9 Overexpression of HIP12 in Human Embryonic Kidney (293T) Cells Several studies have demonstrated that overexpression of HIPl in cultured cells decreases cell viability (Hackam et al., 1998; Hackam et al, 2000). HIP 12 was transiently transfected into HEK 293T cells by R. Singaraja and me to test if overexpression of HIP 12 is also toxic (figure 3.2.9A-B). Interestingly, 48 hours post-transfection, cells expressing HIPl showed a significant decrease in cell viability when compared to cells expressing LacZ whereas overexpression of HIP 12 did not influence cell viability (p> 0.05, n=6). Cells expressing the first 540 amino acids of huntingtin and containing 128 CAG repeats (1955-128) was used as a positive control and displayed reduced cell viability relative to cells expressing lacZ (pO.001, n=6). Caspase-3 activation assays were performed by R. Singaraja and me in order to determine whether the decreased viability of HEK 293T cells overexpressing HIPl or 1955-128 may be due to the activation of the pro-apoptotic enzyme caspase 3. At 24 hours post-transfection, cells expressing either HIPl or 1955-128 showed significantly elevated levels of caspase 3 activity when compared to cells expressing LacZ (pO.001, n=6) whereas overexpression of HIP 12 did not influence caspase 3 activity (p> 0.05, n=6). 85 HIP12 + HIP12pGBT9 HIP1 + HD15pGBT9 PGAD424 + HD15pGBT9 ^ ^ HIP1 + HIP1pGBT9 HIP12 + pGBT9 HIP1 + pGBT9 HIP12 +HIP1pGBT9 HIP12 + HD15pGBT9 Figure 3.2.8. Yeast 2-Hybrid interactions of HIP1 and HIP12 with various GAL4 DNA-binding domain constructs. Chromogenic (3 -galactosidase filter assay showing the interaction between HIP1 with the first 540 amino acids of huntingtin with 16 CAG repeats and with HIP1. HIP1 did not interact with GAL4-BD vector alone. The pGBT9-AD vector alone did not interact HIP1 pGAL-BD vector. HIP12 interacted with HIP1 but did not interact with a GAL4-BD vector containing HIP12. HIP12 did not show any interaction with GAL4-BD vector alone. pGBT9-AD vector alone did not interact HIP12 pGAL-BD vector. 86 Figure 3.2.9. MTT assays as a measure of cell viability. (A) Transfection and tamoxifen treatment of vector expressing p-galactosidase (LacZ; n=6) served as controls. Expression of huntingtin and HIP1 but not HIP12 constructs results in increased cell death relative to control vector LacZ (n=6). Absorbance values (y-axis) reflect the proportion of viable cells. Increased absorbance is equivalent to increased viability. Caspase-3 activation assays as a measure of cellular Apoptosis. (B) Transfection and tamoxifen treatment of vector expressing P-galactosidase (LacZ; n=6) served as controls. Expression of huntingtin and HIP1 but not HIP12 constructs results in increased levels of activated caspase-3 relative to control vector LacZ (n=6). Absorbance values (y-axis) reflect the level of caspase 3 activation. Increased absorbance is equivalent to increased level of apoptosis. 87 3.3 Discussion In this study we have demonstrated that HIPl is a member of a family of cytoskeletal-associated proteins through the isolation of a novel protein termed HIPl 2. HIP 12 is 1068 amino acids in length and shows structural similarity with not only the two HIPl splice variants, HIP1-1 and HIP 1-2, but also Sla2p and ZK370.3. HIP 12 shows an overall 47% identity and 64%> amino acid conservation with HIPl. The highest degree of sequence similarity between both proteins occurs at the carboxy-terminal region, which shows considerable homology to talin, an actin filament binding protein which is found at focal cell adhesions (Moulder et al, 1996). HIPl has been shown to interact with huntingtin in a CAG length-dependent manner, the larger repeat conferring weaker interaction. Thus, it could be expected that in cells expressing mutant huntingtin, there would be more unbound HIPl. This free HIPl^ may then initiate biochemical events which may play a role in the pathogenesis of HD. Overexpression of HIPl in HEK 293T cells results in a significant decrease in cell viability as well as a significant increase in caspase 3 activity. We have shown that transfection of HIP 12 into HEK 293T cells does not cause any alteration in cell viability or caspase 3 activity. These findings indicate that HIPl2, in contrast to HIPl, is not a proapoptotic protein. Using the yeast 2-hybrid system we demonstrated that HIPl can interact with huntingtin whereas HIP 12 shows no detectable interaction with huntingtin. Interestingly, HIPl interacts with itself and can interact with HIP 12 in the yeast 2-hybrid system. Western blot analyses indicate that HIPl and HIP 12 are both highly expressed in the brain. Furthermore, immunolocalization studies of both proteins in NT2 cells and ES cell-derived neurons demonstrate a similar intracellular distribution indicating that both 88 proteins may co-localize in specific cellular compartments. Taken together these findings suggest that such interactions may also occur in vivo and that HIP 12 may be able to modulate the toxic effects of HIP 1. The yeast ortholog of HIP 1 and HIP 12, Sla2p, was originally characterised as a null mutation causing temperature-sensitive growth defects related to a general disorganization of the membrane cytoskeleton (Holtzman et al., 1993). Two other alleles of Sla2p have been identified based on their mutant phenotype which have defects in endocytosis and regulation of cell surface H+ ATPase (Raths et al., 1993; Na et al., 1995). More recently, Sla2p mutants have been shown to be defective in their ability to transport secretory vesicles from the Golgi apparatus to the plasma membrane (Mulholland et al., 1997; Blader et al., 1999; Bowman and Kahn, 1995). Thus, Sla2p and, by analogy, HIP1 and possibly HIP 12 may play a role in the regulation of membrane events through interactions with the underlying cytoskeleton and vesicle trafficking. Recently, Engqvist-Goldstein et al. (1999) have demonstrated that the murine homolog of HIP 12 is a component of clathrin-coated pits and vesicles and that it may link the endocytic machinery with the actin cytoskeleton. Interestingly, other huntingtin-interacting proteins may be involved in vesicle transport and the regulation of membrane events. HAP1 interacts with Duo (Colomer et al., 1997) and the pl50Glued subunit of dynactin (Engelender et al., 1997 ; Lippincott-Schwartz, 1998), proteins known to play a role in vesicle trafficking. The huntingtin-interacting protein, SH3GL3, is a member of a novel SH3-containing family of proteins (Sittler et al., 1998). In the central nervous system, certain SH3 containing proteins are involved in vesicle trafficking, signalling at the cell surface and organization of the 89 cytoskeletal matrices (McPherson et al, 1994; Ringstad et al., 1997). Huntingtin is membrane associated, co-localizes with microtubules and may also be involved in vesicle trafficking (DiFiglia et al., 1995; Gutekunst et al., 1995). In addition, huntingtin has also been shown to be localized with clathrin in membranes of the trans-Golgi network and in clathrin-coated and non-coated endosomal vesicles in the cytoplasm and plasma membrane (Velier et al., 1998). Together, these observations suggest a role for HIPl and huntingtin as two intimately associated proteins essential for vesicle transport. Thus, the loss of interaction between HIPl and huntingtin may perturb the organization of the cytoskeleton and vesicle transport, which may ultimately play an important role in the pathogenesis of Huntington disease. 90 Chapter 4: HIP1 Interacts with Clathrin and AP2 The data presented in this chapter contributed to the manuscript: Metzler M, Legendre-Guillemin V, Gan L, Chopra V, Kwok A, McPherson PS, Hayden MR. HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2. J Biol Chem (2001) 276:39271-39276 My contribution towards obtaining the data within the manuscript shall be discussed in the results section 4.2. 91 4.1 Introduction In humans, HIPl belongs to a family of proteins consisting of HIPl and HIP 12. The HIPl and HIP 12 proteins have a similar molecular weight and contain an amino-terminal ENTH (Epsin N-terminal Homology), a central coiled coil and a carboxy terminal talin-like domain (Chapter 3). The ENTH domain is proposed to induce clathrin-mediated endocytosis through binding to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2J-containing membranes (Ford et al., 2001; Itoh et al., 2001). Furthermore, ENTH domains are also present in several other endocytic proteins including epsin 1 and 2, API80 and its homolog, CALM (Ford et al, 2002; Ford et al., 2001; Itoh et al., 2001). The carboxy-terminal domains of the HIPl and HIP 12 proteins share significant sequence similarity to the mammalian membrane cytoskeletal-associated protein, talin (Kalchman et al., 1997). Proteins that bind both PtdIns(4,5)P2 and clathrin are thought to promote the linkage of clathrin triskelia to the lipid bilayer (Hao et al., 1999). Consistent with this idea is the observation that HIP 12 is involved in clathrin mediated endocytosis (CME) and actin cytoskeleton dynamics (Engqvist-Goldstein et al., 1999). For example, HIP 12 has been shown to colocalize with markers of receptor endocytosis in mammalian cells including AP2 and clathrin. In addition, HIP 12 has also been revealed to be associated with clathrin coated vesicles (CCVs; Engqvist-Goldstein et al., 1999). Finally, HIP12 was also found to colocalize with newly endocytosed vesicles containing labeled transferrin. However, as the transferrin-containing vesicles traveled further into the cytoplasm, their co-localization with HIP 12 decreased (Engqvist-Goldstein et al., 1999). Since HIPl shows significant sequence and biochemical similarities, we hypothesize that HIPl may also be 92 involved in CME and actin cytoskeleton dynamics. To address this possibility, we tested whether HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and assessed whether HIP1 is enriched on CCVs purified from brain homogenates. In addition, we examined whether HIP1 and HIP 12 interact directly with components of receptor mediated endocytosis. The findings in this chapter indicate that HIP1 is a component of the endocytic machinery. 