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Identification of proteins that interact with the N-terminal domain of the androgen receptor Quayle, Alandra Nola 1999

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I D E N T I F I C A T I O N OF P R O T E I N S T H A T I N T E R A C T W I T H T H E N - T E R M I N A L D O M A I N OF T H E A N D R O G E N R E C E P T O R B y A L A N D R A N O L A Q U A Y L E B . S c , The University of Victoria, 1996 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E In T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming Tojjae-re^ttired standard T H E U N I V E R S I T Y OF B R I T I S H C O U M B I A July 1999 © Alandra No la Quayle, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of pQjHlOiojj qrA U W ^ t > r ^ \\<A^O0O The University of British Columbia Vancouver, Canada Date JUu 7,0. DE-6 (2/88) Abstract The androgen receptor (AR) plays an essential role in the proliferation of prostate cells, so the progression of prostate cancer to the incurable androgen independent (AI) state l ikely involves alterations in the regulation of AR-mediated gene transcription. One possible route to A I progression is through changes in the activity of coregulator proteins, which interact with A R to contribute further control and specificity to the process of transcriptional regulation. A variety of affinity assays was used to try and identify AR-speci f ic coregulator proteins through their ability to bind to unique sequences in the A R N-terminal domain. Detection of calreticulin (a known AR-binding protein) fol lowing GST-pul ldown with A R - G S T fusion proteins from L N C a P cells (a human prostate cancer cell line), proved that this method was able to selectively pull down human proteins that interact with A R . GST-pul ldown with the N-terminal domain of A R identified two L N C a P nuclear proteins of approximately 40 kDa and 70 kDa and a yeast protein of about 70 k D a that specifically interacted with the N-terminal domain, relative to other regions of A R . Yeast proteins of about 32 kDa, 45 kDa, and 55 kDa that appeared to interact uniquely with the N-terminal domain of A R were co-immunoprecipitated with fragments of the A R N-terminal domain expressed in yeast cells. Further characterization of these protein bands was complicated by high levels of non-specific background associated with these techniques. The yeast 2-hybrid system was used to screen a human prostate c D N A library, util izing the first 232 amino acids of the N-terminal domain of human A R as bait. A positive clone, A R B P 1 , was identified with very strong sequence homology to a known human protein, R a n B P M , that was previously shown to be involved in microtubule nucleation. Hybridization of an A R B P 1 c D N A probe to multiple tissue expression blots showed that the expression of this gene was much higher in testis tissue than in other human tissues. In the future, the interaction between A R and the approximately 70 kDa protein expressed by the A R B P 1 clone can be confirmed using GST-pul ldown or co-immunoprecipitation. Elucidating the role of A R B P 1 in A R function w i l l provide an excellent focus for future experiments. Table of Contents Abstract p. i i Table of Contents p. iv List of Figures p. v i i List of Abbreviations p. v i i i Acknowledgements p. ix Chapter 1. Introduction p. 1 1.1 Prostate Cancer and Tumour Progression p. 1 1.2 Mechanisms Leading to Androgen Independent Progression of Prostate Cancer p. 1 1.3 Structure and Function of the Androgen Receptor p. 4 1.3.1 The Androgen Receptor Gene and Protein p. 4 1.3.2 The Androgen Receptor N-Terminal Domain p. 6 1.3.3 The Androgen Receptor DNA-B ind ing Domain and Hinge Region p. 8 1.3.4 The Androgen Receptor Ligand-Binding Domain p. 9 1.4 Specific Activation of Transcription by the Androgen Receptor p. 11 1.5 Steroid Receptor Coregulators p. 14 1.5.1 Mechanisms of Coregulator Function p. 15 1.5.2 p 160 Family of Steroid Receptor Coactivators p. 18 1.5.3 Other Steroid Receptor Coactivators p. 21 1.5.4 CBP/p300 Coactivators p. 22 1.5.5 Androgen Receptor Coactivators p. 23 1.6 Objectives of this Study p. 29 Chapter 2 Experimental Procedures p. 31 2.1 GST-pul ldown using L N C a P nuclear protein extract or yeast protein extract p. 31 2.1.1 Radioactive labeling of L N C a P cells p. 31 2.1.2 Preparation of L N C a P nuclear extract p. 33 2.1.3 Radioactive labeling of yeast cells p. 33 2.1.4 Preparation of yeast protein extract p. 34 2.1.5 Preparation of G S T fusion proteins p. 34 2.1.6 Preparation of glutathione-sepharose affinity columns p. 36 2.1.7 Binding of radiolabeled proteins to affinity columns p. 36 2.1.8 Elution of proteins bound to affinity columns p. 36 2.1.9 Analysis of protein eluted from affinity columns p. 37 2.2 Co-immunoprecipitation using yeast protein extract p. 38 2.2.1 Radioactive labeling of yeast cells p. 38 2.2.2 Co-immunoprecipitation, elution, and analysis of proteins p. 40 2.3 Yeast 2-hybrid screening p. 40 2.3.1 Preparation of c D N A library p. 41 2.3.2 Transformation of yeast cells for screening p. 43 2.3.3 Screening of yeast transformants p. 44 2.3.4 Confirmation of positive clones p. 45 2.3.5 Sequencing of positive clones p. 47 2.3.6 Analysis of positive clone by Western Blotting p. 47 2.3.7 Northern blot analysis of positive clone p. 48 V Chapter 3. Results p. 51 3.1 Preliminary Experiments p. 51 3.1.1 Incorporation of 3 5S-methionine by yeast cells p. 51 3.1.2 Preparation of G S T - A R affinity columns p. 51 3.1.3 Sequential washing of immunoprecipitates p. 53 3.2 Aff inity pulldown and immunoprecipitation studies p. 57 3.2.1 GST-pul ldown using L N C a P nuclear extracts p. 57 3.2.2 GST-pul ldown using yeast cell extracts p. 61 3.2.3 Co-immunoprecipitation using yeast cell extracts p. 64 3.3 Yeast 2-hybrid Assay p. 70 3.3.1 Sequencing and analysis of positive clones p. 70 Chapter 4. Discussion p. 78 4.1 GST-pul ldown assay and co-immunoprecipitation p. 78 4.2 Yeast 2-Hybrid System p. 83 4.3 Future Directions of Research p. 88 4.4 Conclusions p. 91 Bibliography p. 93 vi L is t of Figures Fig. 1 Sequence homology between certain members of the steroid receptor family p. 5 Fig. 2 Mechanisms of coregulator function p. 16 Fig. 3 Illustration of some coactivators and other proteins known to interact with AR p. 24 Fig. 4 Schematic diagram demonstrating principles of GST pulldown assay p. 32 Fig. 5 Schematic diagram demonstrating principles of co-immunoprecipitation p. 39 Fig. 6 Schematic diagram demonstrating principles of yeast 2-hybrid system p. 42 Fig. 7 Incorporation of 35S-methionine by yeast cells p. 52 Fig. 8 Expression of GST-AR fusion proteins p. 54 Fig. 9 Amount of protein material removed by sequential washes of a GST affinity column p. 55 Fig. 10 Level of 35S-labeled material removed by sequential washes p. 56 Fig. 11 GST-AR pulldown of calreticulin p. 58 Fig. 12 GST-AR pulldown of 40 kDa protein in LNCaP cells p. 60 Fig. 13 GST-AR pulldown of 70 kDa protein in LNCaP cells p. 62 Fig. 14 GST-AR pulldown of 70 kDa protein in yeast cells p. 63 Fig. 15 GST-AR524-649 and GST-AR233-649 did not pulldown some forms of hsc70 p. 65 Fig. 16 Co-immunoprecipitation of a 45 kDa yeast protein with GAL4 DBD-ARLssg p. 67 Fig. 17 Co-immunoprecipitation of a 55 kDa yeast protein with GAL4 DBD-AR,.232 and GAL4 D B D - A R , ^ p. 68 Fig. 18 Co-immunoprecipitation of a 32 kDa yeast protein with GAL4 DBD- A R , . ^ p. 69 Fig. 19 Yeast 2-hybrid clone is homologous to human gene RanBPM p. 71 Fig. 20 RanBPM/ARBP1 is expressed in yeast cells transformed with the library plasmid. p. 74 Fig. 21 RanBPM/ARBP1 is most highly expressed in human testis tissue p. 75 Fig. 22 RanBPM/ARBP1 is most highly expressed in testis tissue p. 77 vn List of Abbreviations Al Androgen Independent AIB1 Amplified In Breast cancer-1 AP-1 Activator Protein-1 AR Androgen Receptor ARA54 Androgen Receptor Activator-54 ARA55 Androgen Receptor Activator-55 ARA70 Androgen Receptor Activator-70 ARBP1 Androgen Receptor Binding Protein-1 ARE Androgen Response Element ARIP3 Androgen Receptor Interacting Protein-3 CBP CREB-Binding Protein CREB cAMP Response Element Binding Protein DBD DNA-Binding Domain ER Estrogen Receptor F-SRC-1 Full-length Steroid Receptor Coactivator-1 Gal4 DBD Gal4 DNA-Binding Domain Gal4 AD Gal4 Activation Domain GR Glucocorticoid Receptor GRIP-1 Glucocorticoid Receptor Interacting Protein-1 GST Glutathione-S-Transferase HA Hemagglutinin HAT Histone Acetyltransferase Hsp Heat Shock Protein IB AP-1 Ig-P Associated Protein-1 LBD Ligand-Binding Domain LNCaP Lymph Node Cancer of the Prostate MMTV Mouse Mammary Tumour Virus MR Mineralocorticoid Receptor MTE Array Multiple Tissue Expression Array MTN Blot Multiple Tissue Northern Blot MTOC Microtubule Organization Center NCoR Nuclear receptor Corepressor p/CAF p300/CBP Associated Factor p/CIP p300/CBP Interacting Protein PR Progesterone Receptor RAC-3 Receptor-Associated Coactivator-3 RAF Receptor Accessory Factor RanBPM Ran Binding Protein in the Microtubule organization center Rb Retinoblastoma RIP 140 Receptor Interacting Protein-140 SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sip Sex-Limited Protein SMRT Silencing Mediator for Retinoid and Thyroid hormone receptors SRC-1 Steroid Receptor Coactivator-1 SRE Steroid Response Element TIF-1 Transcription Intermediary Factor-1 TIF-2 Transcription Intermediary Factor-2 TRIP-1 Thyroid Receptor Interacting Protein-1 viii Acknowledgements I would like to express my thanks to my supervisor Dr. Paul Rennie for his guidance, support, and patience throughout this research. I greatly appreciate his efforts to always find time for this project in his very busy schedule. I would also like to thank Helen Cheng for her willingness to teach me techniques and her endless patience with me while I was learning. I am very grateful for her contributions to this project and particularly for her invaluable assistance with the yeast 2-hybrid system. I would like to express my appreciation to Dr. Rob Snoek and Dr. Colleen Nelson for their advice and their assistance in solving many technical problems. Final ly, I would like to acknowledge the continual support and encouragement I have received from Mike and my family throughout my graduate studies. IX 1. Introduction 1.1 Prostate Cancer and Tumour Progression Prostate cancer has become the most commonly diagnosed lethal cancer in Canadian men, expected to affect an estimated 16,600 new men in 1999 alone, and has grown to be the second greatest cause of cancer deaths in men (1). When discovered in its early stages prostate cancer can often be cured, but unfortunately almost a third of cases are not found until the disease has reached an advanced stage that is presently incurable (2). In the normal prostate, secretory epithelial cells require the presence of male sex hormones, or androgens, for the maintenance of normal structure and function. Androgen withdrawal causes involution of the prostate, due to an inhibition of cellular proliferation and an induction of cell death mechanisms (3). Since the 1940's, the only therapies available for the treatment of advanced prostate cancers involve the withdrawal of androgens or the blockade of androgen action. Whi le almost 80% of advanced prostate cancers initially exhibit a positive clinical response to these androgen deprivation therapies, the median duration of this response is only about 18 months (4). Virtually all prostate cancers w i l l eventually fail hormone therapy and once the tumour begins to grow in the absence of androgens the average survival time for patients is between four and 15 months (5). 1.2 Mechanisms Leading to Androgen Independent Progression of Prostate Cancer Although the mechanisms underlying the progression of prostate cancer to an androgen independent (Al ) state are currently unknown, it is thought that prostate cells may bypass the normal androgen receptor (AR) regulated pathway of prostate growth by a variety 1 of mechanisms, which could include mutations in the A R gene, amplification of the A R gene, and/or recruitment of alternative receptor activation pathways. The A R is activated by binding to androgens such as dihydrotestosterone and then interacts with the D N A to activate transcription of specific genes. Observations in the L N C a P cell line, derived from a human prostate cancer lymph node>metastasis, indicate that A R present in those cells has a mutation in the ligand-binding region. Although the wi ld type A R is normally activated by binding to androgens such as dihydrotestosterone, this mutation can allow A R to bind and be activated by a variety of unusual ligands, including adrenal androgens, metabolites of androgenic compounds, estradiol and progesterone, as wel l as several non-steroidal anti-androgens (6). Mult iple studies have identified this and other A R mutations in cl inical prostate cancers, possibly allowing A R to be stimulated in response to a range of ligands (7,8). However, these mutations have been found at a relatively low frequency, even in advanced cancers. Many studies have examined the frequency of all A R mutations in prostate cancers, with conflicting results. Whi le several studies have found very few or no mutations in early or advanced cancers, others have found frequencies nearing 50% in primary tumours and in A I metastatic disease (7-10). The reason behind these differences is not clear, but many of the studies reporting low frequencies of mutation have examined only a limited region of A R sequence. Although the relative occurrence of A R mutations in prostate tumours remains a matter of debate, it is clear that other mechanisms must be involved as not all prostate cancers contain A R mutations (10,11). Altered expression of A R may be another factor in the progression to A I disease. It has been shown in numerous studies that virtually all prostate cancers express A R and the levels of A R m R N A and protein in distant metastases may be even higher than in primary 2 tumours (12-14). In a situation where pathways leading to the androgen-independent activation of A R are functioning, higher levels of A R protein in prostate cells may allow increased transcription of androgen-regulated genes. Amplif icat ion of the A R gene and increased A R m R N A levels have been demonstrated with progression to A l disease (15,16). In one study, amplification was found in 30% of A l tumours but none of the primary tumours tested (17). Whi le this may be an important mechanism in the progression of some tumours, it is not l ikely to account for all cases. The recruitment of alternative receptor activation pathways is another mechanism that may lead to the development of hormone resistance in prostate cancer cells. Alterations in the signaling pathways upstream or downstream of the androgen receptor may lead to inappropriate transcriptional activation. It is known that steroid receptors interact with a large number of accessory factors when binding to D N A and activating transcription, and it is possible that changes in the expression or activity of these factors could provide a growth advantage by allowing activation of A R in the absence of normal levels of androgenic ligand. These accessory factors include coactivators of A R , such as A R A 7 0 , which may allow increased activation of A R in response to low levels of androgens, non-androgenic, or anti-androgenic ligands (18). One estrogen receptor coactivator, A I B 1 , was recently identified because it was highly expressed in 64% of primary breast tumours, with gene amplification found in 10% of samples tested (19). The role of these coactivators in human prostate cancer progression requires further study before the importance of these factors can be determined. Other changes could occur at the level of interactions between A R signaling pathway and growth factor pathways. Human prostate cancer cell lines have been used to show that the A R signaling pathway can be activated by a variety of growth factors, including insulin 3 l ike growth factor-1, keratinocyte growth factor, and epidermal growth factor, when grown in the absence of androgens (20). Other findings indicate that the protein kinase A pathway can also activate A R in a ligand-independent manner (21). Clearly, the progression of prostate cancer to an androgen-independent state is a complex and multi-factorial process, requiring further research before any conclusions about its causes can be reached. However, it does appear that A R plays a major role in the progression of prostate cancer to the A I state. 1.3 Structure and Function of the Androgen Receptor The androgen receptor belongs to the superfamily of nuclear receptors whose members function as transcription factors. Once activated by binding to the appropriate ligand, they bind to regulatory elements in the D N A and induce transcription of a wide range of genes. Members of this superfamily all share structural and functional similarity and while the ligands for some receptors have not yet been identified, others bind a variety of hormones and lipophilic compounds, including steroids, thyroid hormone, retinoids, and vitamin D. Members that bind to steroids form one subgroup, the steroid receptor family, which includes A R , estrogen receptor (ER), progesterone receptor (PR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) (Fig. 1). Through deletion mapping and mutational studies, several conserved functional regions have been identified in each of these receptors. Most members of the steroid receptor family contain (a) a N-terminal domain involved in transcriptional activation, (b) a DNA-b ind ing domain (DBD) , (c) a hinge region involved in nuclear translocation and dimerization, and (d) a ligand-binding domain (LBD) . 1.3.1 The Androgen Receptor Gene and Protein The single copy gene encoding A R is located on the X chromosome, in the q l 1-12 N-terminal D N A Ligand 100 100 100 h A R 559 624 676 919 <15 80 55 hPR 565 629 685 933 <15 77 50 h G R 419 483 530 777 <15 77 51 h M R 601 665 736 984 <15 57 20 h E R 183 247 313 553 595 Fig. 1 Sequence homology between certain members of the steroid receptor family. The level of sequence homology between the human androgen receptor (AR) and progesterone receptor (PR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR) , and estrogen receptor (ER) is indicated as a percentage value for each of the functional domains (N-terminal domain, DNA-bind ing domain, and ligand-binding domain). For each of the receptors, the length of the various domains is indicated by the size and the amino acid number. region (22). The gene is 90 kb in length and contains 8 exons (23). The A R gene has a single promoter driving the transcription of the 10.6 kb message. O f this message, only 2.7 kb actually encodes the protein, while the 5' untranslated region is 1.1 kb in length and the 3' untranslated region is 6.8 kb in length (24,25). The size of the A R protein is about 919 amino acids, although the size can range between 900 and 920 amino acids, depending on the length of variable homopolymeric amino acid repeats in the N-terminal domain. In many cell types there are A and B forms of the A R protein, the B form is a 110 kDa protein and the A form is 87 k D a (26,27). In some cell types up to 20% of the immunoreactive A R present is found as the N-terminally truncated 87 k D a form, which is produced from an alternative start codon located at amino acid 188 in the B form (26). One study has demonstrated that the truncated A form of A R is functionally similar to the B form with subtle differences in transcriptional activity dependent on the cell and promoter context (27). The only known post-translational modification of the protein is thought to involve phosphorylation of certain N-terminal residues, causing the molecular mass of the B form of the protein to increase to 112 kDa (28). Although there have been many divergent findings as to the subcellular localization of A R , it seems likely that A R protein can be found in the nucleus and cytoplasm in the absence of steroid, but upon the addition of ligand all A R moves to the nucleus (29). 