Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor(s)

Dr. Stephen S. G. Ferguson

Abstract

PDZ (PSD95/Disc Large/Zona Occludens) domain-containing proteins are scaffolding proteins that play important roles in regulating the activity of G protein-coupled receptors. Corticotropin Releasing Factor Receptor 1 (CRFR1) and Serotonin 2A Receptor (5-HT2AR) are two GPCRs that are commonly associated with mental disorders. Both receptors also contain a class I PDZ-binding motif at the carboxyl terminal tail. In the first chapter, we investigate the effects of CAL (CFTR-associated ligand) on regulating the trafficking and signaling of CRFR1. We demonstrate a role for CAL in inhibiting CRFR1 endocytosis, cell surface expression, and CRF-mediated ERK1/2 signaling via the CRFR1 PDZ-binding motif. Additionally, CAL can elicit its effects on CRFR1 by mediating the post-translational glycosylation of the nascent receptor at the Golgi apparatus. The second and third chapters focus on the MAGI (MAGUK with inverted orientation PDZ) proteins; MAGI-1, MAGI-2 and MAGI-3. We observe distinct functions for the MAGI proteins in regulating the trafficking and signaling of CRFR1 compared to that of 5-HT2AR. MAGI proteins can mediate CRFR1 endocytosis via regulating β-arrestin recruitment to the receptor. No effect is observed on the basal plasma membrane expression of CRFR1 or CRF-stimulated cAMP formation in response to overexpression or knockdown of MAGI proteins. On the other hand, MAGI proteins regulate 5-HT2AR-stimulated inositol phosphate accumulation as well as surface expression levels but do not have an effect on the internalization of the receptor. MAGI proteins can also mediate ERK1/2 signaling activated by both CRFR1 and 5-HT2AR. We propose a compensatory mechanism of regulation between MAGI-1, MAGI-2 and MAGI-3 based on their similar functional roles. Our work characterizes the interactions between two different GPCRs and four PDZ proteins, further confirming the importance of this diverse family of scaffolding proteins and providing possible targets for specific drug design.