All aspects of cardiovascular function are regulated by receptors of the 7TM receptor family. The largest and most ubiquitous of all the receptor families, it includes receptors for catecholamines, acetylcholine, angiotensin, adenosine and endothelins. A universal mechanism regulating 7TM receptors is desensitization of heterotrimeric G-protein signaling. Classically, this is mediated by a 2-step process in which activated receptors are phosphorylated by G-protein-coupled receptor kinases (GRKs) leading to the binding of a B-arrestin molecule, which sterically interdicts further activation of the G protein. Over the past several years it has become clear that B-arrestins can also serve as multifunctional endocytic and signaling adaptors, which can ateo activate additional pathways. These include MAP kinases, such as ERK1/2 and JNK3 and physiological outcomes such as chemotaxis and anti-apoptosis, all of which are of great importance in regulating cardiovascular function in atherosclerosis, restenosis, and cardiac hypertrophy. Accordingly, this proposal has 3 closely linked aims, which involve a primary focus on elucidating the molecular mechanisms by which B-arrestins and GRKs mediate signaling by 7TM receptors. Our goals are to elucidate: 1) the roles of GRKs in mediating 7TM receptor signaling, by developing siRNA techniques and 'knock-out"""""""" mouse embryo fibroblasts to reduce or eliminate GRK expression from cells; and by determining the sites of GRK phosphorylation and their functional consequences for several 7TM receptors including the B2-adrenergic receptor and Ang 111A receptor; 2) the roles of B-arrestin 1 and 2 in mediating ERK activation by 7TM receptors and of B-arrestin ubiquitination in this process; 3) the structural and biophysical basis of B-arrestin-mediated signaling by studying the """"""""activated"""""""" conformations of a-arrestins by limited proteolysis, tryptophan fluorescence and atomic structure determination. Our hypothesis is that the phosphorylation of distinct sites on receptors by different GRKs leads to structurally and functionally distinct activated conformations of B-arrestins which mediate distinct signaling outcomes. The resulting understanding of the molecular basis of the newly appreciated GRK and a-arrestin-mediated signaling pathways should point the way toward development of novel therapeutics for cardiovascular diseases.
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