The ubiquitin ligase c-Cbl down-regulates FcgammaRIIa activation in human neutrophils

J Immunol. 2009 Feb 15;182(4):2374-84. doi: 10.4049/jimmunol.0801420.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Down-Regulation
  • Flow Cytometry
  • Humans
  • Immunoprecipitation
  • Microscopy, Confocal
  • Neutrophils / immunology*
  • Neutrophils / metabolism
  • Proto-Oncogene Proteins c-cbl / immunology*
  • Proto-Oncogene Proteins c-cbl / metabolism
  • RNA, Small Interfering
  • Receptors, IgG / immunology*
  • Receptors, IgG / metabolism
  • Signal Transduction / immunology*
  • Transfection
  • Ubiquitination

Substances

  • FCGR2A protein, human
  • RNA, Small Interfering
  • Receptors, IgG
  • Proto-Oncogene Proteins c-cbl