Receptors for the constant region of IgG (FcgammaR) are expressed on the surface of hematopoietic cells where they enable the detection and destruction of IgG coated microbial organisms during infection and of IgG coated erythrocytes, lgG coated platelets and lgG containing immune complexes in immunohematologic and autoimmune disorders. FcgammaRIIA is present on a variety of human hematopoietic cells, including monocytes/ macrophages, and is the only FcgammaR expressed on platelets. FcgammaRllA is unusual among human FcgammaR in that it induces signal transduction in the absence of an associated subunit. Rather, FcgammaRIIA signaling employs unique sequences within its cytoplasrnic domain. Engagement of FcgammaRllA can lead to the clearance of lgG-containing immune complexes by endocytosis in platelets and monocytes/macrophages and to the clearance of lgG-coated particles by phagocytosis in monocytes/macrophages. Endocytosis and phagocytosis are two mechanistically different processes and the complex series of events that influence the occurrence of FcgammaRIIA mediated ingestion and destruction of foreign particles by these two processes is not well understood. The goal of this proposal is to combine fluorescence microscopy imaging and molecular biology techniques to study the mechanisms by which FcgammaRllA induces endocytosis and phagocytosis. Fluorescent proteins will be used to monitor the distribution of signaling components as well as to study protein-protein interactions in living cells. We have demonstrated that FcgammaRIIA phagocytosis requires Syk kinase, that FcgammaRIIA becomes ubiquitinated following receptor crosslinking, that Cbl co-immunoprecipitates with FcgammaRIIA, and that internalization via endocytosis but not via phagocytosis is dependent on the ubiquitination process. Based on these observations and considering the ubiquitin ligase activity of CbI, we hypothesize that the nature of the internalization process triggered by FcgammaRllA depends on the relative signals generated by CbI and Syk. We will: 1) define the role of ubiquitination in FcgammaRlIA mediated endocytosis; 2) study determinants for the formation and maturation of FcgammaRIIA induced phagosomes vs. endosomes; 3) determine the role of CbI ligase in FcgammaRIIA induced signaling in vivo using unique mouse strains deficient in Cbl; and 4) examine the role of a unique FcgammaRllA cytoplasmic motif, which we have recently identified, in phagosomal and endosomal maturation into lysosomes.
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