93 4.2 Results 4.2.1 Co-localization of HIP1 with components of the endocytic machinery. In order to address whether HIP1 may play a role in clathrin-mediated endocytosis, Dr. M. Metzler and I performed immunofluoresence studies in the neuronal precursor cell line NT2. The NT2 cell line was chosen because HIP1 is highly expressed in the brain (Kalchman et al., 1997; Wanker et al., 1997). Within the NT2 cells, HIP1 displayed a characteristic punctate staining pattern throughout the cytoplasm (figure 4.2.1 A). Furthermore, HIP1 co-localized with the clathrin heavy chain (CHC) and the adapter protein AP2 (figure 4.2.1 A). In addition, HIP1 partially co-localized with the endocytic vesicle trafficking protein, Rab5 (figure 4.2.1 A). Rab5 is a guanosine triphosphatase that has been shown to regulate the formation, transport and fusion of clathrin-coated vesicles with target organelles (Zerial and McBride, 2001). These finding suggest that HIP1 may play an important role at the early stages of clathrin-mediated endocytosis. To determine the subcellular localization of HIP 1, tissue from rat brain was fractionated by differential centrifugation (figure 4.2IB). The different cytosolic fractions consisted of the homogenate (H), pellet fraction #1 (PI) containing cell debris and nuclei, pellet fraction #2 (P2) containing the mitochondria and some synaptosomes, supernatant fraction #2 (S2) containing clathrin-coated vesicles and synaptosomes, the supernatant after sucrose gradient centrifugation of the S2 fraction (SGs) containing synaptosomes and membranous debris, and the pellet after sucrose gradient centrifugation of S2 fraction (SGp) containing the purified clathrin-coated vesicles. The clathrin coat was separated 94 A Ot-Adaptiu HUM overlay - r---.r.,,v; j». v * <••• • Clathrin HUM overlay .... . jvr^f Ral>5 HUM overt*) B 116 -97- Blot: mAb HIP1-9 -W-180- Blot: mAb anti-CHC 116-97- — Blot: mAb nnli-AI'2 H PI P2 S2 SGp SGs coats vesicles Figure 4.2.1. HIP1 colocalizes with markers of endocytosis and is enriched in CCVs. (A) Localization of HIP1 (green) with a-adaptin, clathrin, and Rab5 (red) was analyzedin neuronal NT2 cells. Overlays of the images are shown in the right panels. Bars, 10 mm. (B) Purification of CCVs was performed by subcellular fractionation of E18 rat brain homogenates. Aliquots of 100 mg of fraction H (homogenate), P1 (pellet 1), P2 (pellet 2), S2 (supernatant 2), SGp (pellet obtainedafter sucrose gradient centrifugation), SGs (supernatant obtained after sucrose gradient centrifugation), coats and vesicles were analyzed on SDS-PAGE and immunoblotted with anti-HIP1, anti-CHC and anti-a-adaptin. 95 from the vesicle using 0.5M Tris pH 9.0. The proteins in the different cytosolic fractions were separated by SDS-PAGE, transferred to a nylon membrane, and probed with antibodies against HIP1, CHC and the AP2 subunit, a-adaptin. Similar to CHC and a-adaptin, HIP1 was enriched in the SGp (coat and vesicles fraction). These findings indicate that HIP1 is a component of CCVs within the brain. 4.2.2 Identification of HIP1 domains necessary for endocytic targeting. Five different HIP1 expression constructs were generated by L. Gan and me in order to determine the HIP1 domains that are required for localization to the CCV (figure 4.2.2A). The constructs were: 1) full-length HIP1 construct which expressed the HIPl-2 splice variant (see chapter 3) 2) HIPl-Acc which expressed a HIP 1-2 fragment lacking the coiled coil domain (corresponding to amino acids 336-610) 3) HIPl-AN-term which expressed a HIPl-2 fragment lacking the amino-terminal sequences including the ENTH domain (corresponding to amino acids 1-335) 4) HIP1-155-610 which expressed a HIP1-2 fragment lacking the ENTH domain and carboxy terminal sequences including the talin-like domain (corresponding to amino acids 1-155 and 611-1003) 5) HIPl-talin which expressed the talin-like domain of HIP 1-2 (corresponding to amino acids 732-1003). Each of the constructs contained a Flag-tag at their respective carboxy-termini. The protein expression from the constructs was verified by Western blot analyses using transfected human embryonic kidney 293T (HEK293T) cell lines (figure 4.2.2B). To determine which HIP1 constructs interacts with the CHC, the CCVs were isolated from transfected HEK293T cells, transferred to membrane by Western blot and probed with antibodies against HIP1, Flag and CHC. The cells overexpressing full length 96 2. HIPl-Acc: || win }. IIIIM AN term: 4. HIPHH5-6I0): 1 1 B 1* ItU tm J31 1003 1 2 3 4 5 -116 -86 -32 Blot: anti-CHC Biol: anti-Hag D anti-Flag anti-AP2 overlay HIPl Blot: anti-Flag HIPl-Acc Blot: anti-Flag HIPl-AN-term Blot: anti-Flag Blot: anti-Flag HI1M-O55-610) HIPl-talin j Q H PI S2 CCV Figure 4.2.2. HIP1 targeting to CCVs. (A) Domain model of full-length HIP1 and HIP1 mutants (amino acid numbersare indicated above the constructs). (B) Expression of each construct (1-5) was analyzed by Western blot and compared to non-transfected cells (-) using an anti-Flag antibody. (C) 293T cells were transfected with various constructs of HIP1 and CCVs were purified as described in Experimental Procedures. Aliquots of various fractions from the purification (100 mg) were analyzed by Western blot with anti-CHC antibody or with anti-Flag antibody. (D) Expression of transfected HIP1 and various HIP1 mutants (red) was visualized using an anti-Flag antibody following transfection in HeLa cells. Expression of AP2 is shown in green. Overlays of the images are shown on the right panels. Bars, 10 um. 97 HIP 1 did not exhibit as large an enrichment in the CCV as the cell expressing endogenous HIPl (figure 4.2.2C). Cells overexpressing HIPl may contain excess amounts of the protein that is incapable of interacting with the CCV and are therefore found in other cellular fractions. The protein expressed by the HIP1-155-610 construct was found to be enriched in the CCV. However, the protein products of the HIPl-Acc, HIPl-AN-term and HIPl-talin constructs were not found to be enriched in the CCV (figure 4.2.2C). These studies demonstrate that sequences between the ENTH and coiled-coil domain are necessary for targeting HIPl to CCVs. To assess whether the HIPl constructs also localized with AP2, transfected HEK 293T cells were examined by immunofluoresence (by Dr. M. Metzler) using antibodies against AP2 and the Flag sequence. The transfected full-length HIPl displayed extensive colocalization with AP2 (figure 4.2.2D, top panel). However, the proteins expressed by HIPl-Acc, HIPl-AN-term constructs did not colocalize with AP2 (figure 4.2.2D, second and third panel). The protein produced by the HIP1-155-610 construct was exhibit significant co-localization with AP2. Thus, the sequence contained between amino acids 155-610 are required for the colocalization of HIPl with AP2. 4.2.3 HIPl interaction with AP2 and clathrin terminal domain. Sequence analyses demonstrated that HIP 1-2 contains the consensus sequence for a clathrin box sequence, LMDMD, at amino acid residues 298-302 (figure 4.2.3A). The clathrin box has been shown to mediate binding to the clathrin terminal domain (Ramjaun and McPherson, 1998; Drake and Traub, 2001). In addition, HIPl also contained a DPF motif, at amino acid residues 324-326, which is known to interact with AP2 (figure 98 4.2.3A; Ramjaun and McPherson, 1998; Drake and Traub, 2001). In order to assess whether the clathrin box and DPF motifs in HIPl can interact with clathrin and AP2, two GST-fusion proteins constructs containing these motifs were generated. The first construct, GST-HIP 1-(219-616) encompassed HIPl sequence containing the clathrin box, DPF motif and the coiled coil domain (figure 4.2.3B, middle panel). The second construct, GST-HIP 1-(276-335), encompassed HIPl sequence containing the clathrin box and DPF motif (figure 4.2.3B, bottom panel). Affinity purification studies demonstrated that the two fusion proteins were both able to bind clathrin and AP2 from rat brain extracts. Interestingly, interaction with clathrin was significantly greater to the protein product of GST-HIP 1-(219-616) than to GST-HIPl-(276-335) (figure 4.2.3C, top panel). The stronger interaction of GST-HIP 1-(219-616) to clathrin suggests that the coiled coil domain may contain additional site(s) that interact with clathrin. AP2 binding was significantly higher to GST-HIPl-(276-335) than to GST-HIP 1-(219-616) (figure 4.2.3C, bottom panel). The different binding affinities suggest that AP2 interaction occurs independently of clathrin binding. Immunoprecipitation studies also showed that both of the fusion proteins were able to bind His6-tagged clathrin terminal domain (figure 4.2.3C, middle panel). These findings suggest that the interaction between clathrin and HIPl is direct. Finally, AP2 was immunoprecipitated from HEK 293T overexpressing full-length HIPl (figure 4.2.3D). 99 A HIPl 294 E K D D L M D M D A 303 Epsin 1 253 F. E S S L M D L A D 263 ACK1 566 A E V T L I D F 0 E 575 ACK2 580 G Ii V T L I D F 0 E 589 P-arrestin 373 V D T N L r E L D T 282 Arrestin3 369 V D T N L I E F E T 378 Amphiphysin 347 E Q A S L L D L D F 356 p2-adaplin 627 L L G D L L N L O L 636 P-Vudaptin 815 K D V s L L D I. D D 824 1 L^i 1MJ HIP,: II ami I I <c I I w.—D-n»« GST-HIPI-(219-«16): I 1 • " , R nt Ms CST-HIPl-<276-335); (—) c HIPl Figure 4.2.3. HIP1 binds clathrin and AP2. (A) Sequence alignment of HIP1 with various clathrin box-containing proteins. (B) Schematic presentation of Flag-tagged full-length HIP1 and sequences present in different HIP1-GST-fusion proteins. (C) Soluble proteins from brain extracts or purified His6-tagged clathrin TD fusion protein were affinity-purified with equal amounts of GST fusion proteins encoding domains of HIP1 bound to glutathione-Sepharose beads. Proteins specifically bound to the beads were analyzed by Western blotting with anti-CHC, anti-tetra-His, and anti-a-adaptin antibodies as indicated. (D) 293T cells were transfected with Flag-tagged full-length HIP1 and processed in parallel with non transfected cells (-). Proteins were extracted and immunoprecipitated with anti-Flag antibody. Immunoprecipitated proteins were analyzed by Western blotting with anti a-adaptin and anti-Flag antibodies. SM refers to starting material. 100 4.2.4 HIPl plays a role in clathrin-mediated endocytosis. In order to determine if HIPl play a role in endocytosis, transferrin uptake assays were performed by Dr. V Legendre-Guillemin. Four different HIPl constructs were transfected into COS7 cells in order to assess their effect on transferrin uptake (figure 4.2.4A). The ability of the transfected cells to endocytose transferrin was determined by immunofluorescent microscopy. COS7 cells transfected with constructs expressing either full length HIPl or the HIPl coiled coil domain (HIPl-cc) exhibited efficient transferrin endocytosis (figure 4.2.4B, panels /, ii, v and vi). However, cells transfected with constructs expressing either HIP1-155-610 or HIP1-155-335 displayed a markedly reduced transferrin uptake (figure 4.2.4B, panels iii, iv, vii and viii). It is possible that the clathrin box and DFP motif present in the HIP1-155-610 and HIP 1-155-335 constructs act as inhibitors of transferrin uptake. Figure 4.2.4C represents a graph of the percentage transferrin uptake for the different constructs in six separate experiments. These findings are consistent with previous findings indicating that the HIPl homolog, HIP 12, colocalizes with newly endocytosed vesicles containing labeled transferring (Engqvist-Goldstein et al., 1999). 