The objective of this work is to visualize the physico-chemical roles of G proteins (G-alpha, G-beta, and G-gamma) as they pass through the conformational states necessary for receptor-stimulated signal transduction. To complement x-ray crystallographic studies, we will use available structural data, mutagenesis, and biochemical characterization of recombinant proteins to assess quantitatively the role of substructural elements and domains in the alpha subunit that are important for protein-protein interactions and nucleotide hydrolysis and exchange. From analyses of structural and biochemical data sets of mutant and wild type proteins, alpha subunits will be engineered to exhibit poor affinity for nucleotide while retaining affinity for receptor and beta-gamma complex. These mutant G-alpha's should have a dominant negative phenotype in vivo and thereby serve as invaluable tools for studying G protein-receptor interactions at the cellular level. Elucidating the mechanisms of cellular communication and transmembrane signaling at the molecular level is a fundamental prerequisite to understanding cell growth and regulation. In particular, this knowledge will define the abnormalities in these pathways that lead to aberrant and uncontrolled cell growth.
