Dissertation/Thesis Abstract

Molecular analysis of M3 receptor dimerization
by McMillin, Sara Margaret, Ph.D., The George Washington University, 2011, 125; 3432930
Abstract (Summary)

G protein-coupled receptors (GPCRs) make up the largest class of cell surface receptors in mammals. Although historically considered to exist and function as monomeric units, GPCRs are now known to form dimers and/or oligomeric arrays both in vitro and in vivo. Class A receptors comprise the largest subfamily of GPCRs, and receptor dimerization/oligomerization is thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the ‘outer’ (lipidfacing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) using bioluminescence resonance energy transfer (BRET) saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the BRET data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Since class A GPCRs share a high degree of structural homology, our findings should be of broad general relevance.

Indexing (document details)
Advisor: Wess, Jurgen, Hu, Valerie W.
Commitee: Goldstein, Allan L., Jacobson, Kenneth A., Lakshman, Raj, Weinstein, Lee S.
School: The George Washington University
Department: Biomedical Sciences
School Location: United States -- District of Columbia
Source: DAI-B 72/02, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology
Keywords: Dimerization, G-protein coupled receptors, Transmembrane receptors
Publication Number: 3432930
ISBN: 9781124380919
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