Year of Award

2006

Document Type

Thesis

Degree Type

Master of Science (MS)

Degree Name

Pharmaceutical Sciences

Department or School/College

Department of Biomedical and Pharmaceutical Sciences

Committee Chair

Darrell A. Jackson

Commitee Members

Jesse Hay, Sandy Ross

Keywords

arrestin, down-regulation, MEF, muscarinic, trafficking, ubiquitination

Abstract

Wilham, Laura E., M.S., December 2006 Pharmaceutical Sciences THE ROLE OF â–ARRESTIN IN AGONIST-INDUCED DOWN-REGULATION OF THE M1 mAChR Chair: Darrell A. Jackson G protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors. They mediate diverse cell signaling pathways and therefore are currently the target for 50% of all prescribed drugs. Cells utilize these receptors to transduce extracellular stimuli across the plasma membrane and activate intracellular G proteins, which then initiate second messenger system cascades. GPCR signaling is the focus of much research. One member of this large family of membrane receptors is the muscarinic acetylcholine receptor (mAChR). Five mAChR subtypes exist and are designated as M1-M5. The goal of this project was to determine whether â-arrestin is critical in mediating agonist-induced down-regulation of the M1 mAChR. Further, the objective of this proposal is to examine whether ubiquitination of â-arrestin is necessary for mediating agonist-induced down-regulation of the M1 mAChR. Substantial research has focused on the cytosolic protein â-arrestin for its key role in the desensitization of most GPCRs. More recently â-arrestin has been indicated in regulating other signaling within the cell. Ubiquitination of proteins, in particular â-arrestin, has been emerging as another major player for targeting receptors for not only degradation but other signaling pathways as well. This project employed mouse embryonic fibroblast (MEF) cells for investigating how â-arrestin is involved with the down-regulation of M1 mAChR. MEF cells were chosen for this study because they do not have endogenous mAChR expression. The MEF cells lacking both isoforms of â-arrestin (KO1/2) were unable to down-regulate the receptor, but single knockouts (KO1 and KO2) could still down-regulate the receptor. Constitutively ubiquitinated â-arrestin facilitated agonist-induced down-regulation. These results indicate that agonist-induced down-regulation of the M1 mAChR is â-arrestin dependent and is mediated by ubiquitination of â-arrestin.

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© Copyright 2006 Laura Elizabeth Wilham