My career plans are in academic biomedical research. I have completed my postdoctoral training and I have attained a tenure-track Assistant Professor position. I have received broad training in the approaches and methodologies used to evaluate G protein-coupled receptor (GPCR) signaling and trafficking. However, a major focus of my independent research is to identify the molecules that dictate sorting of GPCRs to lyosomes. The requires a new area of expertise in gene cloning, protein purification and protein-protein interaction based strategies. My sponsor Dr. T. K. Harden is an especially attractive mentor because of this experience in training researchers, his time commitment to this endeavor and his proven ability to address important questions in GPCR biology. Members of my advisory committee, Drs. Parise and Milgram are also committed to my training and have considerable experience in approaches and methodologies pertinent to my proposed research. In addition I will take full advantage of the rich scientific environment offered at UNC. This includes interactions with senior faculty members, participation in seminars and coursework. This training will provide me with greater adaptability for my future research career and exposure to mentoring and management of trainees so that I may serve as an effective teacher and mentor. The overall goal of this proposal is to understand the mechanisms of thrombin- regulated signaling. Thrombin's effects on cells are essential in thrombosis, atherogenesis and vascular development; therefore understanding thrombin signaling may provide new strategies for prevention and treatment of thrombosis and other cardiovascular diseases. Thrombin signals through at least three protease-activated GPCRs: Protease-activated receptor-11 (PAR), the prototype of this family, mediates thrombin signaling in human platelets, endothelial, fibroblast and smooth muscle cells. PAR is irreversibly activated by thrombin, then internalized and sorted rapidly to lysosomes. Our recent studies strongly suggest that sorting of activated PAR1 to lysosomes is critical for termination of receptor signaling and for temporal fidelity of thrombin signaling. The molecular mechanisms by which PAR1 is internalized and sorted to lyosomes are largely unknown. We will examine whether PAR1 internalizes via the same route as recycling GPCRs and whether arrestin mediates internalization and lysosomal sorting of activated PAR1. Sorting of activated PAR1 to lysosomes is rapid and robust and information residing in PAR1's cytoplasmic carboxyl tail (C-tail) specifies this trafficking pattern. Thus we will use the C-tail of PAR1 as a probe in protein-protein interaction based strategies to identify new molecules that recognize and sort PAR1 to lysosomes.
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