101 Figure 4.2.4. Transferrin uptake in C0S7 cells. (A) Schematic of full-length HIP1 and HIP1 mutants. (B) The uptake of Cy3-labelled transferrin into COS7 cells (v-viii) was analyzed in the presence of various constructs of HIP1. Transfected cells were revealed by immunofluorescence with an anti-Flag antibody (i-iv). Bars, 10 mm. (C) The graph represents the mean ± SEM from 6 separate experiments of the percentage of transferrin uptake for the different constructs. The white and black bars represent non-transfected and transfected cells, respectively. 102 4.3 Discussion In this study we have demonstrated that HIP1 is a major component of the clathrin-mediated endocytic machinery. HIP1 was found to interact with CHC via its type 1 clathrin box sequence LMDMD at amino acid position 318 to 322. These findings have been confirmed by site-directed mutagenesis studies performed by Mishra et al. (2001). They demonstrated that the conversion of the HIP1 LMDMD sequence to AAAMD completely abolished CHC binding. The consensus sequence for the type 1 clathrin box is L(L/I)(D/E/N)(L/F)(D/E) (Ramjaun et al., 1998; Drake et al., 2001). Variations of this sequence has been identified in a several endocytic machinery proteins including API80, amphiphysin, epsins, and (3-arrestin (Ford et al., 2001; Slepnev et al., 2000; Drake et al., 2000; Laporte et al., 1999). In addition, we have shown that HIP1 interacts with AP2 in part through its consensus AP2 binding sequence DPF at amino acid position 324 to 326. Variations of the AP2 binding sequence are present in the endocytic proteins epsin (DPW) and epsl5 (DPF) (Mishra et al., 2001). Our findings have been confirmed by binding studies showing that the HIP1 DFP sequence interacts directly with the a-adaptin subunit of AP2 (Mishra et al., 2001; Waelter et al., 2001). Interestingly, Mishra et al (2001) also identified additional AP2 binding motifs FDNKP, FDDIF, FGSSF at amino acids 309 to 321. The consensus sequence FXDXF (where X represents any amino acid) is also present in the endocytic proteins amphiphysin 1 and API80 (Slepnev et al., 2000; Hao et al.,1999). Examination of our HIP1 expression constructs that bound to AP2 contained the DPF and the FDNKP, FDDIF, FGSSF sequences. Thus, it is possible that both these motifs interact with AP2. The tandem arrangement of the clathrin box and AP2-binding 103 motifs present in HIPl has also been previously observed in epsin (Drake et al., 2000). The arrangement of these two binding sites has been postulated to help stabilize the relatively weak interaction between AP2 and clathrin. We have demonstrated that, similar to the adapter proteins AP2 and API80, HIPl was also highly enriched in clathrin-coated vesicles (CCVs) isolated rat brain homogenate. AP2 has been shown to play an important role in the recruitment and polymerization of clathrin subunits which ultimately form clathrin-coated vesicles (Brodsky et al., 200). The brain-specific protein API 80 has been shown to interact with AP2 and clathrin heavy and is postulated to assist in the formation of CCVs (Kay et al., 1999; Ford et al., 2001; Itoh et al., 2001). Furthermore, the carboxy-terminal region of API 80 contains several copies of the amino acid sequence, DLL, which is thought to function in clathrin coat assembly (Slepnev and DeCamilli, 2000). Interestingly, the HIP 1-2 central coiled coil domain contains a single DLL motif at amino acid position 450-452. At this time it is unclear whether the DLL motif present in HIPl plays a direct role in clathrin assembly. HIPl was initially identified on the basis of its interaction with huntingtin (Kalchman et al., 1997; Wanker et al., 1997). The strength of the interaction has been shown to be inversely correlated with the length of the polyglutamine tract present in huntingtin. HIPl and huntingtin have been shown to be highly enriched within the brain and display a similar subcellular distribution (Kalchman et al., 1997; Wanker et al., 1997; Chopra et al., 2000). The present study indicates that HIPl is a component of the clathrin-mediated endocytic machinery. Similarly, huntingtin has been shown to interact with the endocytic proteins a-adaptin and endophilin A3 (Faber et al., 1998; Sittler et al., 1998). In 104 addition, huntingtin is also associated with clathrin-coated vesicles and thought to function in vesicle trafficking in the secretory and endocytic pathways (Velier et al., 1998). Taken together, these findings suggest that interaction of huntingtin with HIP1 may play a role in endocytosis. Within the neurons of the brain it may be postulated that these proteins may be involved in synaptic vesicle endocytosis. 105 Chapter 5: Differential Binding of HIP1 and HIP12 to Components of the Endocytic Machinery The data presented in this chapter contributed to the manuscript: Legendre-Guillemin V, Metzler M, Charbonneau M, Gan L, Chopra V, Philie J, Hayden MR, McPherson PS. HIP1 and HIP 12 display differential binding to F-actin, AP2, and clathrin. Identification of a novel interaction with clathrin light chain. J Biol Chem. (2002) 277:19897-19904. My contribution towards obtaining the data within the manuscript shall be discussed in the results section 5.2. 106 5.1 Introduction Accumulating evidence suggests that HIPl and HIP 12 both play an important role in endocytosis and regulation of the cortical actin cytoskeleton. As mentioned previously, both these proteins contain an ENTH (epsin N-terminal homology) domain which can bind to phosphatidylinositol 4,5-bisphosphate-containing membranes and thought to induce clathrin-mediated endocytosis (Kay et al., 1999; Ford et al., 2001; Itoh et al., 2001; DeCamilli et al., 2002). The central portion of these proteins contains a coiled-coil domain which is thought to mediate protein-protein interactions. Finally, these proteins contain a C-terminal talin-homology domain that has been shown to bind F-actin (Engqvist-Goldstein et al., 1999; McCann and Craig, 1997). These findings suggest that HIPl and HIP 12 may play a role in linking membrane attachment and clathrin-coated vesicle (CCVs) formation with actin dynamics (Engqvist-Goldstein et al., 1999; Bennet et al., 2001). Recent studies have shown that both HIPl and HIP 12 are enriched at discrete cytoplasmic membrane sites that are undergoing clathrin-mediated endocytosis. Furthermore, HIPl and HIP 12 co-localize with markers of endocytosis including clathrin, AP2 andRab5 (Engqvist-Goldstein et al 1999, Metzler et al., 2001; Mishra et al., 2001; Waelter et al., 2001). The HIPl protein contains a type 1 clathrin box motif (LMDMD) which is thought to mediate the direct interaction of HIPl with clathrin heavy chain (CHC; Chapter 4, section 4.2.3; Metzler et al., 2001; Mishra et al., 2001; Waelter et al., 2001). In addition, HIPl also possesses the consensus AP2 binding sequences FDNKP, FDDIF, FGSSF and DPF which have been shown to interact with the AP2 subunits a-107 adaptin A and C (Chapter 4, section 4.2.3; Metzler et al., 2001; Mishra et al., 2001; Waelter et al., 2001). The previous studies mentioned have not confirmed whether HIP 12 can also interact directly with clathrin or AP2. However, sequence analyses have demonstrated HIP 12 also contains the clathrin box motif (LIEIS) but does not contain any AP2 binding sites (Mishra et al.; 2001). In this chapter the protein interaction and functional properties of HIP1 and HIP 12 were analyzed in order to elucidate their roles in clathrin-mediated endocytosis. We demonstrated that HIP1 strongly interacts with clathrin and AP2 but does not interact with F-actin. However, relative to HIP1, HIP 12 displayed weaker affinity for clathrin and AP2 but did interact with F-actin. In addition, HIP1 and HIP 12 were both shown to bind directly to the clathrin light chain (CLC) through their central helical domain. These results suggest related but distinct functions for HIP1 and HIP 12 in clathrin-mediated endocytosis. 108 5.2 Results 5.2.1 Comparison of HIP1 and HIP12 protein expression 5.2.1.1 HIP1 and HIP12 protein expression in adult and embryonic rat brain To further characterize the functions of HIP 1 and HIP 12, their developmental expression profiles were assessed in adult and embryonic rat brain. These studies were performed in collaboration with Dr V. Legendre-Guillemin. Western blot analyses indicated that HIP1 was highly expressed at embryonic day 13 (El3) and El6 (figure 5.2.1A, top panel). However, HIP12 protein levels were highest in post-natal day 2 and adult rat brain but relatively low in all the embryonic days examined (figure 5.2.1 A, bottom panel). Thus, HIP1 is expressed earlier in rat brain development than HIP 12. 