Functional separation of pseudopod extension and particle internalization during Fcγ receptor-mediated phagocytosis

Receptors for the Fc portion of immunoglobulin (Ig)G (Fc gamma C) mediate phagocytosis of IgG-opsonized particles by a process that can be divided into four major steps: receptor-ligand binding, pseudopod extension, internalization, and lysosomal fusion. We have expressed single classes of Fc gamma R in COS fibroblasts to examine the structural determinants necessary to complete the four steps of phagocytosis. Using phase contrast, fluorescence, confocal, and electron microscopy we have demonstrated that Fc gamma R-expressing COS cells can phagocytose in a manner similar to that of professional phagocytes. Wie have further analyzed the capacity of the three classes of Fc gamma R to phagocytose, placing special emphasis on the Fc gamma RIA-gamma chain complex, which allowed us to examine independently the roles of che ligand-binding unit (Fc gamma RIA) and the signaling unit (gamma chain). We found that receptor complexes containing a conserved tyrosine activation motif(ITAM), as found in the cytoplasmic domain of Fc gamma RIIA and in the gamma chain associated with Fc gamma RIA and Fc gamma RIIIA, readily internalized target particles. In contrast, Fc gamma RIA alone, having no ITAM, was unable to internalize target particles efficiently, but did mediate pseudopod extension. Cotransfection of gamma chain with Fc gamma RIA restored the ability of the receptor to internalize target particles, A mutant Fc gamma RIA in which the cytoplasmic domain had been deleted was also capable of mediating pseudopod extension, showing that neither the gamma chain nor the cytoplasmic domain of Fc gamma RIA were required for this step. Cytochalasin D, an inhibitor of actin polymerization, blocked particle internalization by all Fc gamma R, but did not block pseudopod extension. Staining the Fc gamma RIA COS cells for F-actin and for tyrosine phosphoproteins, we found that actin did not polymerize during Fc gamma RIA-mediated pseudopod extension, nor were tyrosine kinases activated. Our data suggest that pseudopod extension and internalization are functionally distinct steps mediated through different pathways.