Semester

Spring

Date of Graduation

2003

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Stephen Graber.

Abstract

A big diversity in heterotrimeric G proteins and their cognate receptors raises many questions about the mechanisms that regulate the specificity of signaling along G protein-mediated pathways. The major goal of this project was to understand how the selectivity of G protein-receptor coupling is achieved at the level of the G protein-receptor interface. Additional goals of this project were to assess the generality of molecular mechanisms controlling G protein-receptor coupling selectivity and to investigate how agonist may regulate G protein-receptor interactions. Molecular determinants of G protein-receptor coupling were studied by reconstitution of exogenously supplied, purified G protein heterotrimers with receptors of interest expressed in Sf9 cell membranes. The receptor coupling activity of native G proteins was compared with that of chimeric G proteins, containing chimeric Galpha subunits composed from different regions of Gi1alpha, Gqalpha and Gtalpha. We found a prominent role for the N- and C-terminus and alpha4 helix-alpha4/beta6 loop domain of Galpha for receptor coupling and speculate that the N- and C-termini cooperate to couple receptors. Data demonstrate that multiple and distinct determinants of selectivity exist for individual receptor families and suggest that receptors recognize specific patterns formed by the amino acids of Galpha on the receptor recognition surface. We also show that individual receptors distinguish themselves by the EC50 with which they interact with Gi1, indicating that G protein concentration may regulate G protein-receptor coupling. We obtained evidence that agonist concentration can regulate the selectivity of G protein-receptor coupling by demonstrating that chimeric G proteins unable to couple receptors at agonist concentrations used for native G proteins, were able to form the ternary complex at higher agonist concentration and, with some exceptions, exchange guanine nucleotides. Additionally, results suggest that G proteins play a more active role in receptor coupling than previously thought. Furthermore, results supporting the existence of multiple active receptor states indicate that the two-state receptor activation model needs to be changed. This project presents interesting findings regarding the molecular mechanisms that regulate G protein-receptor coupling and contributes to better understanding of GPCRs function.

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