5.2.1.1 Subcellular fractionation studies. To compare the subcellular localization of HIP 1 and HIP 12, tissue from rat brain was fractionated by differential centrifugation (figure 5.2.2B). The different cytosolic fractions consisted of the homogenate (H), pellet fraction #1 (PI) which contained cell debris and nuclei, pellet fraction #2 (P2) containing the mitochondria and some synaptosomes, supernatant fraction #2 (S2) containing clathrin-coated vesicles and synaptosomes, the supernatant after sucrose gradient centrifugation of the S2 fraction (SGs) containing synaptosomes and membranous debris, and the pellet after sucrose gradient centrifugation of S2 fraction (SGp) containing the purified clathrin-coated vesicles. The clathrin coat was separated from the vesicle using 0.5M Tris pH9.0. The proteins in the different cytosolic fractions were separated by SDS-PAGE, transferred to a nylon membrane, and probed with antibodies against HIP1, HIP 12, CHC and the AP2 109 subunit, a-adaptin. HIPl and HIP 12 were both found to be enriched in the clathrin-coated vesicle fractions (CCV; figure 5.2.IB, top two panels). As expected , CHC and the clathrin associated protein subunit, a-adaptin, were also present in the CCV fractions (figure 5.2.IB, bottom 2 panels). The clathrin coat proteins were separated, from the embryonic day 18 tissue (El 8) and adult rat brain CCVs using 0.5M Tris pH 9.0 and fractionated by linear 5-20% sucrose gradients (figure 5.2.1C, bottom 2 panels). The proteins in the different cytosolic fractions were separated by SDS-PAGE, and either stained using Coomassie Blue or transferred to a nylon membrane, and probed with antibodies against HIPl or HIP 12. Coomassie Blue staining indicated that HIPl and HIP 12 were present on both El8 and adult rat brain CCVs. 5.2.2 Interaction of HIPl with HIP12 5.2.2.1 Co-immunoprecipitation As mentioned previously, yeast two-hybrid studies have demonstrated that fragments of HIPl and HIP 12 can interact and form heterodimers. Furthermore, the fragments of HIPl and HIP 12 which mediate interaction both contain their central coiled-coil domains (Chapter 3, Chopra et al., 2000). To confirm that the full-length HIPl and HIP 12 proteins interact, co-immunoprecipitation studies were performed in HEK 293T cells using the monoclonal HIPl antibody, HIPl #9. The immunoprecipitation of endogenous HIPl led to co-immunoprecipitation of HIP12 (figure 5.2.2A). Furthermore, transfection of HEK 293T cells with HIP 12 cDNA resulted in a greater amounts of HIP12 co-immnopreciptated by the endogenous HIPl (figure 5.2.2A). Negative controls 110 Figure 5.2.1. HIP1 and HIP12 are coat proteins of CCVs and display distinct developmental expression profiles. A, post-nuclear supernatants (100 (ig) from brain extracts of various rat developmental stages were analyzed by Western blot with anti-HIP1 or anti-HIP12 antibodies as indicated. E, embryonic day; P, post-natal day; A, adult. B, purified CCVs were obtained by subcellular fractionation of adult rat brain homogenates. Aliquots (100 jitg) of the various fractions were analyzed by Western blot with antibodies against clathrin heavy chain (CHC), the cc-adaptin subunit of AP2 (AP2), HIP1 and HIP 12, as indicated. H, homogenate; P, pellet; S, supernatant; SGp, sucrose gradient pellet; SGs, sucrose gradient supernatant; C, coats; V, vesicles. The coats and vesicles correspond to the supernatant and pellet fractions, respectively, following incubation of the purified CCVs with 0.5 M Tris, pH 9.0. C, purified CCVs from adult and El 8 brains were stripped of their coats in 0.5 M Tris, pH 7.0, 2 mM EDTA. Aliquots of the coat fractions were analyzed on SDS-PAGE and Coomassie Blue staining (panels at left). The coat proteins were then fractionated on continuous 5-20% sucrose gradients. The gradients were collected in 20 equal fractions and aliquots from fractions 7-18 were analyzed by SDS-PAGE with Coomassie Blue staining (Coomassie). The migration of HIP1 and HIP 12 was determined by Western blot as indicated. The bracket on the El 8 gradient demonstrates the migratory positions of the large subunits of the adaptins, whereas the arrow indicates the migratory position of HIP1. Ill 116-Blot: mAb IIIPIOT Blot: IIIPI2I [' KH 116 KIK l>2 A B 116-97-116- WKr^r fr-J k± iio-i 97 -Blot: inAli HIPI#9 Blot: pAbHIPI2FP Blot: mAb anti-CHC Blot: mAb anli-AP2 H PI P2 S2 SGp SGs C V 205 116-97-68-45-<»-CHC 4-HIP12-fe ad apt ins 116-Coomassie Blot: pAb HIP12FP 7 8 9 II 12 13 14 15 16 17 18 CLC Adull CHC 1 HIPI/ r J ad apt ins L 116 mmm Wm^Wmw — mm t 1— • • • ^IBBBBBBW i Coomassie Blot: mAbHIPl#9 7 8 9 10 11 12 13 14 1$ 16 17 18 CLC 112 H1P12 IP: mAb HIPI#9 Blot: pAb HIP12FP IP -AB SM IP HIPI2-HA HIP1 overlay V ft**. HIPI Blot: mAb HIPI#9 SM GST H1P12 HIPI2 302-348 349-644 H1P12 1 Blot: pAb HIP12FP SM GST HIPI HIPI 276-335 336-610 Fig. 5.2.2. HIP1 and HIP12 interact via their helical domains. A, HEK 293T cells were transfected with full-length HIP12 cDNA (HIP12) or were left untransfected (). Cell lysates were prepared and immunoprecipitations were carried out in the presence or absence (AB) of mouse mAb HIP1#9. Cell lysate (SM) and immunoprecipitated protein (IP) were analyzed by SDS-PAGE and Western blot with anti-HIP12FP antibody. B, co-localization of transfected HIP12-HA(red) and endogenous HIP1 (green) was analyzed in neuronal NT2 cells. Overlays of the images are shown in the panel on the right (overlay). Scale bar =10 uM. C, HeLa cells were transfected with either HIP1 or HIP12 full-length cDNAas indicated. Cell extracts were prepared and tested for binding to GST alone or various HIP12 and HIP1. 113 in which no HIPl #9 was added resulted in a lack of immunoprecipiptation of HIP 12 (figure 5.2.2A). 5.2.2.2 Confocal microscopy To assess the intracellular locations where HIPl and HIP 12 may interact, confocal microscopy studies were performed in collaboration with Dr M. Metzler. An hemagglutinin-tagged HIP 12 (HIP12-HA) expression construct was generated and transfected into the neuronal cell line NT2. The intracellular localization of HIP12-HA and endogenous HIPl was determined using a monoclonal antibody against hemaagglutinin and a polyclonal antibody against HIPl. Within the NT2 cells, HIP 12-HA exhibited punctate staining throughout the cell and displayed significant co-localization with HIPl (figure 5.2.3B). The HIPl and HIP12 staining pattern was similar to the distribution of clathrin-coated pits and vesicles 5.2.2.3 Affinity purifications using deletion constructs To confirm whether the coiled-coil domains of HIPl and HIP 12 are required for their interaction, pull-down assays were performed using HIPl and HIP 12 deletion constructs. Four different deletion constructs were generated by L. Gan and I and pull down assays were performed by Dr V. Legendre-Guillemin. The constructs were: 1) HIPl 336-610 which expressed an HIPl fragment containing the central coiled coil domain (corresponding to amino acids 336-610) 2) HIPl 276-335 which expressed an HIPl fragment expressing the sequences between the coiled coil and ENTH domains (corresponding to amino acids 276-335) 3) HIP12 349-644 which expressed an HIP12 114 276 301 PVVVIPAEASSPDSEPVLEKDD PVVVIPEEAPEDE EPEN 306 QNL|FDNK|F|DDI F G S S F| 326 PAGEPVVVA[DJL f_ DQTF D P F L M D M D L I E I S A S Q T G P NFNSQNGVN P N G S V Hipl Hipl2 Hipl Hipl2 g 1 1003/1068 HIP1/HIP12: II ENTH I  helical I I talin II 276 335 GST-HIPl-(276-335): • 302 348 GST-HIP12-(302-348): • Blot: mAb anti-CHC Blot: mAb anti-AP2 Fig. 5.2.3. HIP1 and HIP12 display differential binding to clathrin and AP2. A, sequence alignment of a region of HIP1 and HIP12 demonstrating consensus clathrin and AP2-binding sites. B, schematic representation of various GST fusion proteins used for the binding assays. C, soluble proteins from brain extracts were affinity purified with equal amounts of HIP1 or HIP12-GST fusion proteins bound to glutathione-Sepharose beads. Proteins specifically bound to the beads were analyzed by Western blot with antibodies against CHC or the a-adaptin subunit of AP2 (AP2) as indicated. 115 fragment containing the central coiled coil domain (corresponding to amino acids 349-644) 4) HIP 12 302-348 which expressed an HIP 12 fragment expressing the sequences between the coiled coil and ENTH domains (corresponding to amino acids 302-348) Each of the proteins contained a glutathione s transferase-tag (GST-tag) at their respective carboxy-termini. It was determined that HIPl 336-610 and HIP12 349-644 were able to specifically "pull-down" HIP12 and HIPl respectively (figure 5.2.3C). Thus, the coiled-coil domains are necessary for HIPl-HIP 12 interaction. 5.2.3 Differential binding of HIPl and HIP12 to Clathrin HIPl has previously been shown to contain the consensus sequence for a clathrin box sequence, LMDMD, at amino acid residues 298-302 (Metzler et al., 2001; Chapter 4, section 4.2.3). In addition, HIPl also possesses the consensus AP2 binding sequences FDNKP, FDDIF, FGSSF (at amino acids 309 to 321) and DPF (at amino acids 324 to 326) ( Mishra et al., 2001; Metzler et al., 2001; Chapter 4, section 4.2.3). Sequence analyses by Mishra et al. (2001) have shown that HIP 12 also contains the clathrin box sequence LIEIS (at amino acids 318-322) (figure 5.2.3A). The HIP12 protein sequence did not contain adjacent AP2 binding motifs. To determine whether the clathrin box and AP2 binding motifs present in HIPl and HIP 12 can interact with clathrin and AP2, two deletion constructs were generated by me. The deletion constructs were: 1) HIPl 276-335 which contained the HIPl clathrin box and its adjacent AP2 binding sequences (corresponding to amino acids 276-335) 2) HIP12 302-348, which contains the HIP12 clathrin box sequence. These studies were performed in collaboration with Dr. V. 116 Legendre-Guillemin. The HIPl 276-335 and HIP12 302-348 fusion proteins were bound to glutathione-sepharose, and incubated with rat brain material and washed several times. The bound proteins were eluted from the columns, separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and probed with antibodies against clathrin and AP2. The HIPl 276-335 fusion protein was found to bind strongly to both CHC and AP2 (figure 5.2.3C, top panel). However, HIP12 302-348 protein displayed relatively weak clathrin binding and no AP2 binding (figure 5.2.5C, bottom panel). 5.2.4 Differential binding of HIPl and HIP12 to F-actin . 5.2.4.1 Co-sedimentation studies As mentioned previously, the talin-like domain in HIP 12 has been shown to interact with F-actin (Chapter 1; Engqvist-Goldstein et al., 1999). Since the HIPl talin-like domain displayed high sequence identity to that of HIP 12, co-sedimentation studies were performed in order to determine whether the HIPl talin-like domain also interacts with F-actin. To perform these studies two different constructs were generated by L. Gan and me. The constructs were: 1) GST-HIP 1-talin which expressed the HIP 1-2 talin-like domain (corresponding to amino acids 731-1003) 2) GST-HIP 12-talin which expressed an HIP 12 fragment expressing its talin-like domain (corresponding to amino acids 765-1068). Both of these proteins contained a GST-tag fused to their respective carboxy-termini. As previously reported, a substantial proportion of GST-HIP 12-talin, 87.6% +6.0 (mean +S.E., n=3), bound to F-actin (figure 5.2.4A). Unexpectedly, a significantly reduced amount of GST-HIP 1-talin bound to the F-action 19.3% +3.7 (mean +S.E., n=3) 117 A HIP12-GST-talin HIPI-GST-talin S P S P SPSPSPSPSP () 5 Actin ||iM| o 1.25 2.5 5 10 Fig. 5.2.4. HIP1 does not bind actin in contrast to HIP12. A, binding of the HIP1-talin homology domain expressed as a GST fusion protein (HIP1-talin) to filamentous actin was analyzed by cosedimentation in the presence of various concentrations of assembled actin (0-10 uM). Binding of the HIP12-talin homology domain GST fusion protein (HIP12-talin) was used as positive control. The arrows indicate HIP1 or HIP12 fusion proteins in the supernatant (S) and pellet (P) fractions. B, the percentage of GST-HIP1-talin bound to actin is shown as mean ± S.E. of three independent experiments. C, HeLa cells expressing a FLAG-tagged construct encoding the HIP1-talin homology domain (HIP1 -talin-Flag) were immunostained with an anti-FLAG antibody shown in green and with Texas Red-phalloidin shown in red to reveal F-actin. Overlays of the confocal images are shown in the right. Scale bar = 10 uM. 118 (figure 5.2.4B). These findings were consistent when tested under different parameters including pH and ion concentration (data not shown). 