The ubiquitin ligase c-Cbl down-regulates FcgammaRIIa activation in human neutrophils.
Little is known about the mechanisms that arrest FcgammaRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. In our previous studies, we observed a loss of immunoreactivity of Abs directed against FcgammaRIIa following its cross-linking. In this study, we report on the mechanisms involved in this event. A stimulated internalization of FcgammaRIIa leading to the down-regulation of its surface expression was observed by flow cytometry and confocal microscopy. Immunoprecipitation of the receptor showed that FcgammaRIIa is ubiquitinated after stimulation. MG132 and clasto-lactacystin beta-lactone inhibited the loss of immunoreactivity of FcgammaRIIa, suggesting that this receptor was down-regulated via the proteasomal pathway. The E3 ubiquitin ligase c-Cbl was found to translocate from the cytosol to the plasma membrane following receptor cross-linking. Furthermore, c-Cbl was recruited to the same subset of high-density, detergent-resistant membrane fractions as stimulated FcgammaRIIa itself. Silencing the expression of c-Cbl by small interfering RNA decreased FcgammaRIIa ubiquitination and prevented its degradation without affecting the internalisation process. It also prolonged the stimulation of the tyrosine phosphorylation response to the cross-linking of the receptor. We conclude that c-Cbl mediates the ubiquitination of stimulated FcgammaRIIa and thereby contributes to the termination of FcgammaRIIa signaling via its proteasomal degradation, thus leading to the down-regulation of neutrophil signalisation and function (phagocytosis) through this receptor.
Marois
Louis
L
Centre de recherche en rhumatologie et immunologie, Centre de recherche du Centre hospitalier universitaire de Québec, Department of Medicine, Faculty of Medicine, Laval University, Quebec, Canada.
Vaillancourt
Myriam
M
Marois
Sébastien
S
Proulx
Sophie
S
Paré
Guillaume
G
Rollet-Labelle
Emmanuelle
E
Naccache
Paul H
PH
D015153:Blotting, Western; D015536:Down-Regulation; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000276:immunology* / Q000378:metabolism; D034741:RNA, Small Interfering; D017452:Receptors, IgG / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D014162:Transfection; D054875:Ubiquitination
C445323:FCGR2A protein, human; D034741:RNA, Small Interfering; D017452:Receptors, IgG; D050721:Proto-Oncogene Proteins c-cbl