The cytoplasmic domain of FcgammaRIIA (CD32) participates in phagolysosome formation.
Signaling 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 FcgammaRIIA 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 FcgammaRIIA lacking its cytoplasmic domain (tail-minus form). This system has allowed us to study Fcgamma receptor-dependent phagocytosis and phagolysosome fusion in the presence and absence of the cytoplasmic domain of FcgammaRIIA. Fluorescent dextran was used to label lysosomes. After target internalization, wild-type FcgammaRIIA-mediated phagolysosome formation was observed as indicated by colocalization of fluorescent dextran and the phagosome. In addition, when studying mutants of FcgammaRIIA 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 FcgammaRIIA, 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 FcgammaRIIA but not by tail-minus FcgammaRIIA and CR3. Thus, the tail of FcgammaRIIA contributes to phagolysosome fusion by a mechanism that does not require a functional ITAM sequence.
Worth
R G
RG
Department of Biological Sciences, Wayne State University, Detroit, MI, USA.
Mayo-Bond
L
L
Kim
M K
MK
van de Winkel
J G
JG
Todd
R F
RF
Petty
H R
HR
Schreiber
A D
AD
D000135:Acid Phosphatase / Q000032:analysis; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000502:physiology*; D016466:CHO Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry*; D003911:Dextrans; D005456:Fluorescent Dyes; D015870:Gene Expression; D008247:Lysosomes / Q000201:enzymology / Q000502:physiology / Q000648:ultrastructure; D008561:Membrane Fusion; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010587:Phagocytosis; D010588:Phagosomes / Q000502:physiology* / Q000648:ultrastructure; D017452:Receptors, IgG / Q000235:genetics / Q000502:physiology*; D012235:Rhodamines; D014162:Transfection
D015703:Antigens, CD; D003911:Dextrans; C080568:Fc gamma receptor IIA; D005456:Fluorescent Dyes; D017452:Receptors, IgG; D012235:Rhodamines; C051311:rhodamine dextran; D000135:Acid Phosphatase