Signal Transduction Pathways of Smooth Muscle

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The overall Aim of this Project is to determine the physiologically relevant mechanisms of signal transduction that regulate contractility by pathways not dependent on cytoplasmic Ca concentration. The domain dependent functions of the upstream regulators of Ca?+-sensitization by the RhoA-Rho kinase pathway, the guanine nucleotide exchange factors (GEFs) PDZ.RhoGEF and LARG, will be determined in conjunction with the structural identification and recombinant expression of GEF domains in Project 2. The role of a membrane associated protein (p120 catenin) as a potential RhoA-binding partner guanine nucleotide dissociations inhibitor (GDI) will be determined. The kinetics of myosin phosphatase regulation by Rho-kinase and its dependence on the expression level of the endogenous phosphatase inhibitor, CP1-17 will be quantitated in order to determine their relative physiological relevance. The dependence of myosin phosphatase activity on the specific long and short splice isoforms of the regulatory subunit (MYPT1) expressed in mammalian smooth muscle will be evaluated, based on the hypothesis that expression levels of the ratio of the two isoforms determine localization of the phosphatase and the mechanism of its regulation through differences in their phosphorylation sites and myosin binding properties. A major, novel, objective of this Project is to determine the functions of a protein, LPP, newly discovered to be selectively expressed in smooth muscle, on cellular phenotypes, including morphology, adhesion to substrate, cytoskeletal structure and motility. Localization and stimulus induced translocation of signaling proteins, co-localization of proteins, such as p120 catenin with RhoA or CP1-17 with myosin phosphatase or telokin and telokin and LPP partners identified in two-hybrid screens will be performed in Project 3. p120 catenin, LPP, recombinant RhoA, telokin, RhoA- and other protein-mutants will be supplied by Core B. Abnormalities of the RhoA pathway are thought to be involved in asthma and hypertension.