5.2.4.2 Confocal microscopy To confirm that HIPl does not interact with F-actin, GST-HIP 1-talin was expressed in HELA cells and examined using confocal microscopy. Using the F-actin stain, phalloidin, and an antibody against GST, it was determined that GST-HIP 1 -talin does not co-localize with the cortical actin cytoskeleton (figure 5.2.4C). These findings are in constrast to previous findings clearly demonstrating that the talin-like domain in HIP 12 can interact with F-actin (Chapter 1; Engqvist-Goldstein et al., 1999). 5.2.5 The Coiled-Coil Domains of HIPl and HIP12 Bind to Clathrin It has previously been demonstrated that the HIPl clathrin box can interact with clathrin heavy chain (CHC; Metzler et al., 2001; Chapter 4 section 4.2.3). Furthermore, the adjacent coiled coil domain seems to enhance HIP interaction with CHC (CHC; Metzler et al., 2001; Chapter 4 section 4.2.3). To determine whether HIPl (and potentially HIP 12) contain clathrin binding domains within their respective coiled coil domains, affinity purification studies were performed using HIPl and HIP 12 expression constructs. The constructs used were: 1) GST-HIPl-(336-610) which expressed the HIP1-2 coiled coil domain (corresponding to amino acids 336-610) 2) GST-HIP 12-(348-644) which expressed an HIP 12 fragment expressing its talin-like domain (corresponding to amino acids 348-644). Both of these proteins contained a GST-tag fused to their respective carboxy-termini. The HIPl-(336-610) and HIP12-(348-644) fusion proteins 119 were bound to glutathione-sepharose, and incubated with rat brain material and washed several times. The specifically bound proteins were eluted from the columns, separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and probed with antibodies against clathrin and AP2. The HIPl and HIP 12 fusion proteins were both capable of interacting with clathrin but not to AP2 (figure 5.2.5A-B). Thus, the coiled coil domains present in both HIPl and HIP 12 contain sequence which mediate interaction with CHC. 5.2.6 Clathrin Coat Assembly using the Coiled-Coil Domains of HIPl and HIP12 Sequence analyses have demonstrated the presence of DLL motifs within the coiled coil domains of both HIPl and HIP 12. The DLL motifs are present on a several adapter proteins (including AP2 and API80) and are required for clathrin cage formation (Morgan et al., 2000). Therefore, it is possible that HIPl and/or HIP12 may promote clathrin coat assembly. This suggestion is supported by studies which demonstrated that full-length HIP 12 can induce the formation of clathrin lattice structures (Engqvist et al., 2001). The fusion protein produced by the constructs GST-HIPl-(336-610) and GST-HIP 12-(348-644) were used to determine whether the coiled coil domain of HIPl and HIPl2 could stimulate clathrin assembly in vitro. These studies were performed in collaboration with Dr V. Legendre-Guillemin. As expected, the negative control, GST protein, was unable to induce clathrin lattice formation in vitro. The GST-HIP 1-(336-610) and GST-HIP 12-(348-644) protein products were both capable of stimulating clathrin assembly in a protein concentration-dependent manner (figure 5.2.6). 120 HIP1/HIP12: II ENTH I GST-HIPl-(336-610): GST-HIP12-(349-644): helical 336 610 helical I 349 644 helical 1003/1068 talin B Brain extract Brain extract Blot: mAb anti-CHC Blot: mAb anti-AP2 SM GST HIPl HIP12 336-610 349-644 Fig. 5.2.5. The helical domains of HIP1 and HIP12 bind to clathrin but not AP2. A, schematic representation of various GST fusion proteins used for the binding assays. B, soluble proteins from brain extracts were affinity purified with equal amounts of HIP1 or HIP12 GST fusion proteins bound to glutathione-Sepharose beads. Proteins specifically bound to the beads were analyzed by Western blot with antibodies against CHC or the a-adaptin subunit of AP2 (AP2) as indicated. 121 205 116-97-68-45-<— CHC «i— GST 0 0.25 0.5 1 2 u.M <— CHC HIP1-336-610 0.12 0.25 0.5 1 2 uM <i— CHC <— HIP12-349-644 SM 0.12 0.25 0.5 1 2 uM Fig. 5.2.6. HIP1 and HIP12 promote clathrin assembly through their helical domains. An aliquot of the purified clathrin used as a starting material (SM) for the clathrin assembly assays was resolved on SDS-PAGE and stained with Coomassie Blue. Clathrin assembly assays were performed with increasing amounts of fusion proteins GST, GST-HIP1-(336-610), and GST-HIP12-(348-644) as indicated. The GST fusion proteins and the clathrin remaining in the supernatant after high-speed centrifugation following initiation of clathrin assembly were analyzed by SDS-PAGE and Coomassie Blue staining. 122 5.2.7 HIPI and HIP12 Binding to the Clathrin Light Chain Although the coiled coil domains of HIPI and HIP 12 can interact with clathrin from rat brain; sequences examinations have not identified any clathrin box motifs within these domains. These findings suggest that either the coiled coil domains contain a novel clathrin binding motif or, these domains bind indirectly with the CHC. In order to confirm the interaction of the coiled coil domains with CHC, affinity purification studies were performed using the GST-HIP 1-(336-610) and GST-HIP 12-(348-644) fusion proteins. As expected, the fusion proteins were able to bind to CHC from rat brain extracts HIPl-(336-610) and GST-HIP 12-(348-644) (figure 5.2.7A, top panel). However, GST-HIPl-(336-610) and GST-HIP 12-(348-644) were unable to interact with purified His6-tagged CHC terminal domain (figure 5.2.7A, bottom panel). These findings suggest that either the coiled coil domains of HIPI and HIP 12 do not interact directly with the CHC or, these domains interact with other segments of the CHC. Since clathrin triskelia are composed of three CHC interacting with three CLCs, it may be possible that the HIPI and HIPI2 coiled coil domain interacts with CLC. Affinity purification studies were performed using HIPI-(336-610) and GST-HIP 12-(348-644) fusion proteins to determine whether HIPI and HIP 12 could interact with in vitro synthesized CLC b. Both HIP1-(336-610) and GST-HIP 12-(348-644) were found to interact with CLC b (figure 5.2.8B, bottom panel). These findings are consistent with previous findings indicating that CLC is required for HIP 12 interaction with the CHC (Engqvist et al., 2001). 123 A Blot: mAb anti-CHC Blot: anti-tetra-HIS Blot: anti-tetra-HIS Fig. 5.2.7. HIP1 and HIP12 bind directly to the clathrin light chain through their helical domains. A, soluble proteins from brain extracts or purified His6-tagged clathrin terminal domain (TD) fusion protein were affinity purified with equal amounts of HIP1 or HIP12 GST fusion proteins bound to glutathione-Sepharose beads. Proteins specifically bound to the beads were analyzed by Western blots with antibodies against CHC or the His epitope (tetra-HIS) as indicated. B, purified His6-tagged clathrin light chain b (LCb) fusion protein was affinity purified with equal amounts of HIP1 or HIP12 GST fusion proteins bound to glutathione-Sepharose beads. Proteins specifically bound to the beads were analyzed by Western blot with the anti-His epitope antibody (tetra-HIS). 124 5.3 Discussion In this study we have demonstrated that HIPl and HIP 12 are components of the endocytic machinery. HIPl and HIP 12 were found to be enriched in purified clathrin-coated vesicles (CCVs) at similar levels to clathrin and the adapter proteins a-adaptin and API80. Furthermore, like the AP2 subunit, a-adaptin, HIPl and HIP 12 was stripped from the coated vesicles using Tris pH 9.0. The buffer Tris pH 9.0 has previously been shown to detach coat components from CCVs (Keen et al, 1979). The association of HIPl and HIP 12 with the CCVs, indicate that these proteins are clathrin coat components and may play a role in the recruitment and assembly of the clathrin lattice structure. Indeed, HIPl was found to interact directly with clathrin and AP2. HIPl was found to contain a type 1 clathrin box sequence LMDMD. The consensus sequence for the type 1 clathrin box is L(L/I)(D/E/N)(L/F)(D/E) (Ramjaun et al., 1998; Drake et al., 2001). The endocytic machinery proteins AP180, amphiphysin, epsins, and p-arrestin have previously been shown to contain a type 1 clathrin box (Ford et al., 2001; Slepnev et al., 2000; Drake et al., 2000; Laporte et al, 1999). HIPl also contains the AP2 binding motifs DPF and FDNKP, FDDIF, FGSSF. The consensus AP2 binding motifs DPF/W and FXDXF (where X represents any amino acid) are also present in the endocytic proteins epsin, amphiphysin 1 and API80 (Mishra et al, 2001; Slepnev et al., 2000; Hao et al.,1999). Unlike HIPl, HIP 12 exhibited weak binding to clathrin and no interaction with AP2. Sequence analyses indicated that HIP 12 contained the clathrin box sequence but did not contain any known AP2 binding motifs. The HIP 12 clathrin box was similar to the endocytic protein P-arrestin and to the CDC42-interacting proteins ACK1 and ACK2 125 (Mishra et al.; 2001; Teo et al., 2001; Yang et al, 2001). As mentioned previously, Cdc42 is known to play an important role in the regulation actin cytoskeletal dynamics, endocytosis and cellular transport (Chapter 1, section 1.9.6; Ramesh et al., 1999). These studies have also demonstrated that HIPI and HIP 12 are not only components of CCVs but also play a role in the assembly of the clathrin coat. Specifically, the coiled coil domains present in both HIPI and HIP 12 were capable of inducing the assembly of the clathrin lattice in vitro. Furthermore, these coiled coil domains did not interact with CHC but rather bound to CLC. Thus, the interaction of HIPI and/or HIP 12 with the CLC may play a role in clathrin assembly. Indeed, CLC has been shown to negatively regulate clathrin assembly (Ybe et al., 1998). The generation of the polyhedral clathrin lattice structure requires the interaction of clathrin triskelions units via salt bridges and electrostatic interactions. Ybe et al. (1998) have demonstrated that CLC can inhibit clathrin assembly by preventing the CHCs from forming salt bridges. More recently, Wilde et al. (1999) have demonstrated that Src-mediated phosphorylation of CHC, at a tyrosine residue located within the site of CHC-CLC interaction, results in both decreased