Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Physiology

Supervisor

Stephen SG Ferguson

Abstract

The seven transmembrane-spanning G Protein-coupled Receptor (GPCR) super family is the largest family of cell-surface receptors, comprising greater than 650 members. GPCRs represent the primary targets of most therapeutic drugs. Desensitization, endocytosis and recycling are major mechanisms of receptor regulation and intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. In the present study, we examined whether multiple Rab GTPase regulate receptor trafficking through endosomal cellular compartments as a consequence of their direct association with GPCRs. We find that Rab4, Rab7 and Rab11 all bind to the last 10 amino acid residues of the angiotensin II Type 1 (AT1R) carboxyl-terminal tail. We show that the Rab GTPases compete with one another for receptor binding and that Rab4 effectively displaces Rab11 from the receptor. In contrast, Rab11 overexpression does not prevent Rab4 binding to the AT1R. Overexpression of wild-type Rab4, but not Rab11, facilitates AT1R dephosphorylation, and a constitutively active Rab4-Q67L mutant reduces AT1R desensitization and promotes AT1R resensitization. We also find that Rab8, a RabGTPase involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton and cell polarity, interacts with the carboxyl-terminal tail of metabotropic glutamate receptor 1 (mGluR1a) and attenuates mGluR1a signalling and endocytosis in a protein kinase C-dependent manner. Finally, we have examined several previously uncharacterised but naturally occurring single nucleotide polymorphisms (SNPs) in mGluR1a that have been associated with cancer that may alter mGluR1a signalling. We find that SNPs found within the ligand binding domain of mGluR1a result in both decreased cell surface expression and basal inositol 1,4,5, trisphosphate formation and bias mGluR1a signalling via the ERK1/2 pathway. Additional mGluR1a SNPs localized to the mGluR1a glutamate binding site, intracellular regulatory domains and Homer binding site also result in changes in mGluR1a subcellular localization, signalling and cell morphology. Taken together, these results indicate that GPCR signalling is significantly modulated by the association of intracellular regulatory proteins that can be influenced by receptor structure.

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