1.3.2 The Androgen Receptor N-terminal Domain The amino-terminal portion of steroid receptors is often referred to as the N-terminal domain and plays an important role in the transactivation function of steroid receptors. O f all the functional domains of A R the N-terminal is the largest, comprised of amino acids (a. a.) 1 to 559 and encoded by exon 1 of the A R gene (30). This region is postulated to be important 6 in cell- and receptor-specific transcriptional regulation, as it is the most poorly conserved domain between members of the steroid receptor family, varying considerably in length and sequence (31). A R is the only human steroid receptor to contain long homopolymeric amino acid repeats in the N-terminal domain, including repeats of glutamine (17 to 29 residues starting at a.a. 59), glycine (24 residues starting at a.a. 449), and proline (nine residues starting at a.a. 372) (25). The function of these repeats is unknown, but the polyglutamine tract may act as a transcriptional repressor, as expansion of this tract to greater than 40 residues is associated with decreased transactivation by A R and the condition known as Kennedy's disease or X- l inked spinal and bulbar muscular atrophy (32,33). It has also been proposed that these stretches may be important for interacting with coregulator proteins that can modulate the ability of A R to activate transcription, as many transcription factors contain similar sequences. In addition, polyglutamine or polyproline tracts fused to a D N A binding domain have been shown to activate transcription in vitro (34). The N-terminal domain is known to be very important for the transactivation function of A R , with deletions in this region disrupting transcriptional function (29). A recent study has shown that the abilities of the N-terminal domain and the L B D to activate transcription can vary with both cel l- and promoter-specificity (35). Within the N-terminal domain there are at least two separate regions containing ligand-independent transcriptional activating function (36). The exact location of these regions is still a matter of debate, as several reports have defined slightly different regions (28,37,38). When either one of these regions is deleted the transcriptional activity of A R is reduced by at least half and when both are removed the activity of the mutant A R is less than 10% of the wi ld type receptor. The deletion of a third region from a.a. 411 to 531 has been observed to further reduce the transcriptional activity of A R and has 7 also been identified as the binding site for a possible coactivator protein, known as receptor accessory factor (RAF) , that can enhance the binding of A R to D N A (38,39). It is thought thai A R may be able to use different N-terminal regions for transactivation depending on a variety of external factors, such as cell and promoter context, which may affect the recruitment of various unique coactivators that remain to be identified. It is also interesting to note that only one transactivation region of the N-terminus is required for full transcriptional activation when the L B D of A R has been deleted, again suggesting that A R is able to use distinct transactivation regions under different circumstances (37). The N-terminal domain of A R has also been shown in several reports to interact with the L B D , both in biochemical studies and in functional studies showing that a region of the N-terminal domain can affect ligand dissociation from the L B D (40,41). This interaction has been shown to be very important for the overall transactivation function of A R and may involve two regions of the N-terminal domain (amino acids 3 to 36 and 371 to 494) (42,43). 1.3.3 The Androgen Receptor DNA-Binding Domain and Hinge Region The DNA-b ind ing domain (DBD) of the human A R follows directly after the N-terminal domain, from amino acids 560 to 624 (44). This region shares the greatest similarity between members of the steroid receptor family, with 80% amino acid identity to PR , 77% identity to M R and G R , and 57% to E R (30). This region has also been strongly conserved throughout evolution of A R , as the amino acid sequence for the D B D is very similar in human, mouse, rat, and rabbit (45). There are two conserved zinc finger motifs within this region, which are essential for interactions with D N A regulatory elements known as steroid response elements (SREs) located within promoters of steroid-regulated genes. The zinc finger located closest to the N-terminal domain interacts specifically with the S R E 8 sequence in the major groove of the D N A and particular amino acids within this region determine the S R E binding specificity of the receptor (46). The second zinc finger is thought to be important in stabilizing the receptor-DNA interaction and may also provide discrimination in reference to S R E half-site spacing (47). The D B D is also thought to be involved in several multi-domain functions, including receptor dimerization and nuclear localization (48-50). The hinge region of A R , located between the D B D and L B D , contains sequences essential for nuclear localization of A R (28). A R appears to have a bipartite nuclear localization signal located in the D B D and hinge region, consisting of two clusters of basic amino acid residues separated by ten amino acids (50). There may be another nuclear localization signal within A R as deletion of the bipartite region in the D B D and hinge still allows A R to move into the nucleus in a ligand-dependent manner (29). Some reports indicate that the L B D , in the absence of ligand and in the presence of an intact N-terminal domain, actively inhibits nuclear transport (50). This may indicate that regions in the N-terminal domain interact with the L B D to inhibit nuclear translocation in the absence of ligand. 1.3.4 The Androgen Receptor Ligand-Binding Domain The ligand-binding domain (LBD) is located at the carboxyl-terminus of A R and is the second most conserved region of steroid receptors. The similarity in the L B D between A R and other members of the family ranges from 20% to 55% and this region has been highly conserved throughout evolution of A R in human, rat, and mouse (30,45). Androgens bind, with high affinity and specificity, to a hydrophobic pocket within this domain, causing a conformational change that traps the ligand and creates new protein interaction sites on the 9 molecule (51). This conformational change triggers receptor transformation, dimerization, D N A binding, and transcriptional activation. In general, short deletions of the L B D cause loss of transactivation function while large deletions in this region create ligand-independent receptor function, suggesting that the L B D suppresses the activity of A R in the absence of ligand (29,52). The L B D contains a ligand-dependent transactivation function, which is conserved among steroid receptors and has been demonstrated to activate transcription in an in vitro system (53,54). Mutations at other sites in the L B D can also disrupt transcriptional activation without affecting ligand-binding, probably by preventing binding of various coactivator proteins that augment the activity of A R (55). Regions of the L B D may also be important in receptor dimerization, as fragments of A R containing the L B D alone can form homodimers, while those containing only the D B D have weaker interactions (56). The N-terminal domain is thought to play a role in dimerization as wel l , by inhibiting dimerization in the absence of ligand (48). A R associates with various heat shock proteins, including hsp90, hsp70, and hsp56, and the L B D is known to be the site of interactions between A R and hsp90 (57-59). Heat shock proteins are chaperone proteins that are widely expressed in species ranging from bacteria to eukaryotes and are known to be important in steroid receptor function. These proteins are involved in the proper folding of the L B D to produce the correct steroid-binding conformation and are important in repressing transcriptional activity of steroid receptors in the absence of ligand (60). There are also sequences within the L B D responsible for binding to components of the nuclear matrix (61). The nuclear matrix is associated with active gene transcription and is thought to be the major site of steroid receptor binding in the nucleus (62). Interactions between A R and the nuclear matrix are ligand-dependent and also appear to be tissue-10 specific, as nuclear matrix from the liver has been found to contain less than 20% of the number of A R interaction sites seen in the prostate nuclear matrix (63). It is clear that there are complex interactions between all the functional domains of the androgen receptor, with all three major regions involved in receptor specificity and capable o f restraining the others under certain circumstances (64). The interactions between the N-terminal region and the L B D appear to be important in the overall transactivation function of A R and seem to affect interactions with ligand and receptor stabilization (41,42,65). These interactions are thought to be stabilized by ligand binding and by interactions with coregulator proteins (66,67). 1.4 Specific Activation of Transcription by the Androgen Receptor One step in the regulation of gene transcription by A R is binding to D N A sequences known as androgen response elements (AREs) , or more generally as steroid response elements (SREs). A R E s are usually found in the promoter regions of androgen regulated genes and are composed of palindromic sequences. A R binds as a dimer to the consensus sequence G G T A C A n n n T G T T C T , which is also known to be the consensus binding sequence for G R , PR , and M R (68). The recognition of a single consensus binding sequence by all these receptors could be due to the high level of amino acid homology in the D B D of these receptors. The fact that these four steroid receptors all recognize the same D N A sequence for binding is inconsistent with the high degree of specificity of target gene activation seen with each of these steroid hormones in vivo. Several hypotheses have been proposed to explain the differential ability of steroids to induce transcription of specific genes, despite the fact that the steroid receptors can 11 theoretically bind to very similar, i f not identical D N A sequences. One idea is that the availability of certain receptors and ligands in cells may allow specific gene activation. However, this is unlikely to be a major factor, as many cell types contain two or more receptor types and their ligands simultaneously (69,70). It was also thought that subtle differences between the consensus S R E and the SREs of naturally occurring androgen-regulated promoters may contribute to specificity. One recent study has indicated that subtle deviations in individual nucleotides within the D N A sequences where steroid receptors bind may play a significant role in dictating steroid-receptor specificity (71). However, the study of promoters from several different androgen-regulated genes has further complicated the issue, suggesting that while variations from the S R E consensus sequence might impart some degree of receptor specificity, many other factors also contribute to the specificity of transcription activation (72,73). Another theory regarding receptor-specific transactivation proposed that particular configurations of multiple A R E s within a promoter might function in a mutually dependent co-operative manner. While this may be the case for AR-speci f ic activation of transcription of some genes, including those encoding probasin and the mouse sex-limited protein (Sip), the multiple repeats in the mouse mammary tumour virus ( M M T V ) promoter cannot alone discriminate between A R , PR , and G R (68,74-77). Non-steroid receptor transcription factors or other factors may bind to sites proximal to the S R E and enhance the activity of one receptor in preference to another (77). The transcription factor Nuclear Factor-1 binds to sites on the M M T V promoter proximal to several S R E elements (78). However, Nuclear Factor-1 cannot bind and enhance receptor transactivation unless a steroid receptor has previously bound and begun a local remodeling of the chromatin structure. It has been demonstrated that P R and A R are poor destabilizers 12 of chromatin for the M M T V promoter and therefore can only weakly activate transcription from this promoter, while G R can very efficiently activate transcription from this site (79). Sequences outside of the S R E have been found to promote receptor-specific responses, as in the androgen-dependent Sip gene. A fragment containing only the S R E from the promoter of the Sip gene can confer androgen and glucocorticoid inducibility to a reporter gene, while a larger fragment from the promoter containing the S R E is restricted to induction by androgens (80,81). This suggests that additional factors may be binding to the larger fragment of the promoter region thereby restricting inducibility to A R alone. Mutations in sequences outside of the S R E in the M M T V promoter have been found to differentially affect receptor transactivation, and the spacing of SREs in relation to each other and to the promoter can greatly affect the strength of transcriptional activation (82,83). A l l these findings suggest that the involvement of factors other than A R may be important in providing specificity in the regulation of androgen-responsive genes. Another theory that may explain the ability of steroids to induce transcription of specific genes involves the interaction of unique combinations of coregulator proteins with steroid receptors, thus allowing steroid-receptor specific gene transcription. Coregulators are nuclear proteins that interact directly with steroid receptors and other transcription factors to modulate their ability to activate transcription (84). Coregulators can act as coactivators or corepressors to either enhance or inhibit transcription, and the tissue-specific expression and ratios of certain types of coregulators may provide a mechanism for steroid-receptor specific activation of gene transcription. The focus of the research presented here w i l l be the isolation of coregulator proteins that may play an important role in directing AR-speci f ic gene transcription. 13 1.5 Steroid Receptor Coregulators In recent years, the mechanisms by which steroid receptors regulate transcription have been found to be much more complex than previously thought. The action of steroid receptors was originally thought to be quite simple, with the receptor binding to its ligand and then to the D N A to activate transcription of target genes. However, activation of transcription from eukaryotic promoters is a complex process involving the action of many different proteins. The transcriptional process is directed by the enzyme R N A polymerase II and requires the presence of additional factors, known as basal transcription factors, to produce basal levels of expression. Regulated transcription of specific genes requires the direct binding of transcription factors to D N A . The discovery of coregulator proteins, which interact with steroid receptors and other transcription factors to modulate their ability to activate transcription, has provided important insight into the mechanism of action of transcriptional activation by steroid receptors in vivo. B y definition, coactivators are nuclear proteins that do not belong to the basal transcriptional machinery themselves and do not affect levels of basal transcription. Although they can contain transcription activation domains, they cannot act as transcriptional activators themselves as they lack the ability to bind D N A . Instead, coactivators must be recruited via protein-protein interactions to D N A targets, where they are required for enhancement of transcriptional activation induced by sequence-specific transcription factors. Evidence for the existence of coactivators for nuclear receptors was based on observations of squelching, or transcriptional interference, when two different types of receptors were present; suggesting that the receptors were binding to a common limiting factor that was involved in mediating functional responses (85). Many experimental approaches have been 14 used in attempts to identify steroid receptor coregulators including biochemical assays for proteins that bind to expressed receptors or receptor fragments, genetic screening in yeast for proteins that affect the activity of steroid receptors, and yeast 2-hybrid screening for identification of clones that express proteins that can interact with whole or partial steroid receptors. 1.5.1 Mechanisms of Coregulator Function Coregulators are not necessary for basal transcription, but are thought to be essential for enhanced activation of transcription by other transcription factors. Mult iple types of coregulators have been identified that function through a variety of methods, including modulating receptor-DNA interactions, acting as bridging factors between steroid receptors and the basal transcriptional machinery, and remodeling of chromatin by histone acetyltransferase and histone deacetylase activity. Coactivators can enhance interactions between steroid receptors and D N A by producing changes in D N A structure, such as inducing bends in the D N A or causing local unwinding. The high mobil ity group proteins can increase D N A binding of PR , E R , G R , and A R and have been demonstrated to enhance transcription by E R and P R (86,87). Steroid receptors can interact directly, or indirectly v ia coactivators, with the general transcription machinery to enhance transcription activation (Fig. 2A). Sequences within the N-terminal domain of A R (amino acids 142 to 485) have been found to interact with T A T A -binding protein and with the basal transcription factor TFIIF (88). Other transcription factors have been found to bind to TFIIB, causing conformational changes that expose binding sites for other basal factors and stabilize the formation of the transcription complex (89,90). 15 A. TFIIB TFIIf) TF I , F ^SEMI,H 33r ARAR TFIIE TFIID transcription basal transcription machinery Fig. 2 Mechanisms of coregulator function. A. Steroid receptors may interact directly or indirectly via coactivators with members of the basal transcription machinery to stabilize formation of the pre-initiation complex and increase the rate of transcription initiation. B. Steroid receptors may interact with coactivators that have intrinsic HAT activity or with coactivators that can recruit proteins with HAT activity, leading to acetylation of histones and alterations in the chromatin structure of DNA, allowing increased access to the DNA by a variety of transcription factors, for example AP-1; Abbreviations: Androgen receptor (AR), CREB-binding protein (CBP), RNA polymerase II (RNA pol II), p300/CBP associated factor (p/CAF), steroid receptor coactivator-1 (SRC-1), aceylation (Ac), activating protein-1 (AP-1), histone acetyltransferase (HAT). 