Fusion of intracellular vesicles with the cell surface is a common mechanism by which hormones and other humoral agents may effect target cells. Studies in our laboratory have demonstrated that the endosome, which contains the majority of cellular receptors involved in ligand accumulation (i.e. receptors for transferrin, alpha 2macroglobulin protease complexes, and mannose terminal glycoprotein), can fuse with the cell surface. We have demonstrated that fusion is induced by exposure of macrophages to phorbol esters and calcium ionophors, resulting in an increase in surface receptor number and increased surface area. In this application we plan to use biochemical, genetic, and morphological probes to define the mechanism and physiological results of the fusion process in macrophages. We plan to define whether the fusion event is specific for the endosome, or if other intracellular membrane compartments also fuse with the cell surface. Some of the biochemical methods to be employed include radiolabeling of surface membrane and endosomal proteins. Morphological techniques will be used to measure both reductions in endosomal area and increases in cell surface area resulting from the fusion event. Chemotactic factors, antigen-antibody complexes, and activated complement also cause morphologic changes in macrophages. Their mechanism of action may also involve endosome-cell surface fusion, and we will evaluate these agents for this effect. Using a macrophage-like cell line, we will isolate mutants unresponsive to phorbol esters and calcium ionophores. Study of these mutants will permit us to dissect the biochemical steps leading from ligand binding to endosome-cell surface fusion. We will also define effects of endosome-cell surface fusion on membrane functions such as receptor mediated endocytosis and phagocytosis. A thorough understanding of the endosome-cell surface fusion event may establish this as a universal mechanism of target cell response to homoral stimuli.
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