The purpose of this proposal is to train a clinician, specializing in Nephrology, in the concepts and techniques of modern molecular and cellular biology, with a focus on molecular mechanisms of transmembrane signaling. Didactic training will include core courses of the Ph.D. degree program in Biochemistry and attendance and participation in seminars, journal clubs, and conferences. Laboratory training will include exposure to experimental concepts and methods utilizing recombinant DNA, molecular genetics, immunology, tissue culture and membrane biochemistry. The overall goal of the research project for Phase I and the initial portion of Phase II is to elucidate the signaling roles of a group of pertussis toxin (PT)-sensitive G proteins, the Gi and Go proteins. The family of GTP-binding G proteins transduces information from receptor proteins oriented toward the outside of the plasma membrane to effector proteins oriented toward the cytoplasm, and includes Gs, transducin, Gi, Go and Gx. Gi has recently been shown to represent at least three different proteins, Gi1, Gi2, and Gi3. All G proteins are heterotrimers composed of alpha, beta and gamma subunits. Gs and transducin mediate hormonal stimulation of adenylyl cyclase and retinal photoreception, respectively. The specific signaling roles of the other G proteins are unknown. AS a group, the Gi and Go proteins are believed to mediate signaling pathways that are disrupted by PT because the toxin inactivates these G proteins. These signaling pathways include hormonal inhibition of adenylyl cyclase, stimulation of phospholipase C and A2, and regulation of ion channels. cDNA clones that encode the alpha chains of Gi1, Gi2, Gi3, and Go and antisera that can distinguish among the four alpha chains will be used to test the activities of individual alpha chains in particular PT-sensitive pathways, and to assess the specific coupling activities of these alpha chains to the receptor and effector elements of these pathways. These experiments will determine which Gi or Go protein mediates which PT-sensitive signaling pathway and will point to structural features and biochemical mechanisms that determine participation of an individual Gi or Go protein in a specific signaling pathway. During Phase II, the structure/function relationships and cell specific control of these and other G proteins will be further analyzed. Finally the role of G proteins in hormonal control of renal tubule epithelial transport will be studied. The proposed work is releant to normal regulation of renal function and to the pathogenesis of renal disease because G proteins are involved in control of glomerular filtration, solute and water transport, renal vascular tone, inflammatory response, and repair of ischemic and toxic injury.
