The overall goal of this proposal is to understand the mechanisms of thrombin-regulated signaling. Thrombin, a coagulant protease, elicits a variety of cellular effects that are essential for hemostasis and thrombosis, as well as inflammatory and proliferative responses produced by vascular damage. Therefore, understanding the mechanisms of thrombin signaling may provide new strategies for the prevention and treatment of thrombin-related cardiovascular diseases. Protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that mediate most of thrombin responses in cells. PAR1, the prototype of this family, is the predominant mediator of thrombin signaling in human platelets, as well as in endothelial cells, fibroblasts and smooth muscle cells. PAR1 is irreversibly proteolytically activated, intemalized and sorted directly to lysosomes. The mechanisms that contribute to termination of PAR1 signaling are not clearly understood. Betaarrestins bind to most phosphorylated GPCRs to mediate desensitization and internalization. Phosphorylation is important for termination of PAR1 signaling (at least to Gq). We found that Betaarr1 is the predominant mediator of PAR1 uncoupling to Gq signaling. PAR1 couples to Gq, Gi, and G12/13, and the molecular basis of PAR1 uncoupling to these distinct G protein subtypes is not known. We will use mouse embryonic fibroblasts (MEFs) derived from betaarrestin knockouts and COS-7 cells to delineate the role of ?arrestins in PAR1 signaling. Internalization and lysosomal sorting of activated PAR1 are also critical for termination of receptor signaling. PAR1 is internalized via a clathrin- and dynamin-dependent pathway that is independent of arrestins. We provide initial evidence that a tyrosine-based motif and perhaps a novel adaptor protein regulate internalization of PAR1. Finally, our studies demonstrate a novel role for SNX1 and perhaps SNX2 in regulating lysosomal sorting of PAR1 and raise the exciting possibility that SNXs function generally in GPCR trafficking.
The specific aims of this proposal are to: (1) delineate the molecular basis of PAR1 desensitization, (2) define the novel internalization properties of PAR1, and (3) define the molecular mechanisms by which sorting nexins regulate lysosomal degradation of activated PAR1.
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