Nitric oxide (NO) exerts protective cardiovascular effects by inducing vascular smooth muscle cell (VSMC) relaxation and inhibiting platelet activation. NO works by raising intracellular cGMP, and activating cGMP-dependent protein kinase (PKG), which phosphorylates specific intracellular substrates. However, few PKG substrates have been characterized. In addition, how PKG becomes targeted within the cell in proximity to its substrates remains poorly understood. This Competitive Renewal Application explores these two issues based on three new mechanisms by which PKG inhibitsVSMC cellular activation discovered during the first Award period. These include identification of (a) the thromboxane receptor as a PKG-regulated substrate; (b) direct binding of PKG to the major phosphatase regulating VSMC relaxation, and demonstration that this interaction is required for cGMP/PKG-mediated vasorelaxation; and (c) inhibition of thrombin receptor (PAR-1) activation by PKG phosphorylation of RGS2, a member of the Regulator of G Protein Signaling (RGS) family. New data show that PKG binds to and phosphorylates RGS2, resulting in translocation of PKG-RGS2 to the plasma membrane. These mechanisms each involve binding of PKG to unique signaling proteins that localize the kinase to specific cellular regions. This is an emerging theme is signal transduction: anchoring proteins, often with intrinsic enzymatic activities themselves, serve to assemble and target multiprotein signaling complexes to distinct cellular domains where they regulate specific functions. MBS and RGS2 are thus members of a newly emerging family of PKG-anchoring proteins (GKAP). This renewal explores the hypothesis that PKG regulates NO/cGMP-mediated vascular events by binding to a family of GKAP, which assemble PKG-substrate signaling complexes in unique subcellular locations, and thereby regulate specific VSMC functions. This hypothesis will be explored in 3 Specific Aims:
Specific Aim 1 will further explore the mechanism by which PKG interacts with and regulates RGS2 and how this in turn inhibits PAR1 activation.
Specific Aim 2 will characterize the function of two new GKAPs we have cloned that are homologues of MBS.
Specific Aim 3 will explore the localization and function of FHOS, a novel forming homology (FH) domain-containing GKAP we also have just cloned. Studies of these 3 new GKAPs will use a variety of molecular approaches, including gene disruption of GKAP in mice, to explore subcellular localization, other proteins in the various PKG-GKAP complexes, and the mechanism (s) by which each PKG-GKAP interaction influences VSMC function. These studies are expected to contribute to our understanding of PKG, the critical regulatory protein for NO in vascular cells, and thus to our understanding of cardiovascular physiology and disease.
