The Ubiquitin Ligase c-Cbl Down-Regulates FcγRIIa Activation in Human Neutrophils

Abstract

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

FcγRIIIb Triggers Raft-dependent Calcium Influx in IgG-mediated Responses in Human Neutrophils

“…We observed that both Fc␥Rs are required for an optimal phagocytosis of opsonized zymosan. They were similarly essential for the stimulation of tyrosine phosphorylation and degradation of Fc␥RIIa (29). Phagocytosis of IgG-opsonized zymosan by human neutrophils required an extracellular influx of calcium that was exclusively dependent on Fc␥RIIIb engagement.…”

“…Phagocytosis Assay-Alexa 488-conjugated zymosan A bioparticles were opsonized by incubation with 10 mg/ml human IgGs at 37°C for 1 h and washed twice in HBSS as described (29). A ratio of 10 particles/cell was added at 25 l of neutrophils (10 ϫ 10 6 cells/ml) preincubated as described in the corresponding legend to initiate phagocytosis.…”

Signalling in Neutrophils: A Retro Look

“…Internalization of the chemoattractant receptors is transient and followed by a recycling to the plasma membrane while that of CD32a results in its proteasome-dependent degradation [48,49]. The mechanisms regulating receptor recycling differ from those involved in their internalization; for example, recycling, as opposed to internalization, of CXCL8 receptors is phosphorylation independent [50,53].…”

“…Arrestins and dynamin also play a role in the internalization of CXCR1/2 which appears to be independent of the interaction with heterotrimeric G proteins as FPR internalization is not affected by pertussis toxin [54]. Engagement of the opsonin receptor CD32a similarly leads to its internalization and subsequent proteosomal degradation [49].…”

“…This includes receptors for chemoattractants such as fMLF, C5a and IL-8 (e.g., [47]) as well as opsonins (CD32a, CD16b) [48,49]. Different regions of the carboxy-terminus of the receptors have been identified that play a role in these events mediated in part by phosphorylation.…”