16 Indirect interactions between steroid receptors and the transcription machinery have been identified involving coactivators such as the CREB-b ind ing protein (CBP) (91). Many coactivators have recently been shown to have intrinsic histone acetyltransferase (HAT) activity or to recruit proteins with H A T activity, suggesting an important role of histone acetylation in transcriptional activation (Fig. 2B). It is thought that proteins with H A T activity may be able to produce local remodeling of chromatin structure, as hyperacetylation of histones has been correlated with transcriptionally active regions of chromatin (92). Targeted acetylation of histones may be able to decrease the affinity of histone-DNA interactions to allow increased access to the D N A by a variety of transcription factors. For example, the coactivator C B P has been shown to have H A T activity and can recruit other proteins with H A T activity such as p / C A F (p300/CBP associated factor), which can also function as a coactivator by interacting directly with steroid receptors (93-95). The yeast SWI /SNF coregulator complex, as wel l as its mammalian homologue, can counteract the repression of transcription by chromatin and promote D N A binding of transcription factors (96,97). The human homologue of the SWI /SNF coregulator complex has been shown to enhance transcription by several nuclear and steroid receptors (98). It is unclear i f proteins other than histones can be the target of H A T activity, but it has been suggested that coactivators with H A T activity may acetylate basal transcription factors and steroid receptors (99,100). Repression of steroid receptor mediated transcription in the presence of antagonists may also be mediated through chromatin remodeling, via the recruitment of histone deacetylases by corepressors (101). Steroid receptors bound to antagonists may have an altered conformation that can promote association with corepressors such as N C o R (nuclear receptor corepressor) and S M R T (silencing mediator for retinoid and thyroid hormone 17 receptors), which are known to interact with homologues of the yeast Sin3 and RPD3 histone deacetylases (79,101,102). 1.5.2 pi 60 Family of Steroid Receptor Coactivators The first coactivator identified that interacted with the L B D of steroid receptors and enhanced their ability to activate transcription in mammalian cells was termed steroid receptor coactivator-1 (SRC-1) (66). This coactivator was later found to be a member of a large family of steroid receptor coactivators, all of which are about 160 kDa in size, have similar functions and share significant amino acid homology to one another. SRC-1 was first identified in a yeast 2-hybrid screen for proteins that interact with the L B D of the P R and was found to enhance the ability of P R to activate transcription up to 15-fold in yeast and mammalian cells, without altering levels of basal transcription (66). Early studies demonstrated that SRC-1 could interact with the L B D of most steroid receptors in a ligand-dependent manner, with the exception of A R (103). However, later work using the full-length SRC-1 (F-SRC-1) demonstrated that F - S R C - 1 , but not the truncated form of SRC-1 that had been shown to interact with other steroid receptors, could enhance AR-mediated transcription and promote interactions between the N-terminal domain and L B D of A R (42). SRC-1 seems to be a coactivator which is relatively specific for steroid receptors, interacting only weakly with other families of transcription factors (104). It is normally expressed in a variety of tissues and cell lines and is predominantly located within the nucleus (105). Overexpression of SRC-1 has been shown to partially reverse the squelching of transcription that occurs when more than one type of activated receptor is present, suggesting that it is a shared, limiting factor involved in mediating the activation function of the L B D (66). SRC-1 has been shown to have an intrinsic transcription activation function, as it is able to activate 18 transcription when fused to the DNA-bind ing domain of another transcription factor, such as Gal4(104). Other members of the p i 60 coactivator family include transcription intermediary factor-2 (TIF-2) and glucocorticoid receptor interacting protein-1 (GRIP-1). TIF-2 has 40% overall identity to SRC-1 and has been shown to enhance transcription activation by A R , PR , and E R (106). GRIP-1 is a mouse homologue with 94% identity to TIF-2 (107). Ligand-dependent interaction of GRIP-1 with A R , G R , and E R has been shown to enhance transcription activation by these steroid receptors in yeast and mammalian cells (108). It is thought that GRIP-1 may act as a bridging factor between the basal transcription machinery and steroid receptors. Overexpression of GRIP-1 has been observed to inhibit basal transcription, perhaps by binding to and sequestering proteins involved in the basal transcription machinery (107). The p i60 coactivators all contain several conserved functional domains, including a centrally located nuclear receptor interacting domain and a p300/CBP interaction site that overlaps a transcription activation domain. The first 300 amino acids of the p i 60 family of coactivators are highly conserved, yet appear to be dispensable for nuclear receptor interaction and coactivator activity suggesting they may have a separate role in the function of the p i 60 coactivators (104). There may also be a conserved histone acetyltransferase catalytic domain within the C-terminal region of the p i 60 coactivators, although it is not clear i f all the family members contain this domain. SRC-1 contains additional nuclear receptor interacting sequences that are not found in the other family members (103). Within the conserved receptor-interacting region are found three conserved L X X L L amino acid motifs, sometimes termed N R boxes, which have been shown to be necessary and sufficient 1 9 for nuclear receptor binding (103). Point mutations within these motifs can abolish interactions with receptors and an eight amino acid sequence containing one of these motifs is sufficient for interaction with nuclear receptors (109). However, there must be some additional sequence or secondary structure requirements, as not all proteins containing an L X X L L motif can interact with steroid receptors (109). A study of individual N R boxes within different coactivators has demonstrated overlapping, but non-identical preferences for binding to particular steroid receptors (103). This may provide a certain level of receptor specificity for various coactivators and could explain why TIF-2 and G R I P - l can interact with A R at the same strength as other steroid receptors, while SRC-1 only weakly interacts with A R . The multiple N R boxes within each coactivator may be important for simultaneously binding to both L B D s of a receptor dimer, or for coupling receptors bound to tandem SREs (110). Several additional homologous proteins, about 155 kDa in size, have been identified as coactivators belonging to the p i 60 family. One member of this group is known as p300/CBP-interacting protein (p/CIP) and has greater than 30% total identity to both SRC-1 and TIF-2 (111). It is usually present as a complex with CBP/p300 and is required for transcriptional activation by a variety of nuclear receptors (111). Another protein, known as A C T R , was found to enhance transcription mediated by steroid receptors 2-3 fold and is expressed in a tissue- and cell-specific manner (112). A C T R has also been found to interact with other coregulators, including CBP/p300 and p /CAF (113). Receptor-associated coactivator-3 (RAC-3) is another member of this group of steroid receptor coactivators and has been found to have intrinsic histone acetyltransferase activity (112). Other members of the p i 60 family have now been identified as having H A T function, including SRC-1 and 20 A C T R (114,115). The last known coactivator that belongs in this group is known as AIB1 and was identified as a gene which was overexpressed in 64% and amplified in 10% of primary breast cancers tested (19). Several other members of the p i 60 family have been found to be overexpressed in various human cancer cell lines, including R A C - 3 , A C T R , TIF-2, and SRC-1 (115). 1.5.3 Other Steroid Receptor Coactivators Many other proteins that interact with steroid receptors have been identified that do not have sequence homology to the p i 60 family. Several of these, including RIP 140 (receptor interacting protein-140), TIF1 (transcription intermediary factor-1), and TRIP1 (thyroid receptor interacting protein-1), were initially shown to enhance transcription by steroid receptors in yeast, but not in mammalian cells (116-118). This difficulty in demonstrating transcription enhancement in mammalian cells, but not in yeast cells, has also been observed for some members of the p i 60 family of coactivators (119). This effect is thought to be related to the fact that in mammalian cells the endogenous levels of coactivators may be high enough that overexpression of a specific coactivator cannot further enhance the level of transcription, or there could be limiting levels of accessory factors required for the interaction between the coactivator and steroid receptor. RIP 140, TIF1, and TRIP1 all contain L X X L L motifs and interact with the L B D of steroid receptors in a ligand-dependent manner (109). RIP 140 was found to interact with the L B D of E R and later was demonstrated to enhance transcription by several steroid receptors up to ten-fold in yeast cells, and has now been shown to increase the activation of transcription by rat A R in mammalian cells (42,116,120,121). TIF1 was first identified as a mouse protein that could interact with nuclear receptors and a human homologue was later found that enhanced 21 transcription by E R and various nuclear receptors but interacted only weakly with A R , G R , PR , or M R (118,122). The coactivator TRIP1 has sequence homology to a known yeast coactivator, Sug-1 and can interact with nuclear receptors such as thyroid receptor, but is not known to interact with A R (117,123). Another human coactivator, R P F 1 , is known to have a yeast homologue, yRSP5 , both of which can enhance the activity of P R and G R in yeast and mammalian cells (124). 1.5.4 CBP/p300 Coactivators The c A M P response element binding protein ( C R E B ) mediates transcription enhancement of cAMP-responsive genes and is regulated by phosphorylation by protein kinase A . C B P is a CREB-b ind ing protein that has been found to act as a coactivator for a wide variety of transcription factors in many different transcription signaling pathways (125). C B P is thought to act as a co-integrator of various signaling pathways, as it is able to function as a coactivator for many diverse families of transcription factors. It is known to be an essential cofactor for activation of transcription by C R E B and is probably involved in recruitment of the general transcription machinery to the promoter, as it can interact with members of the transcription pre-initiation complex, including TFIIB and the R N A polymerase II holoenzyme (126,127). A protein called p300, which interacts with the adenoviral E l A protein, was found to have extensive sequence homology to C B P and appears to be interchangeable with C B P as a coactivator of transcription. Both C B P and p300 interact with steroid receptors in a ligand-dependent manner through an essential L X X L L motif (109). They also interact with p i 60 family members including SRC-1 and p/CIP (111,128). It appears that SRC-1 and CBP/p300 can bind simultaneously to the L B D of steroid receptors, as the formation of complexes containing steroid receptors, CBP/p300, 2 2 and p i60 coactivators has been demonstrated in vitro (105). Overexpression of CBP/p300 has been shown to enhance transcriptional activation by various nuclear and steroid receptors in mammalian cells and can act synergistically to increase levels of transcription when SRC-1 is also present (111,129). C B P has been found to enhance AR-mediated transcription by three to five times in the presence of ligand (130,131). A recent study claims to have identified a ligand-independent interaction between C B P and A R that requires a portion of A R N-terminal domain (amino acids 300 to 566) and may also involve non-essential interactions with the L B D (131). Several reports have identified a mutual repression of transcription between A R and the transcription factor complex AP-1 (activator protein-1) (131-133). Overexpression of CBP/p300 was found to relieve this repression, suggesting that CBP/p300 may be a common, l imiting factor that enhances transcription by both A R and AP-1 (105). 1.5.5 Androgen Receptor Coactivators A s mentioned previously, CBP/p300 as wel l as some members of the p l 60 family of coactivators, including TIF-2 and GRIP-1 , can interact with A R to enhance its ability to activate transcription. In addition, there are several other A R coactivators that are not members of any well-described coactivator families (Fig. 3). A s almost all of these coactivators interact with the conserved D B D or L B D portions of A R , it is of great interest to try and identify A R coregulators that interact with the relatively unique amino acid sequences found in the A R N-terminal domain. A putative AR-specif ic coactivator has been termed androgen receptor activator-70 (ARA70) and is the only coactivator that has been discovered to date that may be relatively specific for a given steroid receptor (134). This coactivator was identified using a yeast 2-hybrid screen for proteins that interact with the L B D of A R in 23 Rb RAF N-terminal domain DNA-binding domain TFIIF ARIP3 C P Coactivators | Other proteins ARA70 hsps F-SRC1 — -ARA54 Ligand-binding domain J * RA55 Fig. 3 Illustration of some coactivators and other proteins known to interact with AR. Various coactivators and other proteins are illustrated adjacent to the general AR domain with which they interact. 24 a ligand-dependent manner and was found to enhance AR-dependent transcription up to 10 fold in yeast and a prostate cancer cell line while having lesser effects (two to three fold increase) on the level of transcription mediated by E R , PR , or G R . Unfortunately, the ability of A R A 7 0 to enhance transcription of other steroid receptors in non-prostate cells was not assessed, so it is possible that the prostate cell environment was not suitable for the enhancement of transcription by other steroid receptors. Recently, A R A 7 0 has also been found to interact with the human peroxisome proliferator-activated receptor in a yeast 2-hybrid screen (135). It appears that A R A 7 0 is nearly identical to another protein known as E L E 1 and independent studies have found that this protein interacts with A R , mouse E R , and rat G R in the presence or absence of ligand (136). The studies on E L E 1 also found that the enhancement of A R activity was in the range of only two to three fold in several human cell lines (136). However, it is not clear i f the minor differences in sequence between A R A 7 0 and E L E 1 are due to natural polymorphisms or i f they are encoded by unique genes. Therefore, it remains unknown i f A R A 7 0 truly represents a coactivator with specificity for A R . A R A 7 0 has been found to contain an L X X L L domain and appears to interact with the histone acetyltransferase p / C A F and the basal transcription factor TFIIB (136). Examination of interactions between A R and various anti-androgens used in the treatment of prostate cancer, including hydroxyflutamide, bicalutamide, and cyproterone acetate, indicates that the presence of A R A 7 0 can increase the ability of these compounds to act as agonists to enhance transcription by A R under certain conditions (18). Addit ional androgen receptor coactivators called A R A 5 4 and A R A 5 5 have been identified recently that interact with the L B D of steroid receptors in a ligand-dependent manner (137,138). A R A 5 4 was found in a yeast 2-hybrid screen for proteins that interact 25 with the L B D of the mutant A R found in the L N C a P cell line, and is highly expressed in testis tissue. This coactivator could enhance transcription activation in the presence of the anti-androgen hydroxyflutamide 3-5 fold by the mutant A R while not enhancing transcription mediated by the wi ld type A R (137). Androgen-dependent transcription by A R was enhanced about six times by A R A 5 4 , while transcription by P R was enhanced about four times and transcription by G R and E R was not affected (137). A R A 5 5 was also identified using a yeast 2-hybrid screen, with the L B D of the wi ld type A R as bait (138). The interaction between A R A 5 5 and A R is ligand-dependent and has been shown to enhance transcription by steroid receptors in yeast and a prostate cancer cell line, DU145. AR-regulated transcription was enhanced about five times, while transcription activation by G R and P R was only increased by two to three times (138). A R A 5 5 is encoded by a TGF-P inducible gene and shows significant homology to a mouse gene hic5. Whi le these coactivators appear to have some degree of specificity for A R , it is still unclear i f this preference w i l l persist when A R A 5 4 and A R A 5 5 are tested in non-prostate cell lines. Another coactivator that can enhance AR-mediated transcription is the long isoform of the B A G - 1 protein, known as B A G - 1 L . B A G - 1 is known to regulate members of the heat shock protein family and is an anti-apoptotic protein known to bind to Bc l -2 (139,140). B A G - 1 L appears to be preferentially expressed in steroid-responsive tissues and tumour cell lines (141). B A G - 1 L was co-immunoprecipitated with A R from L N C a P cells and was found to increase the ability of A R to activate transcription up to 5-fold in response to ligand (142). It is not clear which domain of A R is involved in this interaction nor has the mechanism involved in the coactivator function of B A G - 1 L been elucidated. A n isoform of B A G - 1 called R A P 4 6 is known to bind several different receptors, including G R , E R , and the 26 retinoic acid receptor (143,144). As the interaction of R A P 4 6 with these other receptors inhibited binding to D N A and the initiation of transcription, the effect of B A G - 1 on steroid receptors requires further study. A protein expressed almost solely in the testis, known as AR-interacting protein-3 (ARIP3), has been found to interact with the D B D of A R (145). AR IP3 appears to be a member of a novel protein family, even though it contains four L X X L L amino acid motifs, as it does not contain any other significant homology to known coactivators. The presence of ARIP3 increased activation of transcription by A R up to four times, but with higher levels of ARIP3 transcription by A R was actually inhibited (145). Addit ion of AR IP3 was also seen to increase interactions between the N-terminal domain and L B D of A R , as has been observed with other coactivators containing L X X L L motifs, such as C B P (42,145). Unfortunately, it is unknown i f ARIP3 preferentially enhances transcription by A R , as its effect was not tested with other steroid receptors. The retinoblastoma (Rb) protein, better known as a tumour suppressor and cell cycle control protein, has recently been identified as a coactivator of A R (146). It was observed that overexpression of Rb enhanced A R activity while loss of Rb inhibited A R function. The interaction between Rb and A R was found to involve the A R N-terminal domain (amino acids 502 to 565) and it is thought that Rb may act as a bridge between A R and the basal transcription machinery as it is also able to interact with the basal factor TAFII250 (146). Intriguingly, it was noted that while overexpression of Rb could enhance G R function, loss of Rb did not affect the ability of G R to activate transcription and Rb did not interact directly with G R , suggesting that A R may have a unique ability to interact with Rb. It has been shown that abnormally low Rb expression is associated with the use of hormonal therapy to 27 treat prostate cancer and with the progression of prostate cancer to A I disease (147,148). While the loss of Rb expression is usually associated with its function as a tumour suppressor, it is not known i f the loss of Rb may also be related to the altered response to androgens seen during prostate cancer progression. A R has also been found to interact with a protein termed receptor accessory factor. (RAF) , which was later found to be identical to insulin degrading enzyme (149). R A F interacts with the N-terminal domain of A R (amino acids 460 to 520) and acts to enhance binding of A R to SREs by 25 fold while only enhancing G R binding to D N A six fold (39). The recent increase in knowledge about A R coactivators and interactions between domains of A R have led to the creation of a useful tool for studying the role of A R in normal and disease states. Recently, an artificial AR-specif ic coactivator was created based on the knowledge that the N-terminal domain and the L B D of A R interact with one another (150). This artificial coactivator was constructed from the L B D of A R fused to a powerful activation domain from V P 16 and enhanced AR-regulated transcription by 4-13 fold. A s expected, this activation was very specific for A R with no effect on P R or G R mediated transcription. This artificial coactivator could provide a novel means of controlling A R activity in vitro and in vivo (150). To date, the mechanisms regulating expression, cellular availability, and activity of coactivators remain largely unknown. It is thought that the cellular ratio of coactivators to corepressors may be important in determining the final level of gene transcription, but additional levels of control probably exist, including tissue-specific differences in expression of coregulators, the structure of the particular promoter, and the type and quantity of available ligand. For steroid-responsive tissues and tumours, the relative levels o f 28 coactivators and corepressors may be particularly important in determining the responsiveness to various steroid antagonists. Although much remains to be learned about coregulator proteins, the existence of such a number and variety of these factors suggests that they must play an important role in steroid receptor function. The identification of A R coregulator proteins may enhance our understanding of A R function in both normal and neoplastic processes. 