Heterotrimeric G-proteins mediate the activity of hundreds of different serpentine receptors (seven pass membrane receptors). They couple the ligand binding event at the extracellular surface to the activity of an intracellular or membrane bound effector. Thus, they play a critical role in an enormous number of physiological processes including sensory perception, hormone action, neurotransmission, the inflammatory response, and perhaps in growth control and morphogenesis. A clear understanding of how they function could result in the development of drugs that block or enhance these processes. Our goals are to analyze the diversity in the heterotrimeric G-protein family and to determine how G proteins function to generate the appropriate connections between receptors and effectors with the correct kinetics to regulate function. The heterotrimeric G proteins are composed of a large family of genes that encode multiple alpha, beta, and gamma subunits. Our hypothesis is that the combinatorial association of these components leads to the formation of a large variety of heterotrimers embodying specificity for different receptors and effectors. In this renewal we propose to: 1. Further explore the diversity of the Galpha, beta, and gamma subunit genes. 2. Study the basis for isoform specific interaction between members of the Gq family and specific phospholipase C isozymes using transient transfection, mutagenesis, and in vitro reconstitution assays. 3. Search for the receptors and effectors that couple to the Galpha12 family of G proteins. 4. Explore the basis of the cell type specificity of expression of certain G proteins. 5. Target the genes encoding the Gq and G(12) family of alpha subunits using homologous recombination in embryonic stem cells (ES cells) and eliminate these gene functions. We will generate mice that lack polypeptides corresponding to specific Galpha and Ggamma subunits. These animals and the ES cell lines will be assayed to determine the effects of the absence of a specific G-protein on physiological and cellular function.
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