Our work is designed to determine how G proteins act to regulate intracellular information processing in neural cells. How do the G proteins coordinate and integrate information regarding chemical and physical changes that occur at the cell surface? How do they generate networks of interconnected receptors and effectors? First we focus on the alpha subunit that corresponds to the Go heterotrimer, since the polypeptide can be produced in an active and abundant form in E. coli. We can use this system to make large numbers of mutants throughout the gene and to screen large numbers of mutant proteins for specific functional defects. We then map the various phenotypes to different portions of the protein and develop a picture of the distribution of functional domains on the protein. furthermore we will generate a number of very useful conditional (temperature sensitive) mutants and dominant negative mutants. In order to understand receptor-G protein interaction and network formation, we will use the Xenopus system to further characterize receptor activation of the Galpha protein. We will coexpress different receptors and different G proteins and develop """"""""synthetic"""""""" networks to assess the relative specificity and information transfer properties of the G proteins. We will further characterize some of the Galphao mutants, particularly with respect to their affinity for specific receptors. We will also use the Xenopus system to explore the specificity of interaction of the novel Galpha subunits with different receptors and effectors. Finally, when cloned ligand gated K+ and Ca2+ channels become available, they can be coinjected with receptors and G proteins, and relatively complex interacting systems can be assembled and studied. In order to further probe G-protein function we will use the mutants that we have generated and characterized to explore signalling systems in PC12 cells. We can make stable cell lines carrying dominant negative mutations in Galphao or temperature-sensitive mutations. These will be used to explore the function of the endogenous signalling system.
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