How a cell responds to an extra-cellular signal and mounts an intracellular response plays a pivotal role in the life cycle of every cell. Signal transduction pathways coupling heterotrimeric G-proteins to upstream seven-helix transmembrane receptors and downstream effectors represent a major class of such cellular signaling cascades. In the following proposal, the visual cascade of the bovine retinal rod cell will serve as the working model for such G coupled signaling cascades. The focus of this proposal is to define the stereochemical events accompanying the passage of a light signal from rhodopsin to the heterotrimeric G- protein transducin. In this proposal two major areas will be addressed by combined crystallographic and kinetic experimental approaches. The stereochemical principles governing transducin alpha's (Gt-alpha) GTPase mechanism will be elucidated by: (a) analyzing the structure of inhibited Gt-alpha.GTP-gamma-S after irreversible ADP-ribosylation of Argl74 by cholera toxin, (b) employing photoactivatable """"""""caged GTP"""""""" to carry out a time resolved crystallographic analysis of the conformational changes accompanying hydrolysis of GTP, and (c) analyzing the structure of a transition state analog complex between Gt-alpha.GDP and vanadate. The interactions of Gt-alpha with rhodopsin will be addressed by: (a) determining the kinetic constants accompanying formation of Gt-alpha.GDP and Gt-alpha.GTP-gamma-S complexes with peptides mimicking rhodopsin's interacting loops and (b) determining the structure of Gt-alpha.GDP and Gt-alpha.GTP-gamma-S complexes with peptides mimicking rhodopsin's interacting loops. The goal of the proposed work is to provide a structural framework for understanding the behavior of G-coupled signaling cascades with a particular emphasis on the mechanism of GTP hydrolysis and the functional coupling between heterotrimeric G-proteins and seven helical transmembrane receptors. The central involvement of such receptors in human physiology and disease will benefit from our combined kinetic and structural approach. The work may also suggest a means of tackling the problem of retinitis pigmentosa that is often correlated with mutations in the photoreceptor rhodopsin.
