RECEPTOR MEDIATED TRANSPORT OF GLYCOPROTEINS

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Receptor mediated endocytosis is a general phenomena that has been found to be relevant to organelle biogenesis, hormone and mitogen physiology and the transport of macromolecules into cells. Alveolar macrophages express a mannose specific receptor that mediates the endodytosis and packaging of glycoconjugates (including lysosomal enzymes) from the extracellular space to the lysosomes of the macrophage. I have developed a formal kinetic mechanism which accurately describes the steady-state uptake mediated by this receptor and developed techniques for studying segments of the endocytic process in isolation. Using these approaches we have been able to show that endosome acidification is rate limiting during steady-state uptake. I proposed to study the mechanism of endosome acidification in intact cells and isolated endosomes using ligands specific for the mannose receptor with covalently attached fluorescent pH sensitive chromophores. The intracellular pathway and mechanism of vesicular transport will be studied using kinetic and fractionation techniques. Labelled ligand and labelled receptor (using a technique I have developed for in situ labelling of the receptor) will be used to follow the progression of these components through the intracellular pathway of receptor mediated endocytosis. The role of acidification of endocytic vesicles in the release of lysosomal enzymes stimulated by zymosan, endotoxin and phorbol myristic acid will be evaluated. The stimulated secretion of lysosomal hydrolases by macrophages will be used as a model for the role of macrophages in chronic inflammation. The mechanism of secretion will be studied and the role of mannose specific endocytosis in the regulation of extracellular hydrolase levels under these conditions will be determined.