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The Ubiquitin Ligase c-Cbl Down-Regulates FcRIIa Activation in Human Neutrophils¹

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

Little is known about the mechanisms that arrest FcRIIa 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 FcRIIa following its cross-linking. In this study, we report on the mechanisms involved in this event. A stimulated internalization of FcRIIa leading to the down-regulation of its surface expression was observed by flow cytometry and confocal microscopy. Immunoprecipitation of the receptor showed that FcRIIa is ubiquitinated after stimulation. MG132 and clasto-lactacystin β-lactone inhibited the loss of immunoreactivity of FcRIIa, 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 FcRIIa itself. Silencing the expression of c-Cbl by small interfering RNA decreased FcRIIa 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 FcRIIa and thereby contributes to the termination of FcRIIa signaling via its proteasomal degradation, thus leading to the down-regulation of neutrophil signalisation and function (phagocytosis) through this receptor.

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¹ This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR). L.M. is the recipient of a Canadian Arthritis Network Graduate Award and a CIHR Doctoral Research Award. P.H.N. holds the Canada Research Chair on the Molecular Physiopathology of the Neutrophil.

² Address correspondence to Dr. Emmanuelle Rollet-Labelle, Centre de recherche en Rhumatologie et Immunologie, 2705 Boulevard Laurier, Room T1-49, Quebec City, Quebec G1V 4G2, Canada. E-mail address: emmanuelle.rollet{at}crchul.ulaval.ca

³ Abbreviations used in this paper: DRM, detergent-resistant membrane; DFP, di-isopropyl fluorophosphate; PP2, 4-amino-5-(4-chlorophenil)-7-(t-butyl)pyrazol(3,4-d) pyrimidine); siRNA, small interfering RNA; dPLB-985, differentiated PLB-985.