1.6 Objectives of this Study Regulation of A R activity is important in the normal prostate and in the progression of prostate cancer to an androgen independent state. One factor that may modulate the ability of A R to activate transcription is the availability of coregulator proteins. The study of coregulator proteins to date has largely been limited to those that bind to the conserved L B D or D B D regions of the steroid receptors. It is not surprising then, that these coactivators have been found to bind to a wide range of receptors and other transcription factors. In order to elucidate the role of coregulator proteins in steroid-receptor specific transcription, further study of coactivators and corepressors that act specifically on certain steroid receptors is required. Thus, the objective of this research is to isolate and identify proteins that interact with the relatively unique amino acids sequences present within the N-terminal domain of A R with the hope of identifying novel coregulator proteins that preferentially interact with A R . Several methods are currently available to identify novel proteins that interact with a protein of interest, such as A R . Most coregulators have been identified using yeast 2-hybrid screening and these interactions have then been confirmed by independent biochemical 29 methods, such as co-immunoprecipitation and GST-pul ldown. In this study, all of these methods were employed to identify novel proteins that interact with the N-terminal domain of A R . Specifically, this research tested the utility of these three different methods in identifying novel protein-protein interactions with the N-terminal domain of A R . The GST-pul ldown is a type of affinity chromatography where the protein of interest is expressed as a fusion protein with glutathione-S-transferase (GST) and coupled to an appropriate matrix. Ce l l extracts can then be added and any proteins that bind to the column can be eluted and analyzed to identify any potential binding partners of the protein of interest. Co-immunoprecipitation involves the incubation of cell extracts with an antibody that recognizes some part of the protein of interest. The antibody can then be precipitated, along with its target protein and any proteins that may be complexed with it. The yeast 2-hybrid assay is a sensitive in vivo system that utilizes two fusion proteins; the first composed of the Gal4 D B D fused to the bait protein, while the second contains the Gal4 activation domain (Gal4 A D ) linked to a c D N A library. Coexpression of these two chimeric proteins in yeast that contain a Gal4-responsive promoter upstream of a reporter gene wi l l result in the expression of the reporter gene only i f the two chimeric proteins interact. Any library clone that encodes a protein that interacts with the bait protein can be easily identified and sequenced, providing information about a novel protein that interacts with the protein of interest. These three methods were utilized for the identification of novel protein interactions. Several unidentified protein bands were detected that specifically bound to the N-terminal domain of A R . Screening of a human prostate c D N A library with the yeast 2-hybrid system identified a clone that interacted with the A R N-terminal domain. 3 0 2. Experimental Procedures 2 . 1 GST-pulldown using LNCaP nuclear protein extract or yeast protein extract The GST-pul ldown assay involves the expression of fusion proteins containing glutathione-S-transferase (GST) linked to various regions of A R (Fig. 4). These fusion proteins were expressed by Escherichia coli transformed with the appropriate plasmid, and could then be easily purified via the ability of G S T to bind to glutathione-coated beads (151). Such G S T - A R fusion proteins have been found to be soluble and capable of interacting with both ligand and D N A in a manner similar to the native A R (152). A radioactively labeled protein mixture, isolated from L N C a P or yeast cells, was added to matrix-bound G S T - A R fusion proteins. Proteins able to interact with the A R fusion protein were collected and analyzed by S D S - P A G E (polyacrylamide gel electrophoresis) and fluorography. The use of a variety of fusion proteins that expressed different fragments of the A R , allowed identification of interactions that were specific for the N-terminal domain. This technique is often used to prove known or suspected protein-protein interactions, but can also be used to detect novel factors binding to the protein of interest. For example, coactivators that interact with the E R and G R have been identified using the GST-pul ldown assay (153,154). 2.1.1 Radioactive labeling of LNCaP cells L N C a P cells were grown at 37°C with 5% CO2 in R P M I medium supplemented with 5% F B S in 175 cm flasks until they reached about 60% confluency. The medium was then removed and replaced with methionine deficient R P M I medium supplemented with 5% F B S and 10 n M R1881, a synthetic androgen. Cells were incubated at 37°C for 20 minutes before 3 5 S-label ing mix consisting of 75% methionine and 25% cysteine (ICN), was added at 31 Protein X Androgen receptor 7 Glutathione-S-transferase glutathione glutathione Fig. 4 Schematic diagram demonstrating principles of the GST-pulldown assay. Fusion proteins containing glutathione-S-transferase (GST) and AR can bind specifically to sepharose beads coated with glutathione. When a protein mixture is added proteins that can bind to AR will also interact with the column and can later be eluted and analyzed. 20 pC i /mL. Incubation was continued at 37°C overnight. The medium was removed and cold P B S containing I m M E D T A was added to harvest the cells. 2.1.2 Preparation of LNCaP nuclear extract Nuclear extracts were prepared using a small scale version of the method described by Dignam et al. (155). L N C a P cells were pelleted at 1000g and washed in cold P B S before being resuspended in the hypotonic Buffer A ( lOmM Hepes p H 7.9, 1.5mM MgCi2, l O m M KC1, 0.5mM DTT) . Cells were left to swell on ice for 10 minutes before being pelleted and resuspended in Buffer A . The cells were then broken up with 25 strokes of a Dounce \ homogenizer and the nuclei pelleted at 14,000 rpm for 35 minutes in a microcentrifuge (IEC). The nuclei were resuspended in Buffer C (20mM Hepes p H 7.9, 25% glycerol, 0.42M N a C l , 1.5 m M M g C l 2 , 0.2mM E D T A , 0.5mM D T T , 0.5mM P M S F ) and incubated at 4°C for 30 minutes. After microcentrifugation (IEC) at 14,000 rpm for 50 minutes, the supernatant was collected and dialyzed against 4000x volume of Buffer D (20mM Hepes p H 7.9, 20% glycerol, 0 .1M KC1, 0.2mM E D T A , 0.5mM DTT , 0.5mM P M S F ) for 2 hours. The sample was microcentrifuged (IEC) at 14,000 rpm for 35 minutes and the supernatant collected. The supernatant was stored at -80°C after being flash-frozen in l iquid nitrogen. 2.1.3 Radioactive labeling of yeast cells A 30 m L overnight culture in Y P D medium (2% peptone (w/v) and 1% yeast extract (w/v)) was inoculated from plated colonies of Saccharomyces cerevisiae (strain Y190). The culture was grown at 30°C with shaking until an O.D . 6 oo of about 0.9 was reached. The cells were pelleted and then washed with minimal SD (synthetic dropout) medium (0.67% yeast nitrogen base without amino acids (w/v), supplemented with the appropriate amino acids) lacking methionine. The cell pellet was resuspended in 300 uL of 33 SD without methionine and was allowed to stand at room temperature for 15 minutes. 330 uC i of J J S-label ing mix (ICN), consisting of 75% methionine and 25% cysteine, was added to each culture and the yeast cells were incubated at 30°C for 2 hours, using the HotBox™ system (Billups-Rothenberg) to prevent escape of radioactive gases. The optimal length of time to incubate the yeast cells in the presence of the 35 35 S-labelmg mix was determined by a S-incorporation assay. After the addition of the 35 S-labeling mix, samples were removed every hour for 4 hours with the last sample taken following overnight incubation. The yeast cells were pelleted and washed three times to remove free radioactivity. The yeast cells were lysed by the addition of 150 uL of I M N a O H , and then the solution was neutralized by the addition of 150 p L of I M HC1. Final ly, the samples were counted using a scintillation counter and the time point that gave the optimal level of incorporation was determined. 2.1.4 Preparation of yeast protein extract The labeled yeast culture was divided into 3 equal parts and to each was added 1 m L of cold yeast lysis buffer (50mM Tris p H 7.5, 250mM N a C l , 50mM NaF , 5 m M E D T A , I m M D T T , I m M P M S F , 0.1% NP-40, Complete™ Protease Inhibitor Cocktai l Tablet (1 tablet/50 mL) (Boeliringer-Marinheim)). The cells were pelleted and washed 2 times with yeast lysis buffer. After final resuspension in the lysis buffer, 100 p L of acid-washed glass beads (125-212p and 425-600p) was added. The mixture was vortexed at maximum speed for 30 minutes at 4°C and then microcentrifuged (IEC) at 13,000 rpm for 30 minutes at 4°C. The supernatant was collected and stored at -80°C. 2.1.5 Preparation of GST fusion proteins The vector p G E X - 3 X (Pharmacia) was used to express fusion proteins consisting of 34 various fragments of the rat androgen receptor fused to glutathione-S-transferase. The N-terminal domain of the rat A R has 76% amino acid identity to that of the human A R , with most differences related to changes in the number and position of the polyglutamine and polyglycine tracts. The constructs of interest in these experiments included one that expressed amino acids 524 to 648 (DBD) of the rat androgen receptor, and one that expressed amino acids 232 to 648 (N-terminal domain plus D B D ) of the rat androgen receptor (provided by Dr. Rob Snoek). The p G E X - 3 X vector alone was used as a control. The use of constructs containing the A R D B D allowed identification of proteins that interacted specifically with the N-terminal domain relative to other fragments of A R . The identification of proteins that interacted with the 232-524 fragment of the N-terminal domain using G S T -pulldown complemented the detection of protein interactions with the 1-232 fragment of the N-terminal domain used in the yeast 2-hybrid system. Inoculation of 10 m L cultures of L-Broth, containing 100 pg /mL of ampicil l in, was done using freezer stocks of E. coli (strain JM109) transformed with the various p G E X - 3 X constructs. After growth at 37°C overnight, the entire 10 m L culture was used to inoculate 400 m L of L-Broth and ampicil l in. This culture was grown at 37°C for 2 hours (O.D.600 about 0.6). Expression of the fusion protein was then induced by the addition of I P T G to 0.1 m M and shaking at room temperature for 2 hours. Cells were pelleted and resuspended in P B S plus protease inhibitors. The cells were lysed by three freeze-thaw cycles, followed by sonication. After the addition of 560 p L of 10% Tri ton-X and 10% Tween-20, the mixture was vortexed and the cell debris was pelleted at 10,000 rpm for 5 minutes (Sorvall SS-34). The supernatant was then collected and stored at -80°C. Production of the fusion proteins was tested by separation of a sample of the lysate by S D S - P A G E , followed by 35 staining with Coomassie brilliant blue (BRL) or Western blotting with a polyclonal rabbit antibody directed against G S T (Santa Cruz). 2.1.6 Preparation of glutathione-Sepharose affinity columns Glutathione-Sepharose affinity columns were prepared by packing disposable polypropylene columns containing plastic filter disks with 266 p L of glutathione-Sepharose beads (Pharmacia), resulting in a bed volume of 200 pL . The beads were washed with 2 m L of cold P B S before the addition of the E. coli lysate. To the columns, 2 m L of lysate from E. coli expressing G S T , GST-AR524-648, or GST-AR232-648 was added. The lysate was allowed to incubate with the beads for 15 minutes at room temperature. The columns were then drained and washed 3 times with 2 m L of cold P B S . The optimal level of washing was determined by measuring the A280 of the wash buffer after each of fourteen washes to assay the amount of protein removed with each wash. 2.1.7 Binding of radiolabeled proteins to affinity columns The labeled protein extract was precleared for non-specific binding to the column by adding it to the column containing bound GST. The flow-through was collected and added to a second column containing bound GST, and then this process was repeated once more. The precleared extract was collected, split into 3 equal parts, and added to the columns containing bound G S T , GST-AR524-648, or GST-AR232-648- The protein extract was allowed to incubate for 15 minutes with the column and was then drained. The columns were washed three times each with 2 m L of Buffer D. These procedures were carried out at 4°C. 2.1.8 Elution of proteins bound to affinity columns 200 uL of glutathione elution buffer (50mM Tris p H 8, 10 m M glutathione) was added to each 10 m L column. The elution buffer was incubated with the column for 15 36 minutes at room temperature before being drained and collected. This was repeated with 200 p L of fresh glutathione elution buffer and this flow-through was also collected. The beads were suspended in 200 uL of SDS sample buffer and heated for 5 minutes at 95°C. The beads were then pelleted and the supernatant collected. 2.1.9 Analysis ofproteins eluted from affinity columns The eluted samples were mixed with sample buffer and heated at 95 °C for 5 minutes before being loaded on a 16 cm S D S - P A G E . Electrophoresis was carried out at 150 V for about 5 hours. If the samples were to be analyzed by fluorography, the gel was fixed in a solution of 30% methanol and 10% acetic acid for 1 hour, soaked in E N H A N C E (NEN) for 1 hour, and washed in distilled water for 30 minutes. The gel was dried with heat under vacuum for 2 hours and then exposed to autoradiographic f i lm at -80°C, with multiple exposures obtained. If the samples were to be analyzed by Western blotting, the proteins were transferred from the S D S - P A G E onto Immobilon-P membrane (Mil l ipore) (pre-soaked in methanol) at 100V for 1 hour. The membrane was blocked for 30 minutes at room temperature in T B S containing 5% low fat mi lk powder (Carnation) and 0.05% Tween-20. The primary antibody was incubated with the membrane for 45 minutes and the membrane was then washed three times for five minutes in T B S (20 m M Tris base, 137 m M N a C l , p H 7.6) containing 0.05% Tween-20. The primary antibodies used included a rabbit polyclonal antibody to human calreticulin (provided by Dr. S. Dedhar) and a mouse anti-Hsp70/Hsc70 monoclonal antibody (StressGen). The membrane was incubated with the appropriate secondary antibody coupled to H R P (Santa Cruz) for 30 minutes and then washed three times for five minutes in T B S plus 0.05% Tween-20 and one time for five minutes in T B S . A n E C L detection kit (Amersham) was used to visualize the secondary antibody and exposure to autoradiographic f i lm was used to record the activity. 2.2 Co-immunoprecipitation using yeast protein extract Another common method of studying protein-protein interactions, co-immunoprecipitation, can also be used to identify novel binding partners of the protein of interest. A radioactively labeled protein extract was prepared from yeast cells that expressed a fusion protein containing all or part of the N-terminal domain of A R linked to the Gal4 DNA-b ind ing domain (Gal4 D B D ) (Fig. 5). This protein extract was then incubated with an antibody that recognized either the A R protein itself or the G a l 4 D B D fusion tag linked to A R . The addition of beads coated with bacterial proteins, such as protein A or protein G , that could bind to immunoglobulin molecules brought A R and any proteins bound to A R out of solution. Similar to the GST-pul ldown analysis, these proteins were analyzed by S D S - P A G E and fluorography. The A R coactivator B A G - 1 L was co-immunoprecipitated with A R and co-immunoprecipitation has been used to confirm interactions between many steroid receptors and coregulators (142). 2.2.1 Radioactive labeling of yeast cells Transformed yeast cells were plated and colonies were picked and used to inoculate 30 m L of either Y P D medium or minimal medium (SD) supplemented with amino acids except tryptophan. Saccharomyces cerevisiae (strain Y190) was transformed with the plasmid p G B T 9 - A R , which expressed fragments of the A R fused to the DNA-b ind ing domain of Gal4. These fragments included a truncated human A R N-terminus (a.a. 1-232) and the full-length N-terminus (a.a. 1-559) of the human androgen receptor (provided by Helen Cheng). Transformed yeast cells were plated on agar plates made with minimal 38 Protein X Anti-Gal 4 DBD A n t i b o d y A R N-terminal domain Protein A / G Fig. 5 Schematic diagram demonstrating principles of co-immunoprecipitation. Antibodies to Gal4 DBD can bind specifically to fusion proteins containing Gal4 DBD and AR. Beads coated with protein G that can bind specifically to IgG molecules can be added to precipitate the Gal4 DBD-AR fusion protein and any other proteins that are interacting with it. These proteins can be eluted from the beads and analyzed. medium supplemented with amino acids except tryptophan to select for the presence of the p G B T 9 plasmid. The labeling of the cells was carried out as described previously (2.1.3). 2.2.2 Co-immunoprecipitation, elution, and analysis of proteins Preparation of the yeast protein extract was carried out as described previously (2.1.4). The labeled protein extract was incubated for 1.5 hours at 4 °C with 50 p L of Sepharose beads coated with protein A (Sigma) or protein G (Sigma), to clear any background binding to the beads. The beads were then pelleted and the supernatant was collected. In these experiments, a rabbit polyclonal antibody directed against amino acids 1-21 of the human androgen: receptor (ABR) or a mouse monoclonal antibody to amino acids 1-147 of the D B D of Gal4 (Clontech) was used. About 2 pg of monoclonal or polyclonal antibody was then added and incubated for 1 hour at 4°C. 50 p L of protein-A Sepharose or protein-G Sepharose was then added and incubated for 1 hour at 4°C. The beads were pelleted and washed four times with yeast lysis buffer. The washing was monitored for radioactivity to ensure completed removal of unbound 3 5 S-labeled protein. After washing, 45 p L of SDS sample buffer was added to the beads and incubated at 95°C for 5 minutes. The beads were then pelleted and the supernatant collected. The samples collected were mixed with SDS sample buffer and heated at 95°C for 5 minutes before being loaded on a S D S - P A G E mini-gel. The gel was run at 150 V for about 1 hour. The 7 cm gel was fixed, treated with E N H A N C E , and dried as previously described, then exposed to autoradiographic f i lm at -80°C until a suitable autoradiograph was obtained. 