The long term goal of the proposed studies is to improve our understanding of lipoprotein-macrophage interactions that may contribute to foam cell formation and atherogenesis. Emphasis will be placed on the acetyl LDL receptor (SRA), hCD36 and its mouse homologue (mDC36), and a 94-97 kDa oxidized LDL (OxLDL)- binding protein from mouse peritoneal macrophages which we have previously characterized and have now identified as macrosialin. Macrosialin and its human homologue, DC68, both previously clones, are found predominantly in the late endosomal fraction and their functions are not known. We propose to study their transport between subcellular compartment, to prepare stably transfected cells to characterize their ligand-binding properties, identify ligand binding sites by preparation of chimeric proteins and by site- directed mutagenesis, and prepare a mcarosialin knockout mouse to ascertain phenotypic consequences with regard to macrophage function, immune function and atherogenesis. CD36, an established OxLDL-binding receptor, functions in the binding and phagocytosis of apoptotic cells in cooperation with the vitronectin receptor and thrombospondin. We will test the hypothesis that binding of OxLDL may be modulated by interactions of this kind. Using cells from the SRA knockout mouse (gift of Dr. T. Kodama), we will assess the role of the receptor in OxLDL metabolism in vitro and in vivo. Finally, we will continue to use expression cloning in Xenopus oocytes to search for additional macrophage proteins that may participate in uptake of OxLDL or/and of damaged or apoptotic cells. Five clones have already been isolated. None of them represents either SRA, mCD36, or macrosialin. These will be sequenced and characterized.
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