This project focuses on the basic molecular mechanisms of transmembrane signal transduction via G-protein coupled receptors. Understanding of how these plasma membrane receptors interact with G-proteins is crucial for a broad range of cellular effects triggered by hormones, neurotransmitters, odorants and light. Practical applications of this knowledge will be important for the development of more specific therapeutics that target G-protein coupled receptors and G-proteins, because it's estimated that more than 40 percent of drugs in use work on G- protein coupled receptors. How exactly receptors activate G- proteins remains unclear, because of the universal technical difficulties in obtaining the high-resolution structural information in receptor/G-protein complexes. This proposal aims at resolving a major question about catalytic signal relay in membrane receptor/G-protein complexes, namely, the molecular role for the G-protein betagamma-subunit complex in the receptor catalyzed activation of the alpha-subunit. We propose to test a hypothesis that the betagamma-subunit complex is actively employed by rhodopsin in order to control the nucleotide-binding site on the alpha-subunit. We have prepared a set of mutant G- proteins in which a rhodopsin interaction domain, the C-terminus of transducin gamma-subunit, is targeted by Alanine replacements, deletions, and sequence reversal mutations. We have shown that some of the mutations severely affect coupling with the light- activated rhodopsin. We will survey extensively the functional properties of these mutants by examining various individual steps of rhodopsin-transducin interactions and transducin activation. Recently, we have developed the model mimetic peptides derived from the surface domains of G-proteins in order to study the dynamics of rhodopsin-transducin interface by NMR spectroscopy. We will study the effect of the inactivating mutations and the lack of farnesylation on the structural features of the C- terminal domain of the gamma-subunit in the light activated rhodopsin-bound state. We will determine whether the conformational switch in the gamma-subunit is affected by mutations. Functional studies of transducin mutants and structural data obtained in parallel with model mimetic peptides will yield valuable information on the role of the betagamma- subunit complex in rhodopsin-catalyzed activation of transducin.
