Vascular smooth muscle (VSM) 12C-adrenoceptors (12C-ARs) are generally retained in intracellular transGolgi compartment, and mobilized to the cell surface in response to conditions of stress including injury or cold temperature. Cell surface 12C-ARs lead to constriction of the blood vessel. These receptors have been implicated in the heightened abnormal vasoconstrictions triggered by cold temperature in individuals with Raynaud's phenomenon, secondary Raynaud's associated with scleroderma, or hand-arm vibration syndrome. Currently, there is no targeted therapy for early control of Raynaud's phenomenon. SIGNIFICANCE: The long term goal of the project is to elucidate key signaling mechanisms controlling intracellular expression and mobilization of 12C-ARs for targeted therapy in early stages of the disease. METHOD: A novel cell culture model of primary human cutaneous arteriolar VSM (microVSM) will be utilized for these studies. These cells retain high expression of 12C-ARs in culture. Preliminary results using this model implicate the cAMP-activated Ras super family small GTP-binding protein Rap1 in orchestrating 12C-AR expression and partial cell-surface mobilization at 37 oC. Rap1 mobilized 12C-ARs through RhoA-Rho kinase ROCK and the actin binding protein filamin-2, similar to receptor mobilization during cooling. The convergence of cAMP and cold-induced activation of signaling to Rho-ROCK may contribute to severity of the disease. In addition, HEK293 cells and murine microVSM with genetic ablation of Rap1 will be utilized in the studies.
Two Specific Aims will be accomplished:
AIM 1 : Will test the HYPOTHESIS of the carboxyl terminus arginine-rich motif (RRRRR) of 12C-AR to be the key region mediating interaction with filamin 2 and receptor mobilization to filaments and cell surface. Structure-function studies are proposed that will allow testing cell-permeable decoy peptides to block interaction and receptor mobilization at 37oC and experimental condition of moderate cooling at 28 oC.
AIM 2 : Will test the HYPOTHESIS of Rap1-Rho-ROCK signaling in facilitating filamin 2 to mobilize 12C- ARs to the cell surface. The role of this signaling pathway in phosphorylating the conserved filamin-2 serine2113 will be examined, and the potential relevance to receptor mobilization at 370C and 280C. ? ? ? ?
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