Student Theses and Dissertations

Date of Award

2001

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Sakmar Laboratory

Keywords

rhodopsin, transducin, G protein, nucleotide exchange, α5 helix, signal transduction

Abstract

The intermolecular interaction between the photoreceptor rhodopsin and the heterotrimeric G protein transducin (Gt) initiates the vertebrate phototransduction cascade. This interaction also serves as a model system for the study of the molecular basis of related G protein-coupled receptor mediated signal transduction systems. Photoactivated rhodopsin (R*) activates Gt by catalyzing the exchange of bound GDP for GTP on its α subunit (Gα). The structure of the R*-Gt complex and the mechanism of nucleotide exchange are unknown. We studied the function of the fourth cytoplasmic loop (C4) of rhodopsin in interactions with Gt. Chimeric mutants of rhodopsin were characterized in which regions of C4 were replaced with amino acid sequences from the β2 adrenergic receptor or the m1 muscarinic receptor. Chimeras in which the amino terminus of C4 was altered were defective in catalyzing Gt activation. A spectroscopic photoregeneration assay was used to demonstrate that mutants of the amino terminus of C4 were defective in binding holo-Gt, a peptide derived from the carboxyl terminus of Gαt, and in certain circumstances a peptide derived from the carboxyl terminus of Gγt. These results suggested that C4 mediated Gt binding and activation and that C4 interacted specifically with the carboxyl termini of Gαt and possibly Gγt. We next studied how R* induces nucleotide exchange by Gt at a distance. We tested the validity of two longstanding hypotheses: 1) that R* induces opening of the interdomain cleft of Gαt , and 2) that R* communicates with the nucleotide binding pocket via the α5 helix of Gαt. We developed an expression and assay system to characterize a large number (>50) of site-directed mutants of Gαt designed to test these hypotheses. The mutants were expressed in vitro in rabbit reticulocyte lysate and the kinetics of both basal and R*-catalyzed nucleotide exchange were determined by quantitative analysis of trypsin digest patterns. Mutations in a series of residues at the interface between the two domains of Gαt had only minor effects on the basal and catalyzed activation rates. In contrast, abstract mutations in a cluster of residues on the buried face of the α5 helix, 0.7-1.5 nm from the nucleotide, greatly (up to 165-fold) accelerated nucleotide exchange. Mutations of residues on the adjacent solvent-exposed surface of α5 disrupted R*-catalyzed activation, as did substitution of α5 residues with prolines. These results provided evidence that R* induced nucleotide exchange primarily by perturbing the structure of buried residues on α5 and not by opening the interdomain cleft. Structural analysis and biochemical data were used to propose a mechanistic model for receptor-catalyzed G protein activation.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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