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UBC Theses and Dissertations

Regulation of functional cell surface expression by oligomerization and N-linked glycosylation of membrane proteins Whitaker, Gina M


Oligomerization and N-linked glycosylation are processes thought to be initiated in the ER during translation and act to regulate the trafficking and functional surface expression of many ion channels and G protein-coupled receptors. HCN channels are known to form tetrameric channels from identical subunits as a prerequisite for functional cell surface expression. Different HCN subunits may also co-assemble to form heteromeric channels with unique properties. Using BRET and immunofluorescence analysis, along with electrophysiology, HCN2 and HCN4 were shown to form functional channels with current properties intermediate of those observed when either isoform is expressed. Furthermore, when expressed in equal amounts in CHO cells, HCN2 and HCN4 did not exhibit preference for homo- versus hetero-oligomerization. Many GPCRs are capable of associating as dimers or higher order oligomers. However the functional and physiological relevance of this type of interaction is not uniform for all GPCRs. The ability of both GIP and GLP-1 receptors to form oligomeric complexes was examined using BRET. The resulting saturation curves suggest that GIPR and GLP-1R are capable of forming receptor homomers and heteromers in CHO cells. The effects of N-linked glycosylation on GPCR trafficking and function are diverse and depend on the receptor studied and whether or not this receptor contains one or more consensus sites for N-glycan binding. Like many family B GPCRs, both the GIP and GLP-1 receptors possess large extracellular N-terminal domains with multiple consensus sites for N-linked glycosylation. Each of these Asn residues was shown to be glycosylated when either human receptor was expressed in CHO cells. Complete removal of N-linked glycosylation severely impaired and completely abolished functional surface expression of GLP-1R and GIPR, respectively. Furthermore, tunicamycin treatment decreased GIPR cell surface number and impaired GIP-potentiated glucose-induced insulin release in an INS-1 pancreatic beta cell line. These results highlight the importance of N-linked glycosylation in regulating the amount of GIPR or GLP-1R at the cell surface. Overall, these results expand upon the diverse roles of oligomerization and N-linked glycosylation in the regulation of membrane protein functional cell surface expression.

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