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

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 FcγRIIA receptor by endocytosis and phagocytosis. A fusion of FcγRIIA to green fluorescent 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 FcγRIIA–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. FcγRIIA–GFP was ubiquitylated in response to receptor cross‐linking. Elimination of the lysine residues present in the cytoplasmic domain of FcγRIIA impaired endocytosis, but not phagocytosis. The proteasomal inhibitor clasto‐lactacystin β‐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.