Phosphorylation-independent Ubiquitylation and Endocytosis of FcγRIIA

Abstract

Endocytosis of the Fc receptor Fc␥RIIA depends on a functional ubiquitin conjugation system, and the receptor becomes ubiquitylated upon ligand binding. Phosphorylation of tyrosines in Fc␥RIIA by Src family kinases is thought to be the initiating event in its signaling. However, although the Src family kinase inhibitor PP1 inhibited both ligand-induced phosphorylation of Fc␥RIIA and phagocytosis in ts20 cells expressing Fc␥RIIA, it did not inhibit receptor ubiquitylation or endocytosis of soluble ligands. Conversely, genistein and the proteasomal inhibitor MG132 did not inhibit receptor phosphorylation but strongly inhibited both receptor ubiquitylation and endocytosis. A region of the receptor lying within the immunoreceptor tyrosine-based activation motif was found to be necessary for both ubiquitylation and endocytosis. Ubiquitylation occurs at the plasma membrane before internalization. Endocytosis of Fc␥RIIA is dependent on clathrin but independent of the adaptor protein AP-2. These findings point to a novel mechanism for ubiquitylation and endocytosis of this immunoreceptor.Although it has long been appreciated that polyubiquitylation of cytoplasmic proteins can target them for degradation by the proteasome, several additional roles for ubiquitylation have emerged. These include functions in the nucleus and in regulating the trafficking of membrane proteins (1). In particular, ubiquitylation of surface receptors can act as a signal both for their endocytosis and their intracellular sorting for degradation in multivesicular bodies (2, 3).Fc␥RIIA, a receptor for IgG-containing immune complexes, is widely expressed in human leukocytes (4). Upon binding of IgG-containing complexes, Fc␥RIIA triggers cell activation and mediates internalization of the immune complexes. We previously showed that cross-linking of Fc␥RIIA induces ubiquitylation of the receptor (5). Furthermore, endocytosis of soluble immune complexes by Fc␥RIIA depends on the presence of a functional ubiquitin conjugation system, whereas actin-driven phagocytosis of large antibody-coated particles does not (5).An important question for understanding the regulation of receptor trafficking is how ligand binding leads to ubiquitylation of the receptor and/or associated proteins. Fc␥RIIA signals for cell activation by virtue of the presence in its cytoplasmic domain of an immunoreceptor tyrosine-based activation motif (ITAM) 2 consisting of the consensus sequence YXXLX (7)(8)(9)(10)(11)(12) YXXL. Such motifs are present in activating Fc receptors, the signaling subunits of the T cell and B cell receptors, and other immunoreceptors (4, 6). The earliest step in signaling from ITAM-containing receptors is thought to be phosphorylation of the tyrosine residues in the ITAM by Src family kinases. The phosphorylated ITAM tyrosines then recruit kinases of the Syk/ZAP-70 family and other effectors to initiate downstream responses such as phagocytosis (4).Furthermore, as ubiquitylation of surface receptors has come to be recognized as a common mechanism for thei...

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

“…3B, ubiquitination of the receptor is also Src kinase dependent. Our results differ from those observed in Fc␥RIIa-transfected CHO-ts20 cells where the delayed ubiquitination of Fc␥RIIa was reported to be independent of tyrosine phosphorylation (62). These differences outline the difficulties of extrapolating from model cell systems to primary cells.…”

“…A stimulated degradation (loss of immunoreactivity) of Fc␥RIIa has previously been noted in HL60 cells (60) and in Fc␥RIIa-transfected CHO-ts20 cells (61). It should be noted that, in the latter model, the degradation was observed only after more than 60 min of stimulation and that, in sharp contrast to the situation in human neutrophils, it was insensitive to inhibition by PP2 (62), thereby suggesting that its molecular basis may be different. We also observed a lack of calcium response to the cross-linking of Fc␥RIIa in cells in which Fc␥RIIa had been cross-linked 5 min previously.…”

Differential Recruitment of Activating and Inhibitory FcγRII during Phagocytosis

“…The Journal of Immunologyendogenous Fc receptors that we have used in previous studies of FcgR-mediated phagocytosis and endocytosis (13,14). When expressed alone in ts20 cells, FcgRIIA-YFP mediated both binding and phagocytosis of IgG-opsonized 3 mm beads, confirming that the tagged protein is functional (Fig.…”

Decoding ubiquitin sorting signals for clathrin-dependent endocytosis by CLASPs

“…The internalization pathway of ubiquitylated cargo proteins Most plasma membrane proteins that undergo ligand-induced or constitutive oligo/polyubiquitylation, including CD4, MHC class I, ␤ 2 adrenergic receptor, ENaC, the anthrax receptor, EGF, GH and PDGF receptors (Bonifacino and Traub, 2003;Hicke and Dunn, 2003) as well as the Fc␥IIRA (Mero et al, 2006) are internalized within clathrin-coated vesicles. Both caveolin-and clathrin-dependent mechanisms, however, have been invoked for uptake of ubiquitylated EGF and PDGF receptors (Chen and De Camilli, 2005;Dupre et al, 2004;Johannessen et al, 2006;Sigismund et al, 2005;Stang et al, 2004).…”