Macrophages, granulocytes, many lymphocytes, and certain epithelia all express receptors for the Fc domain of IgG. Fc receptors (FcR) serve diverse functions in cellular and humoral immunity. In macrophages, they mediate the endocytosis soluble antibody-antigen complexes, the phagocytosis of large IgG-coated particles, and the release of potent cytotoxic and inflammatory agents. FcR on B-lymphocytes, on the other hand, regulate the process of B-cell activation via surface Ig. Recently, this diversity in function has been found to reflect an even greater diversity in structure. FcR comprise a large, multi-gene family of Ig-related molecules. Among the most important of these if FcRII, a receptor class expressed on virtually all FcR-positive cells. However, even this single class exhibits considerable structural heterogeneity due to cell type-specific alternative mRNA splicing that generates receptors with different cytoplasmic domains. In murine cells, the major splice products are mFcRII-B1 (lymphocytes) and -B2 (macrophages). These isoforms are identical except for an in-frame insertion of 47 amino acids in the cytoplasmic tail of mFcRII-B1. Over the past few years, we have begun to establish the functional differences between mFcRII-B1 and -B2, and to determine whether different receptor activities are associated with distinct regions of the FcRII cytoplasmic domain. For example, the presence of the insertion completely blocks endocytosis by preventing receptor accumulation at coated pits. In this proposal, we will extend these investigations using a combination of biochemical, molecular, morphological, and genetic approaches. First, we will better define the unique features of the FcRII-B2 coated pit localization domain. Although coated pit accumulation involves a conserved tyrosine-containing region of the receptor's cytoplasmic tail, more detailed analysis is required since unlike most other plasma membrane receptors the tyrosine residue itself is not required for coated pit localization in all cell types. Using molecular and biophysical approaches, the FcRII coated pit domain will be precisely defined and compared to more """"""""conventional"""""""" coated pit domains. Second, we will define the mechanism by which the alternatively spliced insertion in mFcRII-B1 (and its human homolog) prevents coated pit localization. Preliminary evidence suggests that the insertion functions by actively preventing coated pit entry, perhaps by binding the receptor to the cytoskeleton. Third, we will define the domains involved in the regulation of B-cell activation. The role of tyrosine phosphorylation will be evaluated since this domain appears to overlap with the region required for coated pit localization, and its tyrosine falls within a consensus tyrosine phosphorylation site. Fourth, we will identify sequences required for FcRII-mediated phagocytosis and signal transduction in transfected fibroblasts and macrophage lines. Finally, we will develop the use of a genetically manipulable professional phagocyte, Dictyostelium discoideum, as a genetic system for the study of phagocytosis in general and FcRII function in particular.