CLC binding and induction of clathrin assembly. Taken together, it is possible that the coiled coil domains of HIPI and HIP 12 stimulate clathrin assembly by interacting with CLC and thereby prevent its inhibitory effects. The carboxy-terminal sequences of HIPI and HIP 12 contain domains which have significant sequence identity with the cytoskeletal-associated protein, talin (Chapter 3, section 3.2.4; Chopra et al., 2000; Engqvist-Goldstein et al., 1999; Kalchman et al., 1997). The talin-like domain present in Sla2p, the yeast ortholog of HIP1/HIP12, has previously been shown to interact with F-actin (McCann and Craig, 1997). Similarly, we 126 have confirmed previous findings that HIP 12 can also interact with F-actin (Engqvist-Goldstein et al., 1999). The talin-like domain in Sla2p and HIP 12 function in the localization of these proteins to the cortical actin cytoskeleton (Wesp et al 1997; Engqvist-Goldstein et al., 1999). Unexpectedly, the HIPl talin-like domain did not exhibit any interaction with F-actin. In this study we have confirmed previous yeast two-hybrid data showing that HIPl can interact with HIP 12 (Chapter 3, section 3.2.8; Chopra et al., 2000). It is possible that the formation of this heterodimers may play a role in localization of HIPl to the cortical actin cytoskeleton. Several studies have shown that HIPl, HIP 12 and Sla2p are localized to the cytoplasmic membrane and, in particular, sites undergoing clathrin mediated endocytosis (CME; Holtzman et al, 1993; Raths et al., 1993; Engqvist-Goldstein et al., 1999; Metzler et al., 2001; Chapter 4, section 4.2.2). The amino terminal sequences of HIPl, HIP12 and Sla2p contain ENTH domains which has been shown to play a role in the localization of these proteins to the plasma membrane (Wesp et al, 1997; Engqvist-Goldstein et al., 1999; Metzler et al., 2001; Chapter 4, section 4.2.2). ENTH domains are proposed to stimulate clathrin-mediated endocytosis by binding to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]-containing membranes (Ford et al., 2001; Itoh et al., 2001). ENTH domains are also present in several other endocytic proteins including epsin 1 and 2 and AP180 (Ford et al, 2002; Ford et al, 2001; Itoh et al., 2001). Within neurons, PtdIns(4,5)P2 levels have been found to be increased on the plasma membrane at sites of CME and on newly endocytosed synaptic vesicles (Micheva et al., 2001). It is possible that the localization of HIPl and HIP 12 to the endocytic machinery may be mediated by their ENTH domains. 127 The present studies suggest that HIPI and HIP 12 are components of the endocytic machinery that can link the plasma membrane to the underlying cortical actin cytoskeleton. HIPI was originally identified as an interactor of huntingtin, the protein altered in HD (Kalchman et al., 1997; Wanker et al., 1997). The strength of the interaction is decreased with increased lengths of the polyglutamine tract present in huntingtin. HIPI and huntingtin have been shown to be highly enriched within the brain and display a similar subcellular distribution (Kalchman et al., 1997; Wanker et al., 1997; Chopra et al., 2000). Huntingtin has also been shown to interact with the endocytic proteins a-adaptin and endophilin A3 (Faber et al., 1998; Sittler et al., 1998). In addition, huntingtin is also associated with clathrin-coated vesicles and thought to function in vesicle trafficking in the secretory and endocytic pathways (Velier et al., 1998). Take together, the altered interaction between mutant huntingtin and HIP 1 may result in alterations in vesicle internalization within the neurons of the brain that ultimately lead to the neuropathological changes observed in HD. 128 Chapter 6: General Discussion 129 6.1 Summary The objective of this thesis was to identify and characterize members of the HIPl family of cytoskeletal-associated proteins. HIPl was initially identified as an interactor of huntingtin (Kalchman et al., 1997; Wanker et al., 1997). Increased lengths of the huntingtin polyglutamine tract were associated with decreased interaction with HIPl (Kalchman et al., 1997). By gaining greater insights into the cellular functions of the HIPl family, it may be possible to determine their role in the pathogenesis of HD. In this thesis we have determined that HIPl is a member of a family of cytoskeletal-associated proteins that function in clathrin-mediated endocytosis (CME). In addition, in vitro studies indicated that HIPl (but not HIP 12) can induce programmed cell death by activating the pro-apoptotic enzyme caspase-3. The following sections will discuss the cellular functions of the HIPl family and their significance in the pathogenesis of Huntington Disease. 6.2 HIPl and HIP12 are components of the endocytic machinery In this thesis I have identified a homolog of HIPl termed HIP12. HIPl and HIP12 shared 47% amino acid identity and both proteins contained ENTH, coiled coil and talin-like domains. However, HIP 12 did not interact with huntingtin but could bind to HIPl and form heterodimers. Please refer to figure 6.1 for the location of the different binding sites of HIPl and HIP 12. Tissue distribution studies indicated that HIPl was expressed predominantly within the brain whereas HIP 12 expression was more ubiquitous. Within cells, HIPl and HIP 12 displayed a similar punctate distribution at the plasma membrane and underlying cortical actin cytoskeleton. Specifically, HIPl and HIP 12 were both 130 Figure 6.1 HIPI and HIP12 Domains. The ENTH , coiled coil, and talin-like domains are represented by red, blue and green shading respectively. The red line represents the death effector domain present in HIPl-1 and HIPl-2. The location of the clathrin box sequences and binding sites for phosphatidylinositol-4,5-bisphosphate, AP2, and clathrin light chain are depicted by arrows. 131 ENTH Ptdlns 4,5-P2 Binding Domain ii HIPl-1 1 mSS^QVPNPLPKVLSRRGVGAGLEAAEKKSKKK'rij'I'VSINKAINTQEVAVKEKHARTCILGTHHEKGAQTFWSVVNRL HIPl-2 1 MDVSKMTVSINKAINTQEVAVKEKHARTCILGTHHEKGAQTFWSVVNRHIP12 1 MNSIKNVPARVLSRRPGHS- [ IEAEIR EQFDKTQAI SISKAINTQEAPVKEKHARRIILGTHHEKGAFTFWSYAIGL Ptdlns 4,5-P2 Binding Domain (continued) U I HIPl-1 81 PLSSNAVLCWKFCHVFHKLLRDGHPNVLKDSLRYRNELSDMSRMWGHLSEGYGQLCSIYLKLLRTKMEYHTKNPRFPGNL HIPl-2 5 0 PLSSNAVLCWKFCHVFHKLLRDGHPNVLKDSLRYRNELSDMSRMWGHLSEGYGQLCSIYLKLLRTKMEYHTKNPRFPGNL HIP12 7 5 PLPSSSILSWKFCHVLHKVLRDGHPNVLHDCQRYRSNIREIGDLWGHLHDRYGQLVNVYTKLLLTKISFHLKHPQFPAGL consensus HIPl-1 161 QMSDRQLDEAGESDVNNFFQLTVEMFDYLECELNLFQTVFNSLDMSRSVSVTAAGQCRLAPLIQVILDCSHLYDYTVKLL HIPl-2 130 QMSDRQLDEAGESDVNNFFQLTVEMFDYLECELNLFQTVFNSLDMSRSVSVTAAGQCRLAPLIQVILDCSHLYDYTVKLL HIP12 155 EVTDEVLEKAAGTDVNNIFQLTVEMFDYMDCELKLSESVFRQLNTAIAVSQMSSGQCRLAPLIQVIQDCSHLYHYTVKLL consensus 161 * * * **** **********_ HIPl-1 HIPl-2 HIP12 consensus 241 FKLHSCLPADTLQGHRDRFMEQFTKLKDLFYRSSNLQYFKRLIQIPQLPENPPNFLRASALSEHISPVWIPAEASSPDS 210 FKLHSCLPADTLQGHRDRFMEQFTKLKDLFYRSSNLQYFKRLIQIPQLPENPPNFLRASALSEHISPVWIPAEASSPDS 235 FKLHSCLPADTLQGHRDRFHEQFHSLRNFFRRASDMLYFKRLIQIPRLPEGPPNFLRASALAEHIKPVWIPEEAP--ED 241 ******* ******** ****** Clathrin Heavy-Chain Binding Motif AP2 Binding Motifs Coiled Coil m HIPl-1 321 EPVLEKDDLMDMDASQQNLFDNKFDDIFGSSFSSDPFNFNSQNG HIPl-2 290 EPVLEKDDLMDMDASQQNLFDNKFDDIFGSSFSSDPFNFNSQNG' HIP12 313 E--EP-ENLIEISTGPPAGEPVWADLFDQTFGPP NGS' consensus 321 * * *.*...* HIPl-1 401 HIPl-2 370 HIP12 383 consensus 401 HIPl-1 481 HIPl-2 450 HIP12 463 consensus 481 Death Effector Domain HVSELEADLAEQQHLRQOAADDCEFLRAEI,, | ITEKAQRSLSEIERKAQANEQRYSKLKEKYSELVQNHA ELEADLAEOOHLR0OAADDCEFLRAELDELRR0REDTEKA0RSLSEIERKA0ANEORYSKLKEKYSELVONHA I .EEQRKQKQKALVDNEQLRHELAQI,1 : IQGLREEAERKASATEARYNKLKEKHSELVHVHA Clathrin Assembly Motif _L ^AEVTKQVSMARQAQV! EDSL LLRKNAEVTKQVSMARQAQVDLSREKKELEDSLERISDQGQRKTQEQLEVLESLKQELATSQRELQVLQGSLETSAQSE LLRKNADTAKQLTVTOOSQEEVARVKEOLAFOVEQVKRESELKLEEKSDQLEKLKRELEAKAGELARAQEALSHTEQSK * * * * HIPl-1 I ANWAAEF AEL EKERDSL VSGAAHREEEL SALR •- KELQDTQLKLASTEESMCQLAKDQRK HIPI-2 ANWAAEFAELEKERDSLVSGAAHREEELSALR HIP12 543 SELSSRLDTLSAEKDALSCIAVROREAULLAAOSLVRETEAALSREQORSSQEQGELOGRLAERESQEQGLRQRLLDEQF consensus 561 *..*.*.* **..*.*.. ,*** *.*....*...*... HIPL-1 62 0 LLVGSRKAAEQVIODALNQLEEPI'LISCAGSADHLLSTVTSISSCIEQLEKSWSQYLACPEDISGLLHSITLLAHLTSDA HIPL-2 • LLVGSRKAAEQVIQDALNQLEEP' ..DHLLSTVTSISSCIEQLEKSWSQYLACPEDISGLLHSITLLAHLTSDA HI PI 2 . •• VLRGAAAEAAGILQDAVSKSBLHLRCTSSPDYLVSRAQEALDAVSTLEEGHAQYLTSLADASALVAALTRFSHLAADT consensus 641 .*.* * ***...*..* *..*.*.*.* ** *** *.*.* *...**..*. 132 HIP1-1 700 IAHGATTCLRAPPEPADSLTEACKQYGRETLAYLASLEEEGSLENADSTAMRNCLSKIKAIGEELLPRGLDIKQEELGDL HIPl-2 6 69 IAHGATTCLRAPPEPADSLTEACKQyGRETLAYLASLEEEGSLENADSTAMRNCLSKIKAIGEELLPRGLDIKQEELGDL HIP12 7 03 IINGGATSHLAPTDPADRLIDTCRECGARALELMGQLQDQQALRHMQASLVRTPLQGILQLGQELKPKSLDVRQEELGAV consensus 721 *..*..*...**..***.*...*...*...*.. ..* * ********* ***** HIP1-1 780 VDKEMAATSAAIETATARIEEMLSKSRAGDTGVKLEVNE HIP1-2 749 VDKEMAATSAAIETATARIEEMLSKSRAGDTGVKLEVNE HIP12 783 VDKEMAATSAAIEDAVRRIEDMMNQARHASSGVKLEVNE consensus 801 *************.*,,*** * * ******** Talin-Like I/L VESGRGTASPKEFY VESGRGTASPKEFY VESGRGAATQQEFY * * * * * 1 HIPI-1 860 MiKt^^BsBnSBBBSn AKNSR^H^^^fflBfflHH^^TODLVVOGRGKFffl|^»^^^^^^^^^BADKDSPNLAOLQQASRGV HIP12 863 AKM.qRMWllj^ln^^^P^^BI^L-.nfT ' fflMBBBBB^^^^BHITl ^F""rT" consensus 881 **********************.,*,***.**,,,**,***,********************* * ** * ** ** * HIPI-1 940 NQATAGWASTISGKSQIEETDNMDFSSMTLTQIKRQE HIP1-2 909 NQATAGWASTISGKSQIEETDNMDFSSMTLTQIKRQE HIP12 943 NERAANWASTKSGQEQIEDRDTMDFSGLSLIKLKKQEH consensus 961 *...*.*****.**..***..*.****...*...*.** ELAGVAEGWEEGT ELAGVAEGWEEGT LAGASGSPGEEV HIPI-1 1020 EASPPTLQEWTEKE HIP1-2 989 EASPPTLQEWTEKE HIP12 1023 AIRPSTAPRSVTTKKPPLAQKPSVAPRQDHQLDKKDGIYPAQLVNY consensus 1041 ...*.*....**.*. 133 found to be localized to plasma membrane sites undergoing CME and associated with clathrin-coated vesicles. The findings in this thesis also indicated that HIPI and HIP 12 differ in their associations with components of the endocytic machinery. Please refer to figure 6.2 for the HIPI and HIP 12 domains and their respective interacting proteins. HIPI was shown to interact with the clathrin heavy chain (via its clathrin box sequence) and AP2 (via its DPF and FXDXF motifs). The tandem arrangement of the clathrin box and DPF present in HIPI has been suggested to help stabilize the relatively weak interaction between AP2 and clathrin. Relative to HIPI, HIP 12 displayed weaker binding to clathrin heavy chain (through its clathrin box sequence) and no interaction with AP2. In this thesis it was also shown that the coiled coil domains present in both HIPI and HIP 12 were capable of inducing the assembly of the clathrin coat in vitro. The HIPI and HIP 12 coiled coil domains were also shown to interact with the clathrin light chain. The clathrin light chain has been shown to inhibit the assembly the clathrin lattice structure (Ybe et al., 1998; Wilde et al., 1999). It may be possible that HIPI and HIP 12 stimulate with clathrin coat assembly by binding to clathrin light chain and abrogating its inhibitory activity. Taken together, these results demonstrate that HIPI and HIPI are components of the endocytic machinery that participate in the recruitment, assembly and stabilization of the clathrin lattice structure. 