The long-term objective of this project is to determine newly recognized, specific mechanisms that regulate contraction of vascular, airway and other smooth muscle independently of changes in cellular calcium. The major specific aims are: 1., to determine the mechanism, in vivo kinetics and upstream regulators and downstream effectors through which the Ras family, monomeric GTP-binding protein RhoA sensitizes smooth muscle to calcium by inhibiting the protein phosphatase that de-phosphorylates the regulatory light chain of smooth muscle myosin, an 2., to determine the mechanisms that independently of changes in Ca2+, accelerate dephosphorylation of the regulatory light chain and, thereby, relax smooth muscle.
The specific aims i nclude identification of Rho-related proteins in smooth muscle, the mechanisms through which rho inhibits smooth muscle myosin light chain phosphatase, as well as determining the effects of phosphorylation of telokin and its novel putative role in cyclic GMP- kinase-mediated Ca2+-desensitization. An additional major goal of this project is to relate, in collaborative studies with structural biologists, the function of Rho and Rho-related proteins to their structures as well as the effect of phosphorylation of telokin on its function and crystal structure. The broad, general significance of the project derives from the involvement of RhoA in signalling in, probably, all cell types and the presence and function of the regulatory subunit of smooth muscle myosin phosphatase also in non-muscle cells; the major health related aspects of the research originate from the importance of smooth muscle in regulating the caliber of blood vessels and airways. It is probable that some forms of both hypertension and asthma are due to abnormalities of the regulatory pathways explored in this project that derives special strength from the very close interdisciplinary collaboration of a crystallographer, a molecular biologists and two cell physiologists.
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