The cytoplasmic domain of FcγRIIA (CD32) participates in phagolysosome formation

Abstract

AbstractSignaling motifs located within the cytoplasmic domain of certain receptors contribute to lysosome fusion. Most studies have described lysosome fusion with respect to endocytic receptors. Phagolysosome fusion has not been extensively studied. To test the hypothesis that the tail of FcγRIIA participates in phagolysosomal fusion, a “reverse” genetic complementation system was used. It was previously shown that complement receptor type 3 (CR3) can rescue the phagocytic activity of a mutant FcγRIIA lacking its cytoplasmic domain (tail-minus form). This system has allowed us to study Fcγ receptor–dependent phagocytosis and phagolysosome fusion in the presence and absence of the cytoplasmic domain of FcγRIIA. Fluorescent dextran was used to label lysosomes. After target internalization, wild-type FcγRIIA–mediated phagolysosome formation was observed as indicated by colocalization of fluorescent dextran and the phagosome. In addition, when studying mutants of FcγRIIA containing a full-length cytoplasmic tail with the 2 ITAM tyrosines mutated to phenylalanine, (1) phagocytosis was abolished, (2) CR3 restored phagocytosis, and (3) lysosomal fusion was similar to that observed with the wild-type receptor. In contrast, in the presence of CR3 and the tail-minus form of FcγRIIA, internalized particles did not colocalize with dextran. Electron microscopy revealed that the lysosomal enzyme acid phosphatase colocalized with immunoglobulin G–coated targets internalized by wild-type FcγRIIA but not by tail-minus FcγRIIA and CR3. Thus, the tail of FcγRIIA contributes to phagolysosome fusion by a mechanism that does not require a functional ITAM sequence.