2.3 Yeast 2-hybrid screening The yeast 2-hybrid assay can detect novel protein-protein interactions, and has been 40 used to identify the majority of known coregulators. This is a sensitive in vivo system that utilized two fusion proteins; the first was composed of the first 232 amino acids of the N-terminal domain of the A R fused to the Ga l4DBD, while the second contained the Gal4 activation domain (Gal4AD) fused to a human prostate c D N A library (Fig. 6). Coexpression of these two chimeric proteins in yeast that contained a Gal4-responsive promoter upstream of a reporter gene resulted in the expression of the reporter gene only i f the two chimeric proteins interacted (156). As the Ga l4DBD and the Ga l4AD could not interact directly with each other, transcription of the reporter gene was only initiated i f the A R interacted with one of the proteins encoded by the c D N A library. The reporter genes used in this system were under the control of Gal4-responsive promoters, and included the lacZ gene, which encoded the P-galactosidase enzyme, and the HIS3 gene, which allowed yeast cells to grow in the absence of histidine. The D N A sequence of clones that exhibited transcription of the reporter gene was obtained, the sequence analyzed and compared to known genes. 2.3. J Preparation of the cDNA Library The human prostate c D N A library ( M A T C H M A K E R Clontech) was screened for interactions with amino acids 1 to 232 of the N-terminal domain of human A R . The library consisted of 3.2 x 10 6 independent clones inserted into &Xho\/EcoR\ site in the p A C T 2 vector which were expressed as fusion proteins with the Gal4 activation domain (Gal4 A D ) . Clontech estimated that 88% of the M A T C H M A K E R library clones contained an insert. Upon testing, Clontech found that 0.25% of the library clones hybridized to p-actin, which they claim indicates a high probability of finding rare transcripts. The library clones were plated onto 75 x 150 mm LB-agar plates with 50ug/mL of ampicil l in and incubated at 37°C overnight to amplify the library. The colonies were then scraped off the plates into L-broth 41 A. Bait (AR) r 1 Gal4 No transcription v DBD J GAL1 UAS promoter lacZ (or HIS3) reporter B. Gal4 A D Library clone No transcription GAL1 UAS promoter lacZ (or HIS3) reporter C. Gal4 A D Bait (AR) i - 1 Library clone Gal4 V ^ D B D y GAL1 UAS promoter -•Transcription lacZ (or HIS3) reporter Fig. 6 Schematic diagram demonstrating principles of yeast 2-hybrid system. A . The fusion protein containing Gal4 D B D and the N-terminal domain of A R can interact with the D N A at the Gal 1 upstream activating sequence but cannot activate transcription of the reporter gene because no activating domain is present. B. The fusion protein containing Gal4 A D and the library clone cannot interact with the D N A so activation of transcription of the reporter gene cannot occur. C. The interaction between A R and the library clone can bring the two fragments of Gal4 together to restore Gal4 function, resulting in the expression of the reporter gene. 42 (1% tryptone (w/v), 0.5% yeast extract (w/v), and 85 m M NaCl ) and glycerol was added to prepare the library for long term storage at -80°C. The titer of the library was determined to be 2 x l 0 9 cfu/mL. A portion of the library stock was then used for the isolation of plasmid D N A using cesium-chloride gradient centrifugation (157). 2.3.2 Transformation of yeast cells for screening Competent Y190 yeast cells were prepared by the lithium acetate (L iAc) method. 1 L of yeast culture was grown to an O.D.600 of 0.5 in Y P D medium. After centrifugation, the cell pellet was washed and resuspended in 8 m L of T E (10 m M Tris, I m M E D T A ) and 100 m M L i A c . Transformation of l m L of competent Y190 yeast cells was performed with 25 pg of plasmid D N A from the library and 25 pg of the bait plasmid. The bait plasmid consisted of amino acids 1 to 232 of the human A R inserted into the shuttle vector p G B T 9 at the BamKl site (provided by Helen Cheng). The bait plasmid had previously been tested to ensure that it did not have any intrinsic activation function. Added to the plasmid D N A and competent cells were 5 mg of herring testes carrier D N A (Clontech) and 30 m L of 40%> polyethylene glycol (PEG) plus 100 m M L i A c . This mixture was incubated at 30°C for 30 minutes with shaking. After the addition of 3.8 m L of 100%) dimethyl sulfoxide (DMSO) (Sigma), the mixture was heat-shocked at 42°C for 15 minutes. The cells were pelleted and finally resuspended in 5 m L of T E . To determine the number of successful transformants, an aliquot (10 uL) was plated on agar plates made with minimal medium supplemented with amino acids except tryptophan and leucine to select for the presence of both plasmids. The total number of transformants was estimated to be 5 x 10 5 and they were plated onto 25 x 150 mm agar plates made with minimal medium supplemented with amino acids except tryptophan, leucine, histidine and containing 20 m M 3-aminotriazole (3-AT). Y190 cells 43 contain a HIS3 reporter gene as wel l as a lacZ reporter under the control of the Gal4 responsive promoter. A positive interaction between the bait and the library proteins results in activation of the HIS3 gene, leading to growth in the absence of histidine in the medium. The addition of 3-AT, a competitive inhibitor of the HIS3 protein, to the medium reduces the occurrence of false positives that can occur due to leaky expression of the HIS3 reporter. These plates were incubated at 30°C for eight to ten days. 2.3.3 Screening of yeast transformants The colonies that grew on agar plates made with minimal medium supplemented with amino acids except tryptophan, leucine, and histidine, and containing 20 m M 3-AT were then assayed for P-galactosidase expression using colony filter lifts. A sterile Whatman #5 filter was placed on the surface of the plate containing the transformed colonies. A notch and three asymmetric holes in the filter were used to orient the filter on the agar. After the colonies had adhered to the filter, it was lifted with forceps, transferred to l iquid nitrogen for 10 seconds, and then removed from the liquid nitrogen and allowed to thaw at room temperature before being placed on another filter that was presoaked in Z-buffer (60mM N a 2 H P 0 4 - 7 H 2 0 , 40mM N a H 2 P 0 4 - H 2 0 , l O m M KC1, I m M M g S 0 4 - 7 H 2 0 ) , containing 3.3 mg/mL X-ga l (5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside) and 0.3% P-mercaptoethanol. The filters were incubated at 30°C for up to 8 hours, until the blue colour that signified the production of p-galactosidase was observed. The colonies corresponding to the positive p-galactosidase clones were selected and streaked onto agar plates lacking leucine and tryptophan. These were incubated for 3 days at 30°C, and then the colonies were restreaked onto the same type of medium and incubated again, to try and ensure that each colony corresponded to a single clone. Each time, the 44 colonies were tested with a colony filter lift for |3-galactosidase to ensure the positive reaction was intact. The positive colonies were then transferred to agar plates made with minimal medium (supplemented with amino acids except tryptophan, leucine, and histidine, and containing 20 m M 3-AT) and incubated at 30°C until the colonies reached a suitable size. These colonies were again tested by (3-galactosidase colony filter lift to ensure positive reactions were still present. 2.3.4 Confirmation of positive clones From the 5 x 10 5 transformants screened, 26 positive clones were obtained. For each clone, 3 m L overnight cultures were set up and plasmid D N A was prepared. The cells were harvested by centrifugation and lysed in 0.2 m L of TES buffer ( l O m M Tris pH8, I m M E D T A , lOOmM NaCl ) containing 1% SDS and 2% Triton X-100. Then 0.2 m L of phenol-chloroform (1:1) and 300 uL of acid-washed glass beads (425-600p) were added. This mixture was vortexed at maximum speed for 2 minutes and then microcentrifuged (IEC) for 5 minutes. The aqueous layer was transferred to a new tube and 10 M L i C l (1:1) and CHCI3 (1:2) were added. After mixing, this solution was incubated at -20°C for 20 minutes. The aqueous phase was again transferred to a clean tube and the D N A was precipitated at -20°C overnight, using 2 volumes of 99% ethanol and a one-tenth volume of 3 M N a O A c . The pellet was later resuspended in lOuL of TE . Electrocompetent leuB E.coli (strain HB101) were prepared from a 100 m L culture with an O.D . 6ooof 0.5. The culture was chilled on ice for 20 minutes before the cells were pelleted at 4000 rpm (IEC 243) for 5 minutes at 4°C and resuspended in 10% ice-cold glycerol. This step was repeated twice before the cells were ready for electroporation and could then be stored at -80°C. 45 Electroporation of 50 p L of electrocompetent leuB E. coli HB101 with 2 to 3 p L of yeast plasmid D N A at 1.8 k V , 25 pF, and 200 Q (Biorad Gene Pulser® II) allowed selection of successful transformants by growth on agar plates made from M 9 minimal medium, supplemented with amino acids except leucine. Only the transformants that had taken up the library plasmid were able to grow in the absence of leucine. The lack o f selection pressure for the bait plasmid resulted in loss of that plasmid. For each of the 26 different E. coli clones, 5 colonies were selected and miniprep D N A prepared. Restriction digests using Xhol and EcoRl allowed the insert to be cut selectively out of the library plasmids, provided the size o f the insert, and also allowed for the identification of clones that contained more than one library plasmid. For each unique clone, 0.1 pg of plasmid D N A was used to transform competent Y190 yeast cells (see section 2.3.2 for preparation of Y190 competent cells). The library plasmid D N A was separately co-transformed with each of the following: 0.1 pg of the bait plasmid (pGBT9 plus sequence encoding amino acids 1 to 232 of the human A R ) , 0.1 pg of the empty p G B T 9 vector, or 0.1 pg of the p L A M 5 ' vector (pGBT9 plus sequence encoding the human lamin C protein, provided by Clontech). The empty p G B T 9 vector or the p L A M 5 ' vector encoding a human protein distinct from the A R act as negative controls as neither should be able to interact with the library plasmid to induce transcription of the reporter genes. The plasmid D N A was combined with 100 pg of herring testes carrier D N A and added to 100 p L of Y190 competent cells and 600 pL of P E G / L i A c solution. The cells were incubated at 30°C for 30 minutes, after which 70 uL of D M S O was added and then the cells were heat-shocked at 42°C for 15 minutes. The cells were chil led on ice for 5 minutes and pelleted before being resuspended in 0.5 m L of TE . The transformants were plated on 4 6 agar plates made with minimal medium supplemented with amino acids except tryptophan, leucine, and histidine, and containing 20 m M 3-AT. The plates were incubated for 7-8 days at 30°C before being assayed for P-galactosidase activity. Three clones were found to be positive only when co-transformed with the bait plasmid. These three clones had been observed to have inserts of approximately the same size when they were digested earlier with EcoRl and Xhol, which suggested that they might contain the same insert. 2.3.5 Sequencing ofpositive clones These three clones were sequenced using B igDye Terminator Cycle Sequencing (ABI) using the A D primers supplied by Clontech and were found to have virtually identical sequences. The sequence was then compared to known sequences available in GenBank. Additional primers were designed to continue sequencing from the 5' end and to sequence from the 3' end of the insert. When this sequence was obtained it was also compared to GenBank sequences. 2.3.6 Analysis of positive clone by Western Blotting The size of the protein being expressed by the positive clone was determined by Western blotting using a rabbit polyclonal antibody against a hemagglutinin (HA) tag on the fusion protein (Clontech). Yeast cells that had been transformed with the plasmid encoding the positive clone were used to inoculate 3 m L of minimal medium supplemented with amino acids except leucine and tryptophan and this culture was incubated overnight at 30°C. The yeast cells were pelleted at 2400 rpm (IEC 6560), washed with water, and pelleted again. The cell pellet was resuspended in 300 p L of a solution containing 1.85 M N a O H and 7.5% P-mercaptoethanol. This mixture was incubated for 10 minutes on ice before 300 uL of 50% trichloroacetic acid (TCA) was added. After a further incubation of 10 minutes the sample 4 7 was microcentrifuged (IEC) at 13,000 rpm for 3 minutes. The supernatant was then discarded and the pellet washed with water three times. Centrifugation was repeated and the pellet solubilized in 200 p L of 1 times SDS sample buffer. The sample was separated by electrophoresis on a 10% S D S - P A G E mini-gel for 1 hour at 150 V . Western blotting using the antibody against the HA-tag was performed as described previously (2.1.9) using a rabbit polyclonal antibody against the hemagglutinin tag (Clontech). 2.3.7 Northern blot analysis of positive clone To prepare a radioactively labeled c D N A probe for the positive clone the insert was cut from the library plasmid using the restriction enzymes N o t l and X h o l . The fragment was separated by electrophoresis on a 1% agarose gel, the band cut from the gel, and the D N A fragment spin eluted. The D N A was precipitated at -20°C overnight, using a 1/10 volume of 3 M N a O A c and 2 volumes of 99% ethanol. The pellet was later resuspended in 10 p L of T E . For radioactive labeling of the probe, 100 ng of D N A was suspended in a total volume of 14 p L and boiled for 10 minutes before being placed on ice for 5 minutes. The Oligolabeling kit (Pharmacia) was used for labeling of the probe. Five p L of 5 times Oligo-Labeling buffer was added along with 5 uL of 3 2 P - d C T P (3,000 Ci/mmol) (Amersham) and 1 p L of Klenow. This mixture was incubated at 37°C for 60 minutes. Then 1 p L of 0.5 M E D T A was added and the mixture placed on ice. To remove unincorporated nucleotides, 74 p L of T E was added to the probe D N A and the entire sample added to a disposable column containing Sephadex G-50. The column was centrifuged at 3,000 rpm for 2 minutes (IEC 6560). A 1 p L sample was precipitated using T C A and analyzed in a scintillation counter to determine the specific activity of the probe, which was 4 8 2.5x10 cpm/pg D N A . The remaining probe D N A was boiled for 10 minutes then placed on ice for 5 minutes. The probe was stored at -20°C until it was used for hybridization. The c D N A probe was used to probe the Mult iple Tissue Expression (MTE) Array (Clontech), a positively charged nylon membrane to which poly A+ R N A s from different human tissues and cancer cell lines have been normalized and immobil ized in separate dots. The use of the M T E Array allowed the determination of relative expression levels of a target m R N A in different tissues and developmental stages. The provided hybridization solution, ExpressHyb (Clontech), was prewarmed for 15 minutes at 55°C. This was mixed with 1.5 mg of sheared salmon testes D N A that had been preheated at 95°C for 5 minutes and then quickly chilled on ice. The M T E Array was prehybridized with 10 m L of the ExpressHyb and salmon testes D N A solution for 30 minutes at 65°C in a hybridization oven, l x l 0 7 cpm of the labeled c D N A probe was mixed with 30 pg of C 0t-1 D N A , 150 pg of sheared salmon testes D N A , and 50 uL of 20 times SSC (3M N a C l , 0.3M sodium citrate) in a total volume of 200 uL. This mixture was heated at 95°C for 5 minutes and then at 68°C for 30 minutes. The mixture was then added to 5 m L of the solution containing ExpressHyb and salmon testes D N A . The prehybridization solution was discarded, this new mixture added to the M T E Array, and the M T E Array hybridized at 65 °C overnight. The hybridization solution was then replaced with 20 m L of a solution containing 2 times SSC and 1% SDS and washed for 20 minutes at 65°C. This washing step was repeated 4 times. Two additional washes with 20 m L of a solution containing 0.1 times SSC and 0.5% SDS were performed for 20 minutes at 55°C. The M T E Array was then removed from the wash solution, wrapped in plastic wrap, and exposed to autoradiographic film (Biomax) at -70°C overnight. 49 The c D N A probe was also used to probe a Mult iple Tissue Northern (MTN) Blot (Clontech), a hybridization ready Northern Blot of high-quality poly A+ R N A from several human tissues. The M T N Blot allowed the examination of specificity and level of m R N A expression of a specific gene over a wide range of tissues. The M T N blot was prehybridized with 5 m L of prewarmed ExpressHyb solution for 30 minutes at 68°C. l x l 0 7 cpm of the radioactively labeled probe was denatured at 95°C for 5 minutes, chil led on ice, and added to 5 m L of fresh ExpressHyb solution. The M T N Blot was incubated with the c D N A probe for 1 hour at 68°C. The blot was rinsed several times with a solution containing 2 times SSC and 0.05% SDS and then washed continuously for 40 minutes at room temperature, with the wash solution replaced twice. A solution containing 0.1 times SSC and 0.1 % SDS was used to wash the blot at 50°C for 40 minutes, with one change of fresh wash solution. The M T N Blot was covered in plastic wrap and exposed to autoradiographic f i lm (Biomax) at -70°C overnight. The M T N Blot was also analyzed using a phosphorimaging screen (Biorad). 50 3. Results 3.1 Pre l iminary Exper iments 3.1.1 Incorporation of35S-methionine by yeast cells The incubation time needed to maximize incorporation of 3 5S-methionine by yeast cells was determined by a time-course assay. This assay was done using several different volumes of yeast growth medium lacking methionine, in order to establish whether the volume of medium used affected the incorporation of S-methionine by the yeast cells. Scintillation counting of the yeast cell lysate produced cpm values that were then normalized for cell numbers. Comparison of the amount of radioactive label incorporated by the yeast cell cultures over time indicated that there were clear differences between the cultures that were suspended in different volumes of medium (Fig. 7). Incorporation of 3 5S-methionine by the 1.2 m L culture varied little over time, reaching a maximum after 4 hours of incubation, while the 0.6 m L culture increased slowly to reach a maximum incorporation after 3 hours of incubation (Fig. 7). After 2 hours, incorporation by the 0.3 m L culture reached a maximum that was clearly higher than the levels achieved in the 0.6 m L and 1.2 m L cultures (Fig. 7). In all subsequent experiments requiring labeling of yeast cells, cultures were resuspended in 0.3 m L of minimal medium supplemented with amino acids except methionine and incubated for 2 hours with the S-methionme labeling mix. 3.1.2 Preparation of GST-AR affinity columns To ensure successful! production of G S T - A R fusion proteins by E. coli, the bacterial lysate was tested by Western blotting with an antibody against G S T or by Coommassie staining. The p G E X - 3 X vector only produced G S T , with a molecular mass of 30 kDa 51 Fig. 7 Incorporation of S-methionine by yeast cells . 