Semester

Spring

Date of Graduation

2003

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Stephen G. Graber.

Abstract

A variety of extracellular signals are transmitted into the cell interior by interactions with a superfamily of heptahelical cell surface receptors. Heterotrimeric G proteins mediate the signal transduction by coupling these receptors to intracellular effector proteins. The molecular mechanisms in G protein-receptor coupling processes are still not completely understood. In this project, two aspects of G protein-receptor coupling were examined: (1) the G protein-coupling properties of the five human muscarinic acetylcholine receptors (mAChRs); (2) a novel regulatory mechanism by a newly identified G protein signal regulator, Activator of G-protein Signaling 3 (AGS3). To study the regulation of G protein-receptor coupling, an Sf9 cell membrane-based in vitro reconstitution system was used, in which purified G protein heterotrimers were reconstituted with individually expressed membrane receptors and their coupling was assessed with radioligand binding assays.;Functional G protein coupling was successfully established for M1, M2, M4 and M5 mAChRs in urea-extracted Sf9 cell membranes. Under the same conditions, M3 mAChRs failed to couple with purified G proteins, indicating they may have a unique G protein signaling mechanism. Within the odd- or even-numbered mAChR groups similar apparent affinities for G protein interactions were observed, however, the odd-numbered mAChRs exhibited higher affinity for G protein heterotrimers than did the even-numbered mAChRs. Differences were also observed among the individual receptor subtypes in their affinity states for the agonist, Oxotremorine-M.;Studies on AGS3 revealed that cytosolic AGS3, but not membrane-associated AGS3, can interfere with receptor-Gi protein coupling. Cytosolic AGS3 can remove Gialpha subunits from the plasma membrane and sequester them in the cytosol. Each of the four AGS3 GPR (G protein regulatory) domains was able to interfere with receptor-Gi protein coupling; however, individual domains were less effective than the full-length GPR domain. None of the GPR domains distinguish among the three Gialpha subunits but they all interact more weakly with Goalpha subunits.;These studies demonstrate that five mAChRs have distinct G protein-coupling behaviors in an identical membrane environment and that AGS3 may down-regulate G protein signaling by interfering with receptor coupling. These findings contribute to the understanding of the mechanism and regulation of G protein-receptor coupling.

Download

Share

COinS