Class A scavenger receptors (SR-A) are characterized by their ability to bind acetylated LDL (AcLDL) and to mediate substantial cholesterol ester accumulation in cells. It is generally thought that SR-A is constitutively internalized via endocytosis in clathrin-coated pits. However, the importance of the cytoplasmic portion of SR-A has been relatively neglected such that an internalization motif for the receptor has not been identified and little is known regarding the cytoplasmic signals that regulate SR-A-mediated endocytosis. Moreover, increasing evidence indicates that AcLDL activates pertussis toxin-sensitive intracellular signaling cascades indicating that SR-A-mediated uptake is coupled to activation of a Gi/o protein. The PI and his team have accumulated substantial data demonstrating that in macrophages, uptake of AcLDL is attenuated by pertussis toxin treatment suggesting that inhibiting Gi/o attenuates SR-A function. Mechanistic details regarding the interaction of SR-A with Gi/o proteins and the regulation of lipoprotein uptake by these PTX-sensitive G proteins in macrophages are lacking. This proposal is to test the central hypothesis that AcLDL promotes an interaction between specific cytoplasmic domains of SR-A with Gi/o proteins resulting in Gi/o protein activation and increased lipoprotein uptake. The PI will first determine the mechanism by which inhibiting Gi/o decreases SR-A dependent lipoprotein uptake. He will define the relative contribution of three possible mechanisms for the reduced uptake of modified lipoprotein in PTX-treated macrophages including; a) decreased number of receptors present on the cell surface, b) a reduced ability of SR-A to bind lipoprotein, and c) a decreased rate of SR-A-mediated internalization. The extent to which a contributing mechanism depends on Gi/o -mediated activation of protein kinase will also be determined. The second goal is to use mutational analysis to define the cytoplasmic sequence of SR-A involved in receptor internalization, Gi/o activation, and regulation by Gi/o signaling pathways.
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