The long-term goal of this research program is to understand the mechanisms of thrombin-regulated signaling. Thrombin, a coagulant protease, is generated in response to vascular injury and in thrombotic disease. Thrombin drives fibrin deposition and activates platelet, which are critical for hemostasis and thrombosis. Thrombin elicited cellular responses have also been implicated in inflammation, vascular development and cancer progression. Thus, understanding thrombin signaling, and developing the ability to manipulate it, may provide new strategies for the prevention and treatment of thrombin-related vascular diseases. Protease- activated receptors (PARs) are G-protein coupled receptors (GPCRs) that mediate most, if not all, thrombin responses in cells. Protease-activated receptor-1 (PAR1), the prototype for this family, accounts for the majority of the cellular effects elicited by thrombin in human platelets, endothelial cells, fibroblasts and smooth muscle cells. Thrombin activation of PAR1 occurs through an irreversible proteolytic mechanism that results in the generation of a tethered ligand that cannot diffuse away. Thus, PAR1 signaling must be tightly regulated. The signal termination mechanisms that regulate the magnitude, duration and spatial aspects of PAR1 signaling remain poorly understood. Phosphorylation and arrestins are some important components that contribute to desensitization of PAR1 signaling, but other mechanisms are likely to exist. PAR1 couples to multiple G-protein subtypes including Gq, Gi and G12/13 and the molecular basis of PAR1 uncoupling from these distinct G-protein subtypes is not known. Internalization and lysosomal sorting are also critical for the regulation of proteolytically activated PAR1 and are poorly understood. We showed that activated PAR1 internalization occurs through a phosphorylation-, clathrin- and dynamin-dependent pathway that is independent of arrestins. This led to our hypothesis that our clathrin adaptors function as critical mediators of PAR1 internalization. We recently discovered that ubiquitination regulates PAR1 internalization. We provide initial evidence that the clathrin adaptor epsin regulates internalization of PAR1. The molecular mechanisms by which ubiquitination and epsin contribute to the regulation of PAR1 signaling and trafficking are not known. We will use HeLa and HEK 293 cells ectopically expressing PAR1 and human endothelial cells expressing endogenous PAR1 to delineate the role of ubiquitination and epsin in PAR1 signaling and trafficking.
The specific aims of the proposal are to: 1) define the functional consequences of ubiquitination and epsin on PAR1 cellular signaling, 2) define the molecular basis for the novel regulation of PAR1 internalization by the clathrin adaptor epsin, and 3) delineate the molecular mechanism for the underlying novel regulation of PAR1 internalization by ubiquitination. Public Health Relevance: Thrombin, the key effector protease of the coagulation cascade, is generated in response to vascular injury and in thrombotic disease. The protein protease-activated receptor-1 mediates thrombin signaling in cells and is critical for blood clotting and inflammatory responses associated with vascular injury. Thus, understanding protease-activated receptor-1 signaling, and developing the ability to manipulate it, may provide new strategies for the prevention and treatment of thrombin-related vascular diseases.
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