Date of Award
Doctor of Philosophy (PhD)
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 G D P for G T P on its alpha subunit (Ga). 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 C 4 were replaced with amino acid sequences from the beta2 adrenergic receptor or the m l muscarinic receptor. Chimeras in which the amino terminus of C 4 was altered were defective in catalyzing Gt activation. A spectroscopic photoregeneration assay was used to demonstrate that mutants of the amino terminus of C 4 were defective in binding holo-Gt, a peptide derived from the carboxyl terminus of Galpha t, and in certain circumstances a peptide derived from the carboxyl terminus of Gyt. These results suggested that C4 mediated Gt binding and activation and that C4 interacted specifically with the carboxyl termini of G alpha t and possibly Gyt. We next studied how R* induces nucleotide exchange by Gt at a distance. We tested the validityof two longstanding hypotheses: 1) that R* induces opening of the interdomain cleft of G alpha t , and 2) that R * communicates with the nucleotide binding pocket via the a5 helix of Galpha . We developed an expression and assay system to characterize a large number (>50) of site-directed mutants of Galphat 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 Galphat 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 alpha5 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 alpha5 disrupted R*-catalyzed activation, as did substitution of alpha5 residues with prolines. These results provided evidence that R* induced nucleotide exchange primarily by perturbing the structure of buried residues on alpha5 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.
Marin, Ethan P., "Receptor- G Protein Interactions in the Visual System: A Study of Structures and Mechanisms That Couple the Cytoplasmic Surface of Rhodopsin to the Nucleotide-Binding Pocket of Transducin" (2001). Student Theses and Dissertations. 349.