35S-labeling mix was added to yeast cell cultures that were resuspended in 300 pL, 600 pL or 1200 pL of growth media lacking methionine. Samples were taken at various time points and analyzed using a scintillation counter to determine the optimum conditions for maximum incorporation of the S-methionine by the yeast cells. The cpm values indicate the amount of 3 5S activity present within the yeast cells normalized to account for the differences in yeast cell density in the final cultures arising from the resuspension of similar numbers of yeast cells in a variety of volumes. (Fig. 8, lane 2), similar to that of G S T that had been previously purified using glutathione-coated beads (Fig. 8, lane 1). The G S T - A R 5 2 4 - 6 4 8 protein had a molecular mass of 43 kDa, while that of the GST-AR233-648 protein was 75 kDa (Fig. 8, lanes 3 and 4). Although some degradation of the fusion proteins was seen, most of the protein detected was the mass predicted for the intact GST-tagged proteins. After incubation of E. coli lysate with the glutathione-Sepharose beads to allow binding of the G S T - A R fusion proteins to the beads, the unbound material was removed by washing with P B S . The amount of protein in the wash buffer was measured after several sequential washes to determine the number of washes required to remove the majority of the unbound material. After three washes, there were consistently low levels of material in the wash buffer (Fig. 9). Therefore, three washes were used for washing all G S T - A R columns before addition of the 3 5 S-labeled protein extracts. 3.1.3 Sequential washing of immunoprecipitates It was also important to determine the number of washes needed to remove the majority of unbound S-labeled protein from a sample of S-labeled protein precipitated through affinity interactions without washing away the precipitated protein. Immunoprecipitates derived from 3 5S-labeled yeast protein extracts were washed sequentially 5 times with 1 m L of buffer and following each wash a sample of the wash buffer was preserved for analysis by scintillation counting. The cpm values were used to determine the 35 amount of S-labeled protein being removed with each wash. When the cpm values were plotted it was clear that the vast majority of unbound S-labeled protein was eliminated with the first two washes (Fig. 10). However, some S-labeled protein was also removed in kDa 1 2 3 4 GST-AR233-648 GST-AR524-648 GST F i g . 8 Expression of GST-AR fusion proteins. Lysates o f E. coli expressing G S T - A R fusion proteins were separated by S D S -P A G E and assayed by Western blott ing using an antibody to G S T . Lane 1, pur i f ied G S T ; lane 2, expression o f G S T ; lane 3, expression o f GST-AR 5 2 4_648 ; lane 4, expression o f GST-AR 2 33_648-number o f washes F i g . 9 Amount of protein material removed by sequential washes of a GST affinity column. G S T - A R fusion proteins were incubated w i th glutathione-sepharose beads and then washed sequential ly seven t imes, measuring the A 2 8 0 o f the wash buffer after each wash. The A 2 8 0 value indicates the amount o f protein being removed by each wash. 55 washes F i g . 10 Level of S-labeled material removed by sequential washes. Co- immunoprecipi tates o f S- labeled yeast ce l l protein extracts were washed sequential ly f ive t imes before elut ion, and the wash buffer was analyzed us ing a scint i l lat ion counter. The c p m values indicate the amount o f S-act iv i ty present in the buffer after each wash. the third and fourth washes (Fig. 10). The cpm values leveled out by the fourth wash, indicating that most of the unbound protein had already been removed (Fig. 10). 3.2 Af f in i ty pul ldown and Immunoprecipitat ion Studies 3.2.1 GST-pulldown using LNCaP nuclear extracts G S T - A R fusion proteins expressed in E. coli were immobil ized on glutathione-Sepharose beads and incubated with 3 5 S-labeled nuclear extracts from L N C a P cells that had been precleared through a G S T column to reduce background. The precleared 3 5 S-labeled L N C a P nuclear extract was divided and run through columns containing G S T alone, GST-AR 5 2 4-648, or GST-AR.233-648- The column containing only G S T served as a control to identify any proteins binding to the glutathione-Sepharose beads or G S T portion of the fusion proteins. Proteins bound to the GST-AR 2 33-64 8 column, which contained the A R N-terminus and D B D , could be compared to those bound to the GST-AR 5 2 4-648 column that contained only the A R D B D , to identify proteins that were interacting specifically with the N-terminal domain. To establish that the G S T - A R fusion proteins and conditions used in this assay were capable of identifying proteins that interact with A R , the system was tested for the ability to identify an interaction with calreticulin, a protein known to bind to the D B D of A R (158). Unlabeled L N C a P cells were used in the assay and the nuclear extract and elution products from the G S T - A R columns were probed with an antibody against calreticulin. A band of about 60 kDa, corresponding to the expected mass of calreticulin, was present in the initial nuclear extract and was still present after the extract had been precleared through a G S T column (Fig. 11, lanes 1 and 2). The eluate from the GST-AR.524_648 and GST-AR 233_648 5 7 kDa 1 2 3 4 5 69 4 6 — •<-calreticulin F i g . 11 GST-AR pulldown of calreticulin. Precleared nuclear extracts f rom L N C a P cells were incubated wi th G S T - A R immob i l i zed on glutathione-sepharose beads. E lu ted proteins were assayed by Western blott ing using A b to calret icul in. Lane 1, whole nuclear extract; lane 2, precleared nuclear extract; lane 3, eluate f rom G S T co lumn; lane 4, eluate f rom GST-AR 5 2 4-648 co lumn; lane 5, eluate f rom G S T - A R 2 3 3 _ 6 4 8 column. 58 columns also contained calreticulin (Fig. 11, lanes 4 and 5). Calreticulin was expected to bind to these G S T - A R columns, as both contain the D B D region of A R where calreticulin is known to interact. Binding of calreticulin to the column containing G S T alone was not seen (Fig. 11, lane 3). These results indicated that the GST-pul ldown assay could be used successfully to identify proteins that interact with A R . Even though the nuclear extract had been precleared through a G S T column high levels of background binding were found when 3 5S-labeled nuclear extract was used, as can be seen by the large number of proteins that bound to the column containing G S T alone (Fig. 12, lane 1). However, several proteins were seen interacting with the column that contained GST-AR233-648 (lane 3), that were not present in the columns containing G S T (lane 1) or GST-AR524-648 (lane 2). This suggests that the protein interactions with the A R are occurring within the N-terminal domain, between amino acids 233 and 524. A protein of about 40 kDa, present in L N C a P nuclear extract, appeared to interact specifically with the GST-AR233-648 (Fig. 12, lane 3). This protein was not apparent in the columns containing G S T or G S T - A R 5 2 4 - 6 4 8 (Fig. 12, lanes 1 and 2). Proteins 1 and 2, the two smaller protein bands directly fol lowing the 40 kDa protein in the eluate from the GST-AR233-648 column, also appeared to have a unique binding pattern (Fig. 12, lane 3). Whi le protein 1 did not seem to have a corresponding band binding to the G S T - A R 5 2 4 - 6 4 8 column, a protein of the same mass bound to the G S T column, indicating that this protein probably did not bind specifically to the A R component of the G S T - A R fusion protein (Fig. 12). This apparent failure to bind to G S T - A R 5 2 4 - 6 4 8 could be explained by the fact that the unlabeled G S T - A R 5 2 4 - 6 4 8 protein was about 40 kDa and was present in such large amounts in the column eluate that it could have obscured other proteins that ran at the same position in the 59 kDa 1 2 3 — 40 kDa protein Protein 1 Protein 2 F i g . 12 GST-AR pulldown of a 40 kDa protein in LNCaP cells. 3 5 S- labe l l ed L N C a P nuclear extracts were precleared and incubated wi th immob i l i zed G S T - A R . E lu ted proteins were v isual ized by autoradiography. Lane 1, eluate f rom G S T co lumn; lane 2, eluate f rom G S T - A R 5 2 4 - 6 4 8 co lumn; lane 3, eluate f rom G S T - A R 2 3 3 _ 6 4 8 co lumn. gel. Protein 2 appeared to interact with the GST-AR233-648 and GST-AR 5 2 4-648 columns, but was not seen in the G S T column (Fig. 12), suggesting that this protein could have been interacting with the D B D of the A R (amino acids 524-648). However, the band seen in the GST-AR524-648 column may have been displaced from a slightly higher position in the gel by the unlabeled GST-AR524-648 protein. Alternatively this protein could also have been specifically interacting with the N-terminal domain of the A R (amino acids 233-524). A protein of about 70 kDa from L N C a P nuclear extract bound uniquely to the column containing GST-AR 2 33-648 (Fig- 13 A , lane 3). No corresponding protein of the same size bound to the columns containing G S T or GST-AR524-648 (Fig. 13 A , lanes 1 and 2). Examination of the column flow-through allowed visualization of the unbound 3 5S-labeled proteins. A protein of about 70 kDa was identified that was present in the unbound material from the G S T and GST-AR524-648 columns, but was lost from the unbound material from the GST-AR 233_648 column (Fig. 13B). This serves to confirm that a protein of about 70 kDa was binding specifically to the GST-AR 233_648 column. 3.2.2 GST-pulldown using yeast cell extracts Many coregulators first identified in yeast have been found to have mammalian homologues that can interact with steroid receptors and other transcription factors. The GST-pulldown was also used with S-labeled yeast cell extracts to identify any yeast proteins that could interact with A R . The yeast cell extract was precleared, then divided and run through columns containing G S T alone (lane 1), GST-AR5 24-648 (lane 2), and G S T - A R 2 3 3 . 6 4 8 (lane 3). A protein of about 70 kDa was observed to bind specifically to the column containing GST-AR 233-648 (Fig. 14, lane 3). In addition, two protein bands of slightly lower molecular masses were unique to the eluate from the GST-AR233-648 column (Fig. 14, lane 3). There 61 kDa 1 2 3 protein kDa 1 2 3 Loss of 70 kDa protein F i g . 13 GST-AR pulldown of a 70 kDa protein in LNCaP cells. 3 5 S- labe l l ed L N C a P nuclear extracts were precleared and incubated wi th immob i l i zed G S T - A R . E lu ted proteins were v isua l ized by autoradiography. A . Lane 1, eluate f rom G S T co lumn; lane 2, eluate f rom G S T - A R 5 2 4 - 6 4 8 co lumn; lane 3, eluate f rom GST- A R 2 3 3 . 648 co lumn. B . Lane 1, unbound material f rom G S T co lumn; lane 2, unbound material f rom G S T - A R 5 2 4 . 6 4 8 co lumn; lane 3, unbound material f rom G S T - A R 2 3 3 . 6 4 8 co lumn. 62 kDa 1 2 3 70 kDa protein Fig. 14 GST-AR pulldown of a 70 kDa protein in yeast cells. S-labelled yeast cell extracts were precleared and incubated with immobilized GST-AR. Eluted proteins were visualized by autoradiography. Lane 1, eluate from GST column; lane 2, eluate from GST-AR 5 24-648 column; lane 3, eluate from GST-AR 2 3 3. 6 48 column. were no corresponding protein bands for any of these three proteins in the eluate from the G S T or GST-AR 5 24-648 columns (Fig. 14, lanes 1 and 2). This suggested that all of the three proteins could interact with the A R N-terminal domain (amino acids 232 to 524). The two smaller, fainter bands could represent degradation products or modified forms of the 70 kDa protein, or they could represent unique proteins that interacted with the N-terminal domain of A R . Although members of the heat shock protein family are known to interact with the L B D rather than the N-terminal domain of the A R , we wanted to ensure that this yeast 70 kDa protein was not a member of the heat shock protein family. Western blotting with an antibody against yeast hsp70/hsc70, following GST-pul ldown of unlabeled yeast cell extract, showed that the forms of hsp70/hsc70 recognized by this antibody were present in the initial protein extract and the precleared extract (Fig. 15, lanes 1 and 2). However, no evidence of hsp70/hsc70 was seen in the eluate from the columns containing G S T , GST-AR524-648, and GST-AR 233-648 (Fig. 15, lanes 3 -5) . Unfortunately, the supplier of the antibody could not verify which members of the multiple yeast hsp70/hsc70 families the antibody recognized. Therefore, the only conclusion that could be reached is that the 70 kDa protein that bound to the N-terminal domain of the A R does not belong to any of the yeast hsp70/hsc70 recognized by this antibody. 3.2.3 Co-immunoprecipitation using yeast cell extracts A n alternative to the GST-pul ldown approach for identifying proteins that interact with the A R is co-immunoprecipitation, or the use of antibodies that bind to and precipitate the A R and any proteins complexed with it. Yeast transformed with plasmids expressing Gal4 D B D - A R fusion proteins were used in these experiments. A plasmid expressing only F i g . 15 GST-AR . 524-648 and GST-AR233 .648 did not pulldown some forms ofhsc70. Precleared extracts f rom yeast cel ls were incubated wi th glutathione-sepharose wi th immob i l i zed G S T - A R . E lu ted proteins were assayed by Western blott ing using an antibody to yeast hsc70. Lane 1, whole nuclear extract; lane 2, precleared nuclear extract; lane 3, eluate f rom G S T alone; lane 4, eluate f rom G S T - A R 5 2 4 - 6 4 8 ' lane 5, eluate f rom G S T - A R 2 3 3 . 6 4 8 . Gal4 D B D served as a control for plasmids that contained the first 232 amino acids of human A R or the entire N-terminal domain of A R (amino acids 1 to 559) fused to the Gal4 D B D . The S-labeled yeast protein extracts were precleared for binding to protein G-Sepharose beads, then incubated with an antibody to the Gal4 D B D and precipitated with protein G-Sepharose. The immunoprecipitated proteins were eluted with S D S , separated by S D S - P A G E , and examined by fluorography. A high level of background binding to the protein G-Sepharose beads was present, even after preclearing of the S-labeled protein extract (Fig. 16, lane 1). A 45 kDa yeast protein was co-immunoprecipitated with the Gal4 DBD-AR1.559 and this protein was not seen in the absence of antibody to the Gal4 D B D (Fig. 16). This 45 kDa protein may represent a yeast protein that binds specifically to the N-terminal domain of A R (amino acids 1 to 559). A protein of about 55 kDa in mass was co-immunoprecipitated with Gal4 DBD-AR! .232 and with Gal4 D B D - A R ! . 5 5 9 (Fig. 17, lane 2 and 3). However, this 55 kDa yeast protein did not appear to be co-immunoprecipitated with the Gal4 D B D (Fig. 17, lane 1). This suggested that this yeast protein of about 55 kDa interacted with the N-terminal domain of A R , probably between amino acids 1 and 232. Co-immunoprecipitation also identified a protein about 32 k D a in mass that may have interacted with sequences within amino acids 1 to 232 of A R . This 32 kDa yeast protein was co-immunoprecipitated specifically with the Gal4 DBD-AR1.232 (Fig. 18, lane 2). A protein of similar size was not co-immunoprecipitated with the Gal4 D B D , suggesting that this interaction is specific for A R (Fig. 18, lane 1). However, the 32 kDa protein was not seen to interact with the Gal4 DBD-AR1.559 (Fig. 18, lane 3). This may suggest that differences in conformation between the Gal 4 D B D - A R fusion proteins create modified interaction 66 kDa 1 2 F i g . 16 Co-immunoprecipitation of a 45 kDa yeast protein with GAL4 DBD-AR^559. S- label led yeast extract was precleared, incubated wi th A b to G A L 4 D B D , precipitated wi th protein G-sepharose, and extracted wi th S D S . Lane 1, no A b control ; lane 2, w i th A b to G A L 4 D B D . kDa 1 2 3 F i g . 17 Co-immunoprecipitation of a 55 kDa yeast protein with GAL4 DBD-ARj.232 and GAL4 DBD-AR}.559 . 3 5 S- labe l l ed yeast extract was precleared, incubated wi th A b to G A L 4 D B D , precipitated wi th protein G-sepharose, and extracted w i th S D S . Lane 1, yeast expressing Ga l4 D B D , lane 2, yeast expressing G A L 4 D B D - A R j . 2 3 2 , lane 3, yeast expressing G A L 4 D B D - A R j . s kDa 1 2 3 <-32 kDa protein F i g . 18 Co-immunoprecipitation of a 32 kDa yeast protein with GAL4 DBD-ARj.232- 3 5 S- labe l l ed yeast extract was precleared, incubated w i th A b to G A L 4 D B D , precipitated wi th protein G -sepharose, and extracted wi th S D S . Lane 1, yeast expressing Gal4 D B D , lane 2, yeast expressing G A L 4 DBD-AR1.232, lane 3, yeast expressing G A L 4 D B D - A R i . 5 5 9 . 69 surfaces on A R . The interaction of the 32 kDa yeast protein with the Gal4 DBD-AR4.232 but not the Gal4 DBD-AR1 .559 could be due to the formation of additional interaction surfaces on the Gal4 DBD-AR1 .232, or due to the loss of native interaction surfaces on the Gal4 DBD-ARi_559. Unfortunately, no definitive conclusion can be reached from this data as to the reason that this 32 kDa yeast protein interacted with the Gal4 DBD-AR1 .232 but not the Gal4 D B D - A R i _ 5 5 9 3.3 Yeast 2-hybrid Assay 3.3.1 Sequencing and analysis of positive clones The yeast 2-hybrid system was used to detect proteins that interacted with amino acids 1 to 232 of A R . Larger forms of the N-terminal domain were not used since those fragments had intrinsic transcriptional activity and were not compatible with the yeast 2-hybrid system. Three positive clones, A R B P 1 , A R B P 2 , and A R B P 3 , were detected that could interact specifically with the bait Gal4 DBD-AR1-232 to activate transcription of reporter genes. Sequencing of these clones showed that they all contained inserts about 2.9 kb in size and had virtually identical sequences. Comparison of the sequence of this clone, simply called A R B P 1 , with sequences in GenBank identified a known human gene with 100% homology to the 5' portion of A R B P 1 sequenced (Fig. 19A) and 96% homology with the 3' sequenced portion of A R B P 1 (Fig. 19B). This gene is named R a n B P M as it was originally identified through yeast 2-hybrid screening for proteins that could interact with the human protein Ran. A similarity of 95% was seen to the mouse B-cel l receptor Ig-(3 associated protein ( IBAP1) was also seen. The size of the complete c D N A for R a n B P M was originally established by others to be 2.3 kb. Sequencing of the first 830 nucleotides of 70 A. C l o n e : c a g c g g c g g c t g a a g c g t c t c t a c c c g g c c g t g g a c g a a c a a g a g a c g c c g c t g c c t c g g I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I R a n B P M : 1 9 0 c a g c g g c g g c t g a a g c g t c t c t a c c c g g c c g t g g a c g a a c a a g a g a c g c c g c t g c c t c g g 2 4 9 C l o n e : t c c t g g a g c c c g a a g g a c a a g t t c a g c t a c a t c g g c c t c t c t c a g a a c a a c c t g c g g g t g I III! 111 11 11 1111 111111III 11 1111111111111111 111 111 111 1111 1111 R a n B P M : 2 5 0 t c c t g g a g c c c g a a g g a c a a g t t c a g c t a c a t c g g c c t c t c t c a g a a c a a c c t g c g g g t g 3 0 9 C l o n e : c a c t a c a a a g g t c a t g g c a a a a c c c c a a a a g a t g c c g c g t c a g t t c g a g c c a c g c a t c c a I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I R a n B P M : 3 1 0 c a c t a c a a a g g t c a t g g c a a a a c c c c a a a a g a t g c c g c g t c a g t t c g a g c c a c g c a t c c a 3 6 9 C l o n e : a t a c c a g c a g c c t g t g g g a t t t a t t a t t t t g a a g t a a a a a t t g t c a g t a a g g g a a g a g a I II I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I R a n B P M : 3 7 0 a t a c c a g c a g c c t g t g g g a t t t a t t a t t t t g a a g t a a a a a t t g t c a g t a a g g g a a g a g a 4 2 8 C l o n e : t g g t t a c A T G g g a a t t g g t c t t t c t g c t c a a g g t g t g a a c a t g a a t a g a c t a c c a g g t n I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I R a n B P M : 4 2 9 t g g t t a c A T G g g a a t t g g t c t t t c t g c t c a a g g t g t g a a c a t g a a t a g a c t a c c a g g t t 4 8 7 C l o n e : g n g a t a a g c a t t c a t a t g g t t a c c a t g g g g a t g a I I I I II II I I I I I I I I I I I I I I I I I I I I I I I I I R a n B P M : 4 8 8 g g g a t a a g c a t t c a t a t g g t t a c c a t g g g g a t g a 5 2 1 C l o n e : t g g a c a t t c g t t t t g t t c t t c t g g a a c t g g a c a a c c t t a t g g a c c a a c t t t c a c t a c t g g II 11111 1111111II11111 111111II11111111111111 I!I 111 1111 111 1111 R a n B P M : 5 2 2 t g g a c a t t c g t t t t g t t c t t c t g g a a c t g g a c a a c c t t a t g g a c c a a c t t t c a c t a c t g g 5 8 1 C l o n e : t g a t g t c a t t g g c t g t t g t g t t a a t c t t a t c a a c a a t a c c t g c t t t t a c a c c a a g a a t g g I I I I I I I I I II I I II II II I I I I I I I I I II I I II I I I I I I I I I I I I II I I I I I I I I I I I I R a n B P M : 5 8 2 t g a t g t c a t t g g c t g t t g t g t t a a t c t t a t c a a c a a t a c c t g c t t t t a c a c c a a g a a t g g 6 4 1 C l o n e : a c a t a g t t t a g g t a t t g c t t t c a c t g a c c t a c c g c c a a a t t t g t a t c c t a c t g t g g g g c t I I I I II I I I II I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I R a n B P M : 6 4 2 a c a t a g t t t a g g t a t t g c t t t c a c t g a c c t a c c g c c a a a t t t g t a t c c t a c t g t g g g g c t 7 0 1 C l o n e : t c a a a c a c c a g g a g a a g t g g t c g a t g c c a a t t t t g g g c a a c a t c c t t t c g t g t t t g a t a t MIIIIIIIIIIIIIIIIIIIIIMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIMI R a n B P M : 7 0 2 t c a a a c a c c a g g a g a a g t g g t c g a t g c c a a t t t t g g g c a a c a t c c t t t c g t g t t t g a t a t 7 6 1 C l o n e : a g a a g a c t a t a t g c g g g a g t g g a g a a c c a a a a t c c a g g c a c a g a t a g a t c g a t t t c c t a t 111111111IIII11II111111111 Ll 111111111111111111111111II111111 R a n B P M : 7 6 2 a g a a g a c t a t a t g c g g g a g t g g a g a a c c a a a a t c c a g g c a c a g a t a g a t c g a t t t c c t a t 8 2 1 C l o n e : c g g a g a t c g a g a a g g a g a a t g g c a g a c c a t g a t a c a a a a a a t g g t t t c a t c t t a t t t a g t 1111111111111111II111111111II111111111111111111111111! 