This is a second revision of a new application with the general goal of investigating the mechanisms by which signaling pathways that regulate vascular contractility are coordinated in individual differentiated smooth muscle cells. The application will focus on testing the general hypothesis that the targeting and activation of two important protein kinases, PKC and ERK1/2, require interaction, directly or indirectly, with three potential scaffold proteins: caveolin (CaV), calponin (CaP) and a newly identified protein, smooth muscle archvillin (SmAV). Preliminary and published data demonstrate the involvement of these 3 proteins in the regulation of: (1.) contractility, (2.) signaling and (3.) cortical cell targeting.
The specific aims are to test the hypotheses: 1) that CaP, SmAV and CaV define discrete cortical cellular domains; 2) that SmAV functions as a scaffolding protein in the targeting and activation of ERK1/2 in smooth muscle; 3) that PKC phosphorylation and activation require interaction with CaV, CaP and SmAV; and, 4) that the distinct Ca-dependent and Ca-independent ERK1/2 pathways in this cell type are the result of scaffold-specific regulation. The proposal will use freshly dissociated cells, organ culture and recombinant proteins. Techniques include quantitative confocal and deconvolution microscopy, in situ photoaffinity crosslinking, FRET, membrane fractionation, antisense and murine knockout models, decoy peptides, a range of standard biochemical, physiological and molecular techniques, and all are established in the principal investigator's or collaborator's laboratories. Results are expected to significantly advance our understanding of how scaffold proteins work in an integrated, relevant system. By working on differentiated vascular cells, the results will have direct relevance to cardiovascular disease. Additionally, novel information will be gained on targeting mechanisms that will be of broad relevance to the signaling community. ? ? ?
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