Our current understanding of the processes which regulate cellular mitogenesis is derived largely from the study of receptor tyrosine kinases, transforming viral oncogenes, and their normal cellular homologues. Recently, receptors which couple to heterotrimeric G proteins have also been shown to mediate signals which regulate cellular growth and differentiation. Growing evidence suggests that these receptors, which mediate potent mitogenic responses to a variety of humoral, endothelium- or platelet-derived substances, participate in the regulation of cellular proliferation in both physiologic and pathophysiologic states and in cellular transformation in some, mostly neuroendocrine, tumors. Perhaps surprisingly, many of these G protein-coupled receptor-derived signals are dependent upon the regulation of tyrosine protein kinases, and are thus indicative of complex crosstalk between G protein-coupled receptor and receptor tyrosine kinase signaling cascades. To date, four distinct classes of tyrosine protein kinase; src kinases, focal adhesion kinases (FAK), receptor tyrosine kinases, and Janus kinases, have been implicated in various aspects of G protein-coupled receptor signaling. The molecular mechanisms whereby these kinases are regulated, and their roles in transduction of G protein-coupled receptor-mediated mitogenic signals, is largely unknown. This project tests the hypothesis that G protein-coupled receptors mediate signals which regulate gene expression, and control cytoskeletal rearrangement, via the activation and targeting of tyrosine kinases to specific multi-protein signaling complexes. Employing Gs-, Gi, and Gq/11-coupling receptors in cultured cell model systems as well as primary cultures of cardiac and vascular smooth muscle, the first specific aim of the project is to determine the proximal mechanisms whereby G protein-coupled receptors regulate the activity of src, FAK and receptor tyrosine kinases. A parallel aim is to determine the role of activated c-src/receptor tyrosine kinase, and c-src/FAK complexes in the regulation of mitogen-activated protein kinase cascades. Another aim is to determine the role of these kinase complexes in mediating G protein-coupled receptor-dependent reorganization of the actin cytoskeleton. Understanding the mechanisms whereby G protein-coupled receptors regulate these components of the mitogenic signal may facilitate the identification of targets for the prevention of the proliferative complications of conditions such as diabetes mellitus, atherosclerosis and post-angioplasty restenosis.
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