111111 R a n B P M : 8 2 2 c g g a g a t c g a g a a g g a g a a t g g c a g a c c a t g a t a c a a a a a a t g g t t t c a t c t t a t t t a g t 8 8 1 C l o n e : c c a c c a t g g g t a c t g t g c c a c a g c a g a g g c c t t t g c c a g a t c t a c a g a c c a g a c c g t t c t I I I I II II I I I I II I I I I I I II II I I I I I I I II I I I I II II I I I II I I I I I I I I I I I II I R a n B P M : 8 8 2 c c a c c a t g g g t a c t g t g c c a c a g c a g a g g c c t t t g c c a g a t c t a c a g a c c a g a c c g t t c t 9 4 1 C l o n e : a g a a g a a t t a g c t t c c a t t a a g a a t a g a c a a a g a a t t c a g a a a t t g g t a t t a g c a g g a a g I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I II I I I I I I R a n B P M : 9 4 2 a g a a g a a t t a g c t t c c a t t a a g a a t a g a c a a a g a a t t c a g a a a t t g g t a t t a g c a g g a a g 1 0 0 1 C l o n e : a a t g g g a g a a g c c a t t g a a II I I I I I I I I I I I I I I I I I R a n B P M : 1 0 0 2 a a t g g g a g a a g c c a t t g a a 1 0 2 0 71 B. C l o n e : t g g a t t t t c t t - g g t t t t t a g g a c g - a a g a a a c - - c c c n t c c c c c c t c t c t c c c c t t c c t 1111111 1111 1111111111111 I Ml I II111 1111111 111 111II R a n B P M : 2 2 1 7 t g g a t t t t c t t t g g t t t t t a g g a c g g a a g a a a c a c c c c c t c c c c c c t c t c t c c c c t t c c t 2 2 7 6 C l o n e : t t a c c t t g t t c a a c a c a a a c a c a a t c g t a c a c c c a c t a c a t t t g a a g t t t a a c c c t c t a c 11111 111111 M 11111111 1111111111II111111 11 111 111 111 I 111 111 11 I R a n B P M : 2 2 7 7 t t a c c t t g t t c a a c a c a a a c a c a a t c g t a c a c c c a c t a c a t t t g a a g t t t a a c c c t c t a c 2 3 3 7 C l o n e : a a g g c t g g g g I I I I I I I I I I R a n B P M : 2 3 3 8 a a g g c t g g g g 2 3 4 6 c. C l o n e : 1 Q R R L K R L Y P A V D E Q E T P L P R S W S P K D K F S Y I G L S Q N N L R V H Y K G H G K T P P K D A A S V R A T H 57 C l o n e : 58 P I P A A C G I Y Y F E V K I V S K G R D G Y 82 C l o n e : 83 M G I G L S A Q G V N M N R L P G X D K H S Y G Y H G D D G H S F C S S G T G Q P Y G P T F T T G D V I G C C 1 3 7 R a n B P M : 1 M G I G L S A Q G V N M N R L P G W D K H S Y G Y H G D D G H S F C S S G T G Q P Y G P T F T T G D V I G C C 55 C l o n e : 1 3 8 V N L I N N T C F Y T K N G H S L G I A F T D L P P N L Y P T V G L Q T P G E W D A N F G Q H P F V F D I E D Y M R E 1 9 7 R a n B P M : 56 V N L I N N T C F Y T K N G H S L G I A F T D L P P N L Y P T V G L Q T P G E W D A N F G Q H P F V F D I E D Y M R E 1 1 5 C l o n e : 1 9 8 W R T K I Q A Q I D R F P I G D R E G E W Q T M I Q K M V S S Y L V H H G Y C A T A E A F A R S T D Q T V L E E L A S I 2 5 7 R a n B P M : 1 1 6 W R T K I Q A Q I D R F P I G D R E G E W Q T M I Q K M V S S Y L V H H G Y C A T A E A F A R S T D Q T V L E E L A S I 1 7 5 C l o n e : 2 5 8 K N R Q R I Q K L V L A G R M G E A I E 2 7 7 R a n B P M : 1 7 6 K N R Q R I Q K L V L A G R M G E A I E 1 9 5 Fig. 19 Yeast 2-hybrid clone is homologous to human gene, RanBPM. A clone from a human prostate c D N A library was found to express a protein that interacted with the human A R (amino acids 1 to 232) in the yeast 2-hybrid system. The D N A sequence of the clone indicates that it is identical to a known human gene, R a n B P M . A . sequence from the 5' end of library clone matched to 5' untranslated and coding regions of R a n B P M (start codon A T G indicated in bold capital letters); B. sequence from 3' end of library clone matched to 3' untranslated region of R a n B P M . ; C . translated sequence of clone, including coding region matched to R a n B P M plus the 5' untranslated region 72 A R B P 1 demonstrated 100% homology with nucleotides 1 9 0 - 1020 of R a n B P M (Fig. 19 A) . When A R B P 1 was sequenced from the 3' end, a 96%> homology was found with the last 129 nucleotides of known R a n B P M sequence (nucleotides 2217 - 2346) (Fig. 19B). A R B P 1 contained portions of the 5' and 3' untranslated regions of R a n B P M , as wel l as a translation start codon (Fig. 19A). A n open reading frame existed for the 830 nucleotides of A R B P 1 that were sequenced from the 5' end, including the 5' untranslated region, and the c D N A was inserted in the correct frame to be expressed as a fusion protein with the G a l 4 A D (Fig. 19C). Western blotting using a rabbit polyclonal antibody to a hemagglutinin (HA) tag linked to the Gal4 A D fusion protein identified a protein of about 90 kDa being expressed from the library plasmid (Fig. 20). The mass of the Gal4 A D and H A portions of the protein was predicted to be close to 20 kDa, suggesting that the mass of the expressed insert was about 70 kDa. The insert in A R B P 1 contained the sequence coding for R a n B P M as wel l as a portion of the untranslated region, which was in frame with the fusion protein and contained no stop codons. It was predicted that a protein of about 66 kDa would be expressed, as R a n B P M consisted of 500 amino acids and the untranslated region coded for about 100 additional amino acids. The observed mass of about 70 kDa is close to the predicted mass of 66 kDa, implying that the entire R a n B P M sequence as wel l as the untranslated region was being expressed. A radiolabeled c D N A probe produced from the A R B P 1 yeast 2-hybrid clone was hybridized to a Mult iple Tissue Expression Array to determine the relative level of expression of this gene in different human tissues. The M T E Array contained m R N A from 76 different human tissues as well as several negative controls spotted in a grid pattern on a nylon membrane (Fig. 2 IB) . Although this gene appeared to be expressed in all the human 73 kDa 1 2 97.4 — 66 — 46 — 3 0 — Fig. 20 RanBPM/ARBPl is expressed in yeast cells transformed with the library plasmid. Protein extracts were prepared from Y190 yeast cells transformed with the ARJ3P1 library plasmid. Proteins were assayed using Western blotting with an antibody against the hemagglutinin tag on the fusion protein. Lane 1, protein extract from Y190 yeast cells transformed with library plasmid; lane 2, protein extract from untransformed Y190 cells. A . B. 1 A * B C 3 4 • * D | . H # * 5 6 7 8 9 10 11 I * • # 12 1 2 3 4 5 6 7 8 9 10 11 12 A / whole brain cerebellum, left substantia nigra heart esophagus colon, transverse kidney lung liver leukemia, HL-60 fatal brain \ yeast total RNA B cerebral cortex cerebellum, right accumbens nucleus aorta stomach colon, desending skeletal muscle placenta pancreas HeLa S3 fatal heart yeast tRNA C frontal lobe corpus callosum thalamus atrium, left duodenum rectum spleen bladder adrenal gland leukemia, K-562 fetal kidney E coli rRNA D parietal lobe amygdala pituitary gland atrium, right jejunum thymus uterus thyroid gland leukemia, MOLT-4 fetal liver £ coli DNA E occipital lobe caudate nucleus spinal cord ventricle, left ileum peripheral blood leukocyte prostate salivary gland Burkitt's lymphoma, Raji fetal spleen PolyrlA) F temporal lobe hippo-campus ventricle, right ilocecum lymph node testis mammary gland Burkitt's lymphoma, Daudi fetal thymus human Cgt.tDNA G p. g.*of cerebral cortex medulla oblongata inter-ventricular septum appendix bone morrow ovary colorectal adeno-carcinoma, SW4SC fetal lung human DNA 100 ng H pons k putamen apex of the heart colon, ascending trachea lung carcinoma, AM9 human DNA 500 ng * paracentral gyrus Fig. 21 RanBPM/ARBP1 is most highly expressed in human testis tissue. The Multiple Tissue Expression Array (Clontech) was hybridized with a cDNA probe made from the positive library clone that encoded RanBPM/ARBPl. A. Autoradiograph obtained following hybridization of radiolabeled cDNA probe to M T E Array B. Diagram showing the location where the RNA from each of the tissues was spotted on the array. 75 tissues tested, the expression was highest in testis (Fig. 21A, spot F8). The gene also appeared to be expressed at an increased level in esophagus, skeletal muscle, placenta, adrenal gland, several fetal tissues, certain areas of the central nervous system, and some cancer cell lines (Fig. 21 A) . There was no hybridization of the c D N A probe to the negative controls, which included yeast R N A , bacterial R N A and D N A and human D N A (Fig. 21 A , lane 12). To confirm the results obtained with the M T E Array, a Mult iple Tissue Northern Blot was also performed. This M T N Blot was prepared using m R N A from 8 different human tissues. Hybridization of the M T N Blot with the radiolabeled c D N A probe prepared from the A R B P 1 yeast 2-hybrid clone verified that the expression of this gene was highest in the testis, as compared to other human tissues (Fig. 22, lane 4). Expression of this gene in the other 7 tissues tested was at a much lower level than in the testis (Fig. 22). The size of the band on the M T N Blot indicated that the m R N A for this gene was about 3 kb, which was consistent with the size of the c D N A library clone encoding A R B P 1 (about 2.9 kb). Although the role of this gene in vivo is not understood, the relative levels of expression suggest it may have an important function in the testis. O f the different methods used to identify novel proteins interacting with the N-terminal of A R the yeast 2-hybrid system provided the most direct information about any proteins found to bind A R . Although proteins of various sizes that appeared to interact with A R were identified using techniques such as GST-pul ldown and co-immunoprecipitation, further characterization of these proteins is necessary. 76 kb 1 2 3 4 5 6 7 8 9.5 — 7.5 — 4.4 — 2.4 — 1.35 — Fig. 22 RanBPM/ARBPl is most highly expressed in testis tissue. Hybridization of a cDNA probe for RanBPM/ARBPl to a Mulitiple Tissue Northern Blot (Clontech) showed the relative level of expression for this gene in several different human tissues. After hybridization with the radiolabeled cDNA probe the M T N Blot was exposed to a phosphorimaging screen for analysis. M R N A isolated from human organs is found in lane 1, peripheral blood leuckocyte; lane 2, colon (mucosal lining); lane 3, small intestine; lane 4, ovary; lane 5, testis; lane 6, prostate; lane 7, thymus; lane 8, spleen. 4. Discussion The limiting factor in the survival of prostate cancer patients is the rate of progression to androgen independence (159,160). When this occurs, AR-regulation of genes involved in prostate growth and apoptosis has become abnormal (160,161). One mechanism that could lead to abnormal AR-regulated gene transcription is changes in the expression or activity of coregulators that can modulate the ability of A R to activate transcription. Steroid receptor coregulators are thought to be particularly important for steroid receptor specific gene transcription. Unique combinations of coregulators may interact with different steroid receptors to allow specific activation of steroid-regulated gene transcription. If there are coregulators that interact with only certain steroid receptors, these interactions would likely involve amino acid sequences that are restricted to each type of steroid receptor. Coregulators that interact with the N-terminal domain of steroid receptors are the most l ikely to exhibit some degree of receptor specificity as the N-terminal domain of steroid receptors is highly variable and is largely composed of sequences unique to each receptor. To identify A R coregulators fragments of the N-terminal domain were used with several different experimental methodologies for the detection of proteins that had direct or functional interactions with A R . Testing of the receptor specificity for proteins found to interact with A R was beyond the scope of this project, but can be determined in future experiments. 4.1 GST-pu l l down assay and co-immunoprecipitat ion The GST-pul ldown is most commonly used to confirm interactions between two known proteins rather than to identify novel protein-protein interactions. Using calreticulin, 78 a protein known to interact with the D B D of A R , the ability of the GST-pul ldown assay to successfully identify a known AR-binding protein was tested. The G S T - A R fusion proteins containing the D B D were able to bind calreticulin out of a mixture o f L N C a P nuclear proteins while G S T alone could not. This demonstrates that the GST-pul ldown assay is a useful tool for testing direct protein interactions. Using the N-terminal domain of A R in GST-pul ldown assays and co-immunoprecipitations to detect direct protein interactions, proteins of several different sizes were detected that appeared to interact specifically with the N-terminal domain of A R . In the GST-pul ldown assay, the human prostate cancer cell line L N C a P was used as a source of proteins that were potential binding partners of A R . The L N C a P cell line is often used as a model for prostate cancer as the cells remain fairly well-differentiated and are responsive to androgens. Various fragments of A R were tested with L N C a P nuclear protein extracts to identify proteins that could interact with A R . Coregulators are generally expected to be found in the nuclear compartment of the cell , as they interact with transcription factors that act by binding to D N A in the nucleus. A protein with.a molecular mass of about 70 kDa located in the nuclear compartment of L N C a P cells was found to interact with A R . This 70 kDa human protein was shown to bind specifically to a region of A R between amino acids 233 and 524. A protein of approximately 40 kDa present in L N C a P nuclear extracts was also shown to interact with A R . While this interaction also appeared to be specific for the N-terminal region of A R (amino acids 233 to 524), the site of this interaction may extend to the DNA-b ind ing domain (amino acids 524 to 648). Due to the nature of the GST-pul ldown assay, large amounts of unlabeled G S T - A R fusion proteins were present in the column eluate along with any proteins interacting with the A R . As the GST-AR524-648 fusion protein had a 79 molecular weight close to that of the protein band of interest, the presence of the fusion protein in the column eluate may have caused a shift in the position of the 40 kDa protein on the gel. Although it cannot be definitively concluded that this 40 kDa human protein interacted with the N-terminal domain of A R , the protein was shown to interact specifically with either the N-terminal domain or the D B D of A R and did not bind to GST. The ability of these proteins to interact with other steroid receptors was not tested. Yeast protein extracts were also tested with the GST-pul ldown assay to identify yeast proteins that could bind to the N-terminal domain of A R . Many coregulators that are known to interact with steroid receptors were originally identified in yeast or have yeast homologues (162). A yeast protein of about 70 kDa was identified that interacted specifically with the N-terminal domain of A R (amino acids 232-524). Two slightly smaller proteins also appeared to interact with the A R N-terminal domain and not with the A R D B D or with G S T alone. It is unknown whether these additional proteins represented degradation products or modified forms of the 70 kDa protein or i f they also represented novel proteins that interacted with A R . It is interesting to note that a protein of about 70 k D a was identified using both yeast and L N C a P protein extracts. Unfortunately, the nature of the G S T -pulldown assay does not allow any conclusions to be made about the identity of proteins detected that interact with A R . Therefore, further study would be required to determine i f the 70 kDa yeast and human proteins had any homology. Neither of the 70 kDa proteins are l ikely to be members of the heat shock protein family as other heat shock proteins are known to interact with the L B D of steroid receptors while these proteins interacted specifically with the N-terminal domain (163,164). A s the heat shock proteins interact with a variety of different steroid receptors, it. is unlikely that the interactions would involve the relatively 80 unique sequences found within the N-terminal domain of steroid receptors. Mult iple forms of hsp70/hsc70 are found in yeast and antibodies against hsp70/hsc70 do not recognize all of these different yeast forms. Although some forms of hsp70/hsc70 were shown to be present in the yeast protein extracts and that those forms did not bind to G S T - A R fusion proteins containing the N-terminal domain or D B D , it was not demonstrated conclusively that this 70 kDa protein was not a member of the heat shock protein family. Co-immunoprecipitations of extracts from yeast cells transformed with plasmids expressing fragments of A R as fusion proteins with the Gal4 D B D showed that proteins of several different sizes appeared to be interacting with the N-terminal domain of A R . A yeast protein of about 55 kDa was demonstrated to interact with the A R N-terminal domain. The site on the A R where this 55 kDa protein binds was probably between amino acids 1 and 232 as it was co-immunoprecipitated with A R fragments that contained the entire N-terminal domain as wel l as those composed of only the first 232 amino acids of the N-terminal domain. Another protein of about 45 kDa was co-immunoprecipitated with a protein containing the entire N-terminal domain of A R and may have interacted at a site between amino acids 1 and 559 of A R . Co-immunoprecipitations of yeast extracts with the protein containing the first 232 amino acids of A R were not done in this experiment, so the site of this interaction cannot be further delineated. A protein of about 32 k D a present in yeast extracts may also interact with the N-terminal domain of A R . However, this interaction was only observed with a fragment encompassing the first 232 amino acids of the N-terminal domain. No protein of a similar size was co-immunoprecipitated with the entire A R N-terminal domain. One explanation of these conflicting results could be that there were differences in the conformation of the two fusion proteins being expressed in yeast. 