Signal transduction within the central nervous system involves the recognition of neurotransmitter or neuromodulator by a specific receptor and the subsequent activation (or inhibition) of a cellular event. These cellular events (ion translocation, neurotransmitter release etc.) are often mediated by second messengers, which are evoked as a result of receptor occupancy. This laboratory has been interested in the adenylate cyclase system for some time, and the proposed research represents an attempt to identify aspects of adenylate cyclase regulation which may be restricted to neural cells. One locus of this regulation is the ability of cytoskeletal components (specifically tubulin) to modify the adenylate cyclase system by direct transfer or GTP to the regulatory G proteins. The physical and biochemical link between tubulin and the adenylate cyclase system will be probed in a number of reconstitution studies. These studies will be performed on nitrocellulose or in phospholipid vesicles, and will test for physical interaction and/or nucleotide exchange between tubulin and G proteins. The former studies will be designed to determine the portion of the tubulin molecule responsible for the association with G proteins as well as the domains of tubulin responsible for nucleotide exchange. Specific antibodies, proteolytic digestion and synthetic peptides will be used to make these determinations. The hydrolysis resistant photoaffinity GTP analog, AAGTP will be used to probe the nucleotide exchange process between tubulin and G proteins as well as among G protein species. Physical interaction among G proteins (including tubulin) will be examined in membranes with the use of cross-linking agents, including a novel, nucleotide based crosslinker. The photoreceptor G protein system will be used to probe the kinetics of the nucleotide exchange process within a less complex system, and antibodies against a specific region of the photoreceptor G protein (transducin) will be used as well. If possible a newly discovered 32 KDa protein will be purified and characterized. Finally, the relevance of this protein, as well as G protein interaction and nucleotide exchange established in a permeable cell system. Greater understanding of the mechanisms whereby neurotransmitter signals are mediated will lead to a better understanding of human mental function and dysfunction.
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