134 u ro "C U) sz c ro H5 •c -o -° SZ c c ro ™ ra O -E £ Q) rj> (A ~ ro •2 i O CM CL a .2 fl € -2 «§ 2 o s ^ % -s -ed g >-i eS o & s S 113 < o u ^ C o <u o "5 * .S3 «J o [B g 'o 5. o 42 -S H M H x 3 O M CJ o © C/J M CO Q 3 T3 a S3 = £ 52 5< U X 2 ? I s § 3 fc, g CCJ T3 (U § -5 .S Q | '§ «1 -O •B 4> cd 3 = '3 *2 M m S 6.3 HIPl and HIP12 Interact with the Cortical Actin Cytoskeleton The observation that HIP 12 interacts with F-actin suggests that HIP 12 may interact with the cortical actin cytoskeleton underlying clathrin-coated pits and vesicles. Furthermore, HIPl may be indirectly associated with the actin cytoskeleton by forming heterodimers with HIP 12. Thus, HIPl and HIP 12 may act as a molecular "link" between the endocytic machinery and the cortical actin cytoskeleton. Consistent with this suggestion is the observation that a number of proteins involved in CME also influence cortical actin cytoskeletal dynamics. For example, dynamin has been shown to interact with several actin-binding proteins including profilin, Abpl, and cortactin (Kessels et al., 2001). Cortactin is known to activate actin assembly at sites adjacent to clathrin-coated pits (Schafer, 2002; Uruno et al, 2001). The endocytic protein, intersectin, has also been shown to induce the polymerization of actin filaments at site of CME (Qualmann et al., 2000). In addition, the huntingtin-interacting proteins, pacsin 1 and CIP4 are involved in the stimulation of the actin polymerization machinery at the site of newly formed endocytic vesicles (Modregger et al., 2002; Holbert et al., 2003). The cortical actin cytoskeleton is thought to perform a number of different functions during CME. The actin cytoskeleton may be involved in localizing the endocytic machinery to specific sites on the cytoplasmic membrane by physically interacting with components of endocytic machinery and7 or acting as a barrier that prevents their lateral movement (Qualmann et al., 2000). 136 6.4 In vivo Evidence of HIPl Function in Endocytosis The data presented in this thesis presents immunohistochemical and biochemical evidence that the HIPl family of proteins are involved in endocytosis. Recently, Metzler et al. (2003) have generated mice with a targeted disruption in the HIPl gene (HIPl7") to determine the cellular function of HIPl in vivo. The HIPl"7" mice exhibited gait ataxia, tremor and developed a distinctive "hunchback" phenotype due to the development of a rigid thoracolumbar kyphosis. These changes were not due to muscular or skeletal defects but rather they were the result of neurological dysfunction. Examination of the HIPl7" mouse neurons indicated that these cells had a reduced ability to form the endocytic protein complexes. Furthermore, these mice exhibited a profound dose-dependent defect in clathrin-mediated internalization of glutamate subtype, a-amino-3-hydroxy-5-methyl -4-isoxazole propionic acid (AMPA) receptor. These findings suggest that HIPl regulates AMPA receptor trafficking in the CNS through its function in clathrin-mediated endocytosis. It may be postulated that by association huntingtin may also play in AMPA receptor trafficking. Interestingly, huntingtin also interact with the glutamate receptor-binding protein postsynaptic density 95 (PSD95; Sun et al., 2001). Furthermore, the expanded polyglutamine tract in mutant huntingtin reduces its interaction with PSD95 (Sun et al., 2001). Sun et al. (2001) demonstrated that the lack of PSD95 interaction with mutant huntingtin results in the sensitization and activation of NMD A receptors. PSD95 is a membrane-associated scaffolding protein which binds directly to N-methyl-D-aspartate (NMDA) receptor and indirectly to (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor and has also been shown to bind to the cytoskeleton and cell-137 adhesion molecules (El-Husseini and Bredt, 2002). Through these multiple interactions PSD95 induces clustering of NMD A and AMPA receptors in the postsynaptic membrane and regulates neuronal activities such as long-term potentiation and long-term depression (Sun et al., 2001; El-Husseini and Bredt, 2002). Taken together, huntingtin interaction with PSD95 may play an essential role in the regulation of glutamate receptor activity. Moreover, the loss of interaction between mutant huntingtin and PSD95 may result in a dysregulation of glutamate receptor activity which results in the glutamate-induced cell death seen in HD. Recently, Wang et al. (2003) have demonstrated that excessive activation of NMDA receptors results the activation of apoptosis and the internalization of AMPA receptors via CME. Using inhibitors of CME, Wang et al. (2003) established that inhibition of AMPA endocytosis selectively inhibits NMDA-induced apoptosis. Thus, clathrin-dependent endocytosis of AMPA is required for NMDA receptor-mediated endocytosis. It may be postulated that clathrin-coated AMPA receptor may form a pro apoptotic complex that results in the activation of cell death signals. Since HIPl and huntingtin and HIPl are both components of the endocytic machinery and can induce apoptosis, it may be possible that these proteins play an important role in NMDA-induced apoptosis. 6.5 The Role of Huntingtin in Endocytosis and Transport Several lines of evidence suggest that huntingtin may play a role in endocytosis and transport. Within neurons of the brain, huntingtin has been shown to be present in the axon and nerve terminals where it seems to be associated with microtubules and synaptic 138 vesicles (DiFiglia et al., 1995; Gutekunst et al., 1995; Tukamoto et al., 1997). Furthermore, huntingtin has been shown to be associated with clathrin-coated and non-coated endosomal vesicles in the cytoplasm and along plasma membranes (Velier et al., 1998). In this thesis it has been confirmed that huntingtin interacts with HIPl, a component in the endocytic and transport machinery of the cell. Thus, by association huntingtin may also play a role in endocytosis and transport. Please refer to figure 6.2 illustrating the known functions of several huntingtin-interacting proteins in endocytosis and transport. 6.5.1 Endocytic and Transport Proteins that interact with Huntingtin In addition to HIPl, huntingtin has been shown to interact with other proteins that play a role in endocytosis and transport. Huntingtin interacts with the brain-enriched protein HAP 1 which is thought to play a role in vesicle transport and actin dynamics. HAP1 interacts with pl50glued, a subunit of the microtubule-based transporter complex dynein (Engelender et al, 1997, Li et al., 1998). HAP1 binds to hepatocyte growth factor-regulated tyrosine kinase (Hrs), a protein involved in the regulation of vesicle transport from the early endosomes to the late endocytic compartments (Li et al., 2002). Finally, HAP1 interacts with duo, a guanine nucleotide exchange factor that is a regulator of actin cytoskeletal dynamics (Colomer et al., 1997). Huntingtin also interacts with a-adaptin-C which is a subunit of the adapter protein 2 complex (AP2). AP2 is involved in the recruitment and polymerization of clathrin subunits into clathrin-coated vesicles (Brodsky et al., 2001). AP2 is also thought to coordinate the process of endocytosis with intracellular signaling pathways (Slepnev and DeCamilli, 2000, Brodsky et al., 2001). 139 Huntingtin can also interact with endophillin A3, a protein hypothesized to play a role in the induction of membrane curvature during clathrin-dependent endocytosis (Sittler et al., 1998; Slepnev and De Camilli, 2000). Huntingtin has been shown to interact with the neurospecific protein Pacsin 1. Pacsin 1 is implicated in the recruitment and stimulation of the actin polymerization machinery at the site of newly formed endocytic vesicles (Modregger et al., 2002; Qualmann and Kessels, 2002). Huntingtin also binds to the brain-enriched protein huntingtin interacting protein 14 (HIP 14). Within neurons, HIP 14 has been suggested to play a role in clathrin-coated vesicle formation and vesicle trafficking at the cytoplasmic and Golgi apparatus membranes (Singaraja et al., 2002). Finally, huntingtin can interact with Cdc42-interacting protein 4 (CIP4). CIP4 is involved in several cellular functions including endocytosis, transport and regulation of actin cytoskeleton (Holbert et al., 2003; Aspenstrom et al., 1997; Ramesh et al., 1999; Tian et. al, 2000; Jiang et al, 2002). 6.5.2 Huntingtin-Interacting Proteins that Influence Endocytosis and Transport Interestingly, Huntingtin has been shown to interact with a number of different proteins which may be "loosely" categorized as cellular signaling proteins. Several of these proteins may, in part, be involved in endocytosis and cellular transport. These huntingtin interacting proteins include HSP70, ubiquitin-conjugating enzyme, and calmodulin. The chaperone HSP70 has been shown to interact with huntingtin in a polyglutamine-length dependent manner; the larger the repeat the greater the interaction (Jana et al., 2000). Within the brains of HD patients HSP70 have been shown to co-140 localize to inclusions in the nucleus but not in the cytoplasm (Jana et al. 2000). The cellular function of HSP70 is to prevent aggregation of denatured proteins and aid in their renaturation (Ohtsuka and Suzuki, 2000). It has been suggested that in HD, the induction HSP70 may be a cellular attempt to "clear' the nuclear aggregates containing mutant huntingtin. With respect to endocytosis and cellular transport, HSP70 and its neuron-specific cofactor auxilin are involved in the rapid disassembly of the clathrin lattice structure from newly formed endocytic vesicles (Slepnev and DeCamilli, 2000). Huntingtin can interact with the ubiquitin-conjugating enzyme hE2-25K (Kalchman et al., 1996). The interaction of hE2-25K with huntingtin is CAG length independent. This enzyme belongs to a family of proteins that participate in the linking of C-terminal glycine residues of ubiquitin to specific lysine residues on target proteins including huntingtin and ubiquitin (Kalchman et al., 1996; Hicke, 