81 Unfortunately, it cannot be determined whether the interaction of a 32 k D a yeast protein with the fusion protein containing the first 232 amino acids of the N-terminal domain was enabled by incorrect folding of the fusion protein or i f a non-native conformation of the protein containing the entire N-terminal domain resulted in the loss of binding to the 32 kDa yeast protein. Although the use of both the GST-pul ldown assay and co-immunoprecipitation allowed us to detect direct protein interactions between the N-terminal domain of A R and protein bands of several different sizes present in yeast and L N C a P extracts, any further analysis of the protein bands was complicated by the high level of non-specific binding seen when using these techniques. Non-specific binding of proteins was extensive, making it difficult to obtain adequate separation of the proteins on a gel to produce a pure protein band for sequencing. Preclearing of the protein extracts for binding to G S T columns or protein G/A-Sepharose beads reduced the level of background somewhat but was not able to sufficiently eliminate the background when trying to identify protein interactions by either the GST-pul ldown assay or co-immunoprecipitation. To obtain any more information about the protein bands identified using these methods, further purification of the proteins using other techniques, such as 2-dimensional electrophoresis or chromatography, would be required. Sequencing of these protein bands was also hindered by the fact that only small quantities of these radioactively-labeled proteins were present. Whi le these approaches produced results more quickly than some other techniques currently available for identifying protein-protein interactions, they were less reliable and provided little information about the proteins identified. Although these systems could be used to detect novel yeast and human proteins that interacted with the N-terminal domain of A R , no information about the identity 82 or function of the proteins could be obtained without much additional purification of the proteins. However, this research has shown that these systems for detecting direct protein interactions can be useful for confirming interactions between two known proteins. These methods have been established and can be used to test proteins that are suspected to interact with A R , such as any clones identified using the yeast 2-hybrid system. 4.2 Yeast 2 -Hybr id System The yeast 2-hybrid system is a very sensitive method for identifying novel proteins that interact with A R . Unfortunately, its use with the N-terminal domain of A R is limited to studying interactions with a small fragment at the extreme amino-terminus of the receptor. Fragments of the N-terminal domain that contained more than the first 232 amino acids had the intrinsic ability to activate transcription and were not suitable for use with the yeast 2-hybrid system. The commercial human prostate c D N A library was tested using amino acids 1 to 232 of A R to identify any proteins that were interacting specifically with the bait. While this method required a large investment of time before any results were obtained, the results were informative and further analysis of the positive clone could begin immediately. Once a positive clone was identified, the sequence could be easily obtained and compared to known gene sequences available in GenBank. The c D N A was also already in place in a vector that could be used to express the c D N A in yeast cells for use in further experiments. Three positive clones called A R B P 1 , A R B P 2 , and A R B P 3 were identified that interacted specifically with the bait plasmid that expressed Gal4 DBD-AR1.232. A R B P 1 , A R B P 2 , and A R B P 3 were sequenced and found to contain the same insert that had extremely high homology to a known human gene, R a n B P M . Since these three clones were virtually 8 3 identical they were simply referred to as A R B P 1 . Through comparison with the sequence of R a n B P M , the A R B P 1 insert was established to contain portions of both 5' and 3' untranslated regions and was slightly larger than the size of the complete c D N A for R a n B P M . The insert was in the correct reading frame to be expressed as a fusion protein with the Gal4 A D and an open reading frame existed throughout the 5' untranslated region that continued in frame from the start codon. A R B P 1 was also found to have very high homology to a mouse gene encoding B-cel l antigen receptor Ig-[3 associated protein-1 ( IBAP1). The mouse IB A P I protein has 97% homology to the R a n B P M protein over the entire R a n B P M sequence, however IBAP1 is thought to be composed of 653 amino acids while R a n B P M only contains 500 amino acids. The IBAP1 protein sequence has an N-terminal extension of 153 amino acids that is not seen in the R a n B P M protein sequence. This can be explained by the presence of an alternative start codon in the mouse IB A P I coding sequence found just upstream of the region that corresponds to the portion of R a n B P M thought to encode 5' untranslated region. A s the D N A sequence of the R a n B P M 5' untranslated region corresponds to the sequence coding for the N-terminal extension on I B A P 1 , there may be an additional start codon just upstream of the known sequence for R a n B P M . A R B P 1 begins at a point corresponding to amino acid 72 of the IB A P I N-terminal extension and the predicted amino acid sequence matches exactly throughout the remainder o f this region. Once the site thought to be the translation start site for the R a n B P M protein is reached, an almost perfect sequence match between A R B P 1 , R a n B P M , and IB A P I is observed, suggesting that these proteins are all closely related. There may be another human protein called K B 0 7 that is also related to R a n B P M and I B A P 1 . There is only a partial c D N A available for this gene but it appears to have very high homology to two 84 different regions that are separated by several hundred nucleotides in R a n B P M and IB A P I but that are linked directly in K B 0 7 . This could indicate that there are alternatively spliced forms of R a n B P M and IB A P I or that there is another gene that is also very closely related to the genes encoding these two proteins. Unfortunately, little is known about the functional roles of IB A P I , K B 0 7 or R a n B P M that might suggest the biological significance of the interaction between A R and A R B P 1 . IB A P I was identified as a mouse protein associated with the B-cel l antigen receptor Ig-P while K B 0 7 is only identified in GenBank as a novel gene from a human dendritic cell. A yeast 2-hybrid screen for proteins that interacted with the human G T P A s e Ran led to the discovery of R a n B P M using; a c D N A library made from Epstein-Barr virus transformed peripheral B-cells. In that study, 81 positive clones were obtained from the yeast 2-hybrid screen and 38 of them contained inserts corresponding to R a n B P M (165). Ran is a small Ras-like GTPase that shuttles between the nucleus and cytoplasm and is essential in transporting many proteins and other macromolecules in and out of the nucleus (166-168). Ran may even be involved in the nuclear translocation of steroid receptors, as mutations in Ran led to a dramatic reduction in the accumulation of G R in the nucleus after the addition of ligand (169). Ran is also thought to have roles in cell cycle regulation and ribosomal R N A processing (170). There is a family of human Ran binding proteins that contain a conserved Ran binding domain (171). There are also several yeast homologues that appear to be functionally equivalent to the human Ran binding proteins (172). However, R a n B P M appears to be a novel Ran binding protein that does not contain the conserved Ran binding domain and interacts specifically with GTP-bound Ran (173). R a n B P M was named Ran binding protein in the microtubule organization center ( M T O C ) as it was found to be 85 localized within the centrosome (173). The M T O C initiates microtubule assembly and anchors the growing microtubules (174). A n antibody against R a n B P M detected proteins of two different sizes in some cell lines while other cell lines seemed to only express one form of R a n B P M (173). There are several explanations that could explain the presence of two different forms of R a n B P M including the existence of alternate translation start sites, alternative splicing of R a n B P M transcripts, or post-translational modification of the R a n B P M protein. R a n B P M was demonstrated to co-localize with y-tubulin, a centrosomal protein involved in microtubule nucleation (175). The overexpression of R a n B P M caused reorganization of the microtubule network, with abnormal sites of microtubule nucleation observed (173). The same effect was also seen with overexpression of y-tubulin and the co-localization of R a n B P M with y-tubulin may suggest a role for R a n B P M in recruiting or activating y-tubulin (176). Although it is unknown what effect R a n B P M / A R B P 1 might have on A R in vivo, the information that is available about R a n B P M might suggest some possible roles. As Ran is known to be important in the transport of ligand-bound G R into the nucleus it may also be involved in the nuclear translocation of A R . R a n B P M might act as a bridging factor between A R and Ran for Ran-mediated transport to the nucleus. The involvement of R a n B P M in nuclear structure suggests that R a n B P M could also act as a bridging protein between A R and the nuclear matrix (62). The activity of R a n B P M in the centrosome could provide a means for A R to effect mitotic processes in the cell. R a n B P M may even bind A R in the nucleus and act to sequester A R , preventing AR-mediated activation of transcription. The identification of R a n B P M as an AR-interacting protein provides an excellent focus for continued research and an opportunity to increase our understanding of A R function. 86 When the protein sequence of A R B P 1 was used in a search to find homology to other known proteins there were several other interesting findings. A yeast protein of unknown function was identified that has has greater than 40% identity to A R B P 1 over the amino-terminal half of the protein. There was similarity between A R B P 1 and about 25 different ryanodine receptor sequences from a wide variety of species ranging from human to Drosophila and C. elegans. A small region of these receptors, which varied from about 160 to 280 amino acids in size, had 30-40% identity to the N-terminal portion o f A R B P 1 . Homology to members of the D E A D - b o x protein family was also found, again ranging from human to Drosophila and C. elegans. A region of about 130 amino acids showed 30% amino acid identity between the A R B P 1 and the D E A D - b o x proteins. The portion of ARJ3P1 that showed homology to the D E A D - b o x proteins began upstream of the putative translation start site of R a n B P M . It is not clear i f the simlarities between A R B P 1 and other protein families has any bearing on the functional role of A R B P 1 . Future studies on the biological role of A R B P 1 may demonstrate some functional similarities. The D E A D - b o x protein family members are RNA-b ind ing proteins with ATPase activity that can affect R N A conformation in vitro and may be important in the assembly of the spliceosome (177,178). It is interesting to note that A R has previously been found to associate with a ribonucleoprotein molecule or molecules, although no information is available on the potential role of this association (179). Ryanodine receptors are intracellular calcium ion release channels that contain a conserved S P R Y domain of unknown function that appears to be the source of the homology to R a n B P M (173,180). The ryanodine receptors play a crucial role in calcium ion-mediated signaling and are involved in diverse signaling pathways that may include programmed cell 87 death (181,182). Changes in intracellular calcium ion levels can also affect the levels of A R m R N A and protein in L N C a P cells, with an increased level of calcium ions associated with a decrease in A R expression (183). When additional information about the function of A R B P 1 becomes available, it w i l l be possible to determine i f the sequence similarities to the D E A D -box proteins and ryanodine receptors have any functional relevance. Whi le the original study on R a n B P M did not examine the expression of this gene in various tissues, it has now been shown that A R B P 1 is expressed at some level in all human tissues tested but is most highly expressed in testis. Having identified A R B P 1 as an A R -interacting protein, it is very interesting to find high expression of this gene in testis. Finding the highest level of expression of A R B P 1 in an androgen-responsive tissue may indicate that the biological role of this protein is more dependent on its ability to interact with A R rather than the induction of microtubule nucleation. However, until further research is done to test the effect that the interaction with A R B P 1 has on A R activity, no conclusions can be made about its importance in A R function. 4.3 Future Directions of Research To elucidate the role of the A R B P 1 protein in vivo there are many additional experiments that must be performed. Co-immunoprecipitations can be done to confirm by an independent biochemical method the interaction of A R B P 1 with the N-terminal domain of A R seen with the yeast 2-hybrid assay. The interaction of A R B P 1 with the full-length A R can also be tested by co-immunoprecipitation to ensure that the full-length A R does not interfere with the A R B P 1 interaction. Smaller fragments of A R can also be examined by co-immunoprecipitation to determine i f the site of the interaction with A R B P 1 can be further 88 delineated. When this approach is limited by structural considerations, site-directed mutagenesis can be employed to identify A R sequences involved in the interaction (184). The ability of A R B P 1 to associate with full-length A R can be assayed by co-immunoprecipitation in the presence and absence of ligand to determine i f the interaction is ligand-independent. The receptor specificity of A R B P 1 can be tested by co-immunoprecipitations with other steroid receptors including P R , G R , and E R in the presence or absence of appropriate ligand. Co-immunoprecipitations can also be done with other members of the steroid receptor superfamily such as thyroid hormone receptor and with members of other transcription factor families. If A R B P 1 and A R are co-transfected into a variety of human cell lines, including androgen-responsive and androgen-independent prostate cancer cell lines, any effect of A R B P 1 on the ability of A R to activate transcription of a reporter gene from an A R -responsive promoter can be assessed. Several different promoters could be tested in this system as the effect of A R B P 1 on the ability of A R to activate transcription may be promoter specific. Promoters from androgen-responsive genes such as P S A and probasin can be tested as well as the recombinant androgen-responsive promoter A R R 3 (35). The co-transfections can be done in the presence or absence of ligand, and the effect of anti-androgens on the interaction can be determined. Co-transfections can also be used to examine the effect of A R B P 1 on the ability of other steroid receptors to activate transcription of a reporter gene from an appropriate promoter such as M M T V for A R , G R , and P R or the E R E for E R (185). If A R B P 1 can enhance or repress transcriptional activation by steroid receptors, other coactivators such as S R C or C B P can be added to determine i f there are any synergistic or inhibitory effects. 89 If no effects on transcription activation by steroid receptors are seen then A R B P 1 may have a function other than that of a coregulator. One approach to identify the role of A R B P 1 is to use antibodies to A R and A R B P 1 to examine cellular localization and possible co-localization of the two proteins (173). Determining the location of A R B P 1 in the cell may imply possible mechanisms of A R B P 1 action in the cell. Overexpression of A R B P 1 in different cell lines may allow the identification of phenotypic changes in cellular processes such as nuclear translocation of A R , attachment of A R to the nuclear matrix, microtubule organization, or intracellular calcium ion levels (173). Using antisense oligonucleotides to inhibit the production of A R B P 1 protein in cell lines may also lead to cellular changes that provide clues to the possible functions of A R B P 1 (186). The expression of A R B P 1 can be examined in various cancer cell lines and tumours to reveal i f changes in A R B P 1 expression might be associated with certain types of cancers. Changes in expression can also be examined in relation to the progression of prostate cancer using expression arrays created from the L N C a P tumour model. These arrays can identify changes in expression between the initial androgen-sensitive growth of the prostate tumour, the regression of the tumour upon androgen withdrawal, and the recurrence of the androgen-independent tumour. Expression arrays can also be used to study the effects of overexpression of A R B P 1 on global gene transcription. This technique can also be applied when the expression of A R B P 1 is inhibited by antisense oligonucleotides. The protein bands identified using the GST-pul ldown assay and co-immunoprecipitation may also be further characterized in future experiments. The eluted proteins may be further separated using two-dimensional electrophoresis or size exclusion chromatography to allow the isolation of a pure protein band. It w i l l be difficult to obtain 90 large enough amounts of the L N C a P proteins identified by GST-pul ldown for sequencing but the yeast proteins identified by co-immunoprecipitation might be sequenced by mass spectrometry. Using this method, proteins can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry (187). A s the yeast genome has been sequenced, the analysis of the mass spectrometric sequencing data can be easily done through database searching. If the sequence of any of these proteins can be obtained, mammalian homologues may be found and further analysis can follow in the manner described for A R B P 1 . 4.4 Conclusions The identification of proteins that interact with the N-terminal domain of A R may enhance our understanding of A R function in normal and neoplastic cells. Methods that identify direct protein-protein interactions such as the GST-pul ldown and co-immunoprecipitation are more useful for confirming interactions between two known proteins than for identifying novel proteins. The yeast 2-hybrid system is another method for identifying functional interactions with the N-terminal domain of A R that provides valuable information about the protein interacting with A R . However, once the sequence of the protein is obtained much further analysis of the interaction is required. Techniques such as the GST-pul ldown and co-immunoprecipitation may be very useful in characterizing the nature of the interaction between A R and proteins identified using the yeast 2-hybrid system. Several yeast and human proteins were identified that interacted directly with the N-terminal domain of A R . Nuclear proteins from L N C a P cells of about 70 kDa and 40 kDa were shown to interact with A R using the GST-pul ldown assay. Yeast proteins of 32 kDa, 91 45 kDa, 55 kDa, and 70 kDa were also demonstrated to interact specifically with A R using either the GST-pul ldown or co-immunoprecipitations. 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