This application for a physician scientist award proposes to train a physician specializing in internal medicine to become an independent investigator in molecular pharmacology. Training will include didactic course work, seminars, and research on the molecular mechanisms of G protein structure and function. The applicant has two years of prior laboratory experience, but has been absent from the laboratory setting for two years while completing his residency. He has no previous training in molecular biology. G proteins amplify extracellular signals and sort the flow of signals from multiple receptors to multiple effector systems. Although cDNA cloning has identified a large number of G proteins (Gs, Gt1,2, Gi1,2,3, Go, Gz) only Gt and Gs have been assigned a specific function. The overall goal of the present project is to identify individual G proteins responsible for mediating specific signaling pathways. This goal will be accomplished by expressing mutant G protein alpha chains in mammalian cells and later in transgenic mice. G protein-effector coupling will be studied in cells that express """"""""dominant positive"""""""" mutant G protein that are constituitively active (mimic hormone stimulation of endogenous G proteins). G protein- receptor coupling will be studied by cells expressing """"""""dominant negative"""""""" mutant G protein that prevent activation of endogenous G proteins by monopolizing a rate limiting step such as binding to the hormone receptor. Dominant positive and dominant negative G protein mutants will be used as tools to study the effects of turning on or off specific signaling pathways in cells and later in transgenic animals. Emphasis will be placed on the creation of transgenic animals with cardiac expression of mutant G proteins to study the effects of manipulating specific signaling pathways in the heart without the systemic effects of pharmacologic treatments. Signaling pathways to be examined will include: beta-adrenergic stimulation of adenylyl cyclase (cAMP), m2-muscarinic inhibition of adenylyl cyclase, as well as the alpha1-adrenergic and m1-muscarinic stimulation of phospholipase C (inositol phosphates and calcium release), and of phospholipase A2 (arachidonic acid release).