2001). Recent evidence suggests that the addition of a single ubiquitin residue (monoubiquitylation) to a target protein may result its transport to various intracellular compartments including late endosomes and lysosomal vacuoles (Shih et al., 2002; for a review see Hicke, 2001). Furthermore, monoubiquitylation has been shown to down-regulate activated receptors on the plasma membrane by inducing their endocytosis (Shih et al., 2000). It has been postulated that ubiquitin regulates protein transport at discreet site on the plasma membrane by serving as a sorting signal on protein cargo and by controlling the activity of trafficking machinery (Hicke, 2001). Interestingly, target proteins can also be conjugated with several ubiquitin "units" and form large multi-ubiquitin chains (Hicke, 2001). These multi-ubiquitin-tagged proteins are then directed to the multi-protein complex known as the proteosome where they are degraded (Hicke, 1999). Thus, ubiquitination also plays a 141 major role in the regulated catabolism of various proteins including huntingtin (Hicke, 1999; Wyttenbach et al., 2000). The Ca2+-binding protein calmodulin (CAM) has also been shown to interact with huntingtin in the presence of calcium (Bao et al., 1996; Tukamoto et al., 1997). In addition, the mutant form of huntingtin interacts with CAM in the absence of calcium and with greater affinity than with wild-type huntingtin. A number of different functions have been ascribed to CAM including smooth muscle contraction, transport, endocytosis, proliferation, protein catabolism, and cellular adhesion (Deckel, 2001). Furthermore, CAM has been shown to regulate the activity of several protein kinases and phosphatases, ion channels and nitric oxide synthase (Deckel, 2001). Of particular interest is CAM's ability to activate the calcium-binding phosphatase, calcineurin. During endocytosis calcineurin is required for the dephosphorylation of several components of the endocytic machinery including dynamin, amphiphysin, synaptojanin, epsin ESP 15, AP2 and API80 which are collectively referred to as dephosphin (Cousin et al., 2001; Cousin and Robinson, 2001). The prevention of dephosphorylation of the dephosphins results in an inhibition of endocytosis (Cousin et al., 2001). Since huntingtin interacts with CAM, it is conceivable that huntingtin also indirectly influences endocytosis. Taken together, these findings strongly suggest that huntingtin may play a role in endocytosis and transport. Please refer to figure 6.2 illustrating the known functions of several huntingtin-interacting proteins in endocytosis and transport. Recently, several studies have demonstrated that huntingtin can interact with transcription factors. At this time these interactions have not been shown to have direct consequences on endocytosis and transport. It may be speculated that huntingtin may instead play a role in the transport 142 of the transcription factors to the nucleus. Consistent with this idea is the observation that huntingtin is found in the nucleus (DiFiglia et al., 1997). Furthermore, the huntingtin protein contains HEAT repeats domains which are also nuclear transport protein importin (Andrade and Bork, 1995). Thus, by analogy huntingtin may also play a role in transport. 143 Figure 6.3 Potential roles for huntingtin interacting proteins in endocytosis. HIPl, AP2, HIP 14, endophillin, pacsin, Cdc42-interacting protein 4 (CIP4) and calmodulin play important in the formation of clathrin-coated pits and release of clathrin-coated vesicles during endocytosis. HIP 14 may also be involved in formation of clathrin-coated pits and vesicles at the trans-Golgi network. HSP70 plays an important in the "shedding" of the clathrin coat. Ubiquitin-conjugating enzyme may play a role in targeting endocytosed vesicles to intracellular organelles including lysosomes and early endosomes. HAP1 in conjunction with HRS is involved in the transport of vesicles from the early to late endosomes. HAP1 also interacts with pl50Glued complex and may help transport cargo along microtubules chains. 144 6.6 The Potential Role of Endocytosis Defects in the Pathogenesis of HD In this thesis it has been demonstrated that HIPl is one of several huntingtin-interacting that are involved in endocytosis and transport. It may be hypothesized that altered interactions between these proteins and mutant huntingtin that potentially result in defects in endocytosis. These defects in endocytosis and transport may play a role in the pathogenesis of HD. Even if this is the case, it is still difficult explain how defects in endocytosis and transport could account for the selective ioss of medium spiny neurons (MSNs) within the striatum. One possibility is that the survival of MSNs requires the endocytosis of trophic factors. For example, brain-derived neurotrophic factor (BDNF) is not produced by cells in the striatum. Instead, medium spiny neurons obtain BDNF from cortical connections (Katoh-Semba et al., 1998). Thus, if the MSNs are unable to endocytose BDNF, they may eventually die. Therefore, if HD is a result of defects in endocytosis, it would be expected that the MSNs in affected individuals would have difficulty in obtaining the trophic support of BDNF. Interestingly, BDNF has also been shown to protect cultured striatal neurons from excitotoxic cell death (Nakao et al., 1995). Several studies have reported that excititoxicity may play an important role in the selective neuronal loss seen in HD patients and mouse models of HD (Albin and Greenamyre, 1992; Hodgson et al., 1999; Zeron et al., 2002). It may be hypothesized that if BDNF cannot be taken up by the MSNs, this may result in an inability to respond to excitotoxic insults which could in turn produce changes reminiscent of HD. 146 6.7 Future Directions The isolation and characterization of the HIPl family has helped to elucidate its role in clathrin-mediated endocytosis and suggest its potential role in the pathogenesis of HD. However, a great deal of further investigations is required to extend the findings within this thesis. The following are suggestion for further investigation of the function HIP 1 family and huntingtin. 1) In this thesis 1 have determined that the HIPl gene comprises 32 exons spanning approximately 215 Kb of genomic DNA and gives rise to two alternate splice forms designated as HIP 1-1 and HIP 1-2. At this time the physiological relevance of these two splice forms is unclear. Using reverse transcriptase (RT) PCR it was determined that both these proteins are expressed at similar levels throughout the body. However, the use of quantitative RT-PCR may help to determine if the mRNA expression levels vary between HIPI-1 and HIP1-2. Alternatively, it may be necessary to generate antibodies that can distinguish between these isoforms. Using these reagents it may be possible to determine whether HIP 1-1 and HIP 1-2 have differences in their spatial and temporal expression patterns. These findings (if any) may suggest that these 2 isoforms perform exclusive functions within the cell. 2) In this thesis it was demonstrated that overexpression of HIPl in HEK 293T cells causes both a significant decrease in cell viability and an increase in caspase 3 activity. However, overexpression of HIP 12 into HEK 293T cells does not cause any alteration in cell viability or caspase 3 activity. These findings indicate that HIP 12 in contrast to HIPl is not a pro-apoptotic protein. Since HIP 12 interacts with HIPl it may be possible that 147 HIP 12 modulates the pro-apoptotic activity of HIPl. To address this possibility, it would be useful to perform co-expression studies in HEK 293T cells to determine whether HIP 12 influences HIPl's ability to reduce cell viability and increase caspase 3 activation. 3) The development of a transgenic mouse overexpressing HIPl would serve as a useful reagent to further address the pro-apoptotic function of HIP 1. These mice may help to determine whether certain ceil regions are more vulnerable to HIPl overexpression. 4) The recent development of a HIPl knockout mice has demonstrated that HIPl plays a critical role in plasma receptor internalization. Future studies may require the development of a HIP 12 knockout in order to determine its functions in vivo. The phenotypes of the HIPl and HIP 12 knockouts mice could then be compared in order to gain insights in the function of HIPl and HIP 12. 5) Huntingtin binds to HIPl at location overlapping the binding sites for AP2, clathrin light and heavy chains. At this time it is unclear whether huntingtin may in fact impede the binding of these endocytic proteins to HIPl. It would be useful to develop deletion constructs for HIPl in order to determine the exact location where huntingtin interacts. In addition, HIPl-huntingtin co-immunoprecipitation assays could be performed in the presence or absence of the clathrin subunits to determine whether these proteins compete for binding to HIPl. These studies will help to address whether huntingtin can modulate the interaction HIPl with the endocytic proteins or if huntingtin is a component of the endocytic machinery. 148 6) This thesis presented further evidence suggesting that huntingtin plays a role in endocytosis. It would be useful to perform endocytosis assays (such as transferrin uptake assays) in cell culture to address whether wildtype and mutant huntingtin differ in their ability to perform clathrin-mediated endocytosis. 7) Members of the HIPl family contain a putative amino-terminal ENTH domain. At this time it has not been confirmed that the HIPl and HIP 12 ENTHs can interact with phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]-containing membranes. Future studies examining the binding of HIPl and HIP 12 to purified [PtdIns(4,5)P2]-containing membranes will confirm the function of their ENTH domains. 8) It is unclear whether all of the HIPl and HIP 12 interacting proteins have been identified. It may be useful to perform yeast 2-hybrid assays to screen for any additional interactor(s) that would help to further delineate the functions of the HIPl family of proteins. 149 6.8 Conclusion The findings in this thesis demonstrate that the HIPl family of proteins plays an important role in clathrin-mediated endocytosis. The interaction of huntingtin with other interacting proteins suggests that it may also function in endocytosis and transport. Since mutant huntingtin displays altered interactions with HIPl and other endocytic proteins; it may be speculated that the pathogenesis of HD is due to defects in endocytosis and transport. 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