Macrophages express a cell surface receptor that binds and internalizes glycoproteins and glycoconjugates terminating in mannose and fucose. The receptor also mediates phagocytosis of mannose-coated organisms (e.g., yeast). The receptor is a 175 KD glycoprotein that has been isolated from rabbit macrophages and human placenta. The placental receptor is found in trophoblasts. Within placenta, the receptor has an apical distribution being found associated with the brush border facing maternal blood. The macrophage receptor recycles constitutively from the cell surface to an acid intracellular compartment where receptor ligand dissociation occurs. Before an analysis of receptor function can be undertaken, the structure of the receptor must be elucidated. We propose to isolate cDNA encoding for the mannose receptor using lambda gtll placental and macrophage libraries. The libraries will be screened with oligonucleotide probes, derived from receptor sequence and antibodies, prepared aganist human receptor. Using information from cDNA's encoding for the receptor and other peptide sequence, the orientation of the receptor in the membrane will be established. The localization of N-linked and O-linked oligosaccharides within the receptor will be determined as well as other post-translational modifications. In parallel experiments, the biosynthesis, intracellular transport and turnover of the receptor in monocyte- derived macrophages will be determined. Of particular interest is whether the receptor is assembled as a monomer or an oligomer in the plasma membrane. Glucocorticoids elevated mannose receptor levels while gamma-interferon lowers receptor levels. The biochemcial basis for these responses will be investgated using 35 S-methionine pulse-chase experiments followed by immunoprecipitation and Northern blots to quantitate messenger RNA levels. The intracellular localization of receptor will be determined in macrophages and placenta using frozen-thin section immuno-gold cytochemistry at the EM level. Transfection experiments will be carried out to express the mannose receptor cDNA, and constructs with various deletions, in polarized and non-polarized cells. The questions to be adddressed include whether the same receptor mediates phagocytosis and pinocytosis, what structural information accounts for the polarized distribution of the receptor in placental trophoblasts, and for receptor-ligand recycling. The mannose receptor may be a good model for the study of receptor mediated pinocytosis and phagocytosis and its regulation in macrophages and placenta.
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