Contrasting requirements for ubiquitylation during Fc receptor-mediated endocytosis and phagocytosis

Abstract

Fc receptors on leukocytes mediate internalization of antibody-containing complexes. Soluble immune complexes are taken up by endocytosis, while large antibody-opsonized particles are internalized by phagocytosis. We investigated the role of ubiquitylation in internalization of the human FcgRIIA receptor by endocytosis and phagocytosis. A fusion of FcgRIIA to green¯uorescent protein (GFP) was expressed in ts20 cells, which bear a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme. Uptake of soluble IgG complexes mediated by FcgRIIA±GFP was blocked by incubation at the restrictive temperature, indicating that endocytosis requires ubiquitylation. In contrast, phagocytosis and phagosomal maturation were largely unaffected when ubiquitylation was impaired. FcgRIIA±GFP was ubiquitylated in response to receptor cross-linking. Elimination of the lysine residues present in the cytoplasmic domain of FcgRIIA impaired endocytosis, but not phagocytosis. The proteasomal inhibitor clasto-lactacystin b-lactone strongly inhibited endocytosis, but did not affect phagocytosis. These studies demonstrate a role for ubiquitylation in the endocytosis of immune receptors, and reveal fundamental differences in the mechanisms underlying internalization of a single receptor depending on the size or multiplicity of the ligand complex.

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

“…These last observations indicate that a selective proteolysis of the cytoplasmic tail of the receptor can also be ruled out. 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.…”

The coordination of signaling during Fc receptor-mediated phagocytosis

“…FcR are ubiquitylated in response to cross-linking, and this modification is necessary for receptor-mediated endocytosis of soluble immune complexes but not phagocytosis of particles [22]. It has not been determined whether the FcR are ubiquitylated during phagocytosis.…”

Differential Role of Actin, Clathrin, and Dynamin in Fcγ Receptor-mediated Endocytosis and Phagocytosis

“…The involvement of clathrin in endocytosis is therefore not unexpected, inasmuch as Fc␥ receptor endocytosis depends on ubiquitination. By contrast, phagocytosis of large particles proceeds normally in ubiquitination-deficient cells (13). It remains to be defined how Fc␥ receptor-induced endocytosis and phagocytosis, two processes triggered by clustering of the same receptors, differ so drastically at the molecular level.…”

“…In contrast, phagocytosis involves acute assembly of F-actin and is blocked by inhibitors of class I phosphatidylinositol 3-kinase, such as wortmannin (11,12). A recent study further highlighted the mechanistic differences between the two modes of Fc␥ receptor internalization (13); endocytosis, but not phagocytosis, was found to require receptor-induced ubiquitination.…”