The transduction of biological signals such as light, hormones and neurotransmitters starts by a specific interaction of the ligand or stimulus with a receptor protein. The ultimate cell-specific responses are produced via receptor activation of specific GTP-binding proteins. While the functional aspects of this process are well-defined, the structural basis of G protein function, the regulated interactions between receptor, G protein and effector, and the activation processes are understood only partially. In the last grant period, expression systems for generation of large amounts of wild type and mutant G proteins were worked out. In addition, the three-dimensional structures of alpha and beta/gamma subunits, and heterotrimeric alpha/beta/gamma complex were solved in collaboration with Paul Sigler's laboratory. This structural information, in conjunction with functional studies, suggests detailed hypotheses for the mechanisms that keep G proteins inactive in the absence of activated receptors, that lead to serial activation of G proteins and effectors by activated receptors during signal transduction, and that determine the timing of the active state by controlling GTP hydrolysis rates. These hypotheses will be tested in this proposal, using site-directed mutagenesis and heterologous expression of G protein alpha and beta/gamma subunits in E. coli and Baculovirus-infected SF9 cells. The model systems include a variety of receptors, G proteins and effectors, either native or overexpressed, tested in biochemical assays in reconstituted membranes. The critical amino acid residues involved in these processes will be determined. The collaboration with Sigler's laboratory will continue with crystallization and resolution of structures of receptor-G protein complexes and complexes of G protein alpha and beta/gamma subunits with a number of downstream effectors. The combined structural and functional information will contribute to our understanding of basic mechanisms of cellular activation by a variety of signals, and will also provide insight into diseases that affect G protein function.
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