Functional Separation of Pseudopod Extension and Particle Internalization during Fcγ Receptor–mediated Phagocytosis

Abstract

Receptors for the Fc portion of immunoglobulin (Ig)G (FcγR) 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 FcγR 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 FcγR-expressing COS cells can phagocytose in a manner similar to that of professional phagocytes. We have further analyzed the capacity of the three classes of FcγR to phagocytose, placing special emphasis on the FcγRIA–γ chain complex, which allowed us to examine independently the roles of the ligand-binding unit (FcγRIA) and the signaling unit (γ chain). We found that receptor complexes containing a conserved tyrosine activation motif (ITAM), as found in the cytoplasmic domain of FcγRIIA and in the γ chain associated with FcγRIA and FcγRIIIA, readily internalized target particles. In contrast, FcγRIA alone, having no ITAM, was unable to internalize target particles efficiently, but did mediate pseudopod extension. Cotransfection of γ chain with FcγRIA restored the ability of the receptor to internalize target particles. A mutant FcγRIA in which the cytoplasmic domain had been deleted was also capable of mediating pseudopod extension, showing that neither the γ chain nor the cytoplasmic domain of FcγRIA were required for this step. Cytochalasin D, an inhibitor of actin polymerization, blocked particle internalization by all FcγR, but did not block pseudopod extension. Staining the FcγRIA COS cells for F-actin and for tyrosine phosphoproteins, we found that actin did not polymerize during FcγRIA-mediated pseudopod extension, nor were tyrosine kinases activated. Our data suggest that pseudopod extension and internalization are functionally distinct steps mediated through different pathways.