Structural requirements of the cytoplasmic domains of the human macrophage Fcγ receptor IIa and B cell Fcγ receptor IIb2 for the endocytosis of immune complexes

Abstract

Two isotypes of the monocyte/macrophage as well as B cell Fc gamma receptor type II (FcRIIa and FcRIIb2, respectively) mainly differ in the length (76 vs. 44 amino acids) and amino acid sequence of their cytoplasmic domains. Only the eight amino acids just behind the putative transmembrane region are identical. Despite this marked difference, both FcRII mediate endocytosis of immune complexes. To determine the functional significance of the cytoplasmic domains, we expressed truncated FcRIIa and FcRIIb2 in FcR- BHK-21 cells. Mutants of both receptors containing only one amino acid (tail-minus) of the cytoplasmic domain failed to mediate immune complex uptake. The significance of the cytoplasmic domain of the receptors could be further demonstrated using a chimeric FcRIII-FcRIIa construct. Therefore we expressed an FcRIII lacking the hydrophobic carboxyl terminus (containing the putative phosphatidyl - inositol - glycan anchor site) fused inframe to the transmembrane and cytoplasmic domain of the FcRIIa in BHK-21 cells. In contrast to the wild type FcRIII, this chimeric receptor mediated immune complex uptake indistinguishable from that mediated by the FcRIIa. Receptor mutants with relatively short cytoplasmic domains (FcRIIb2: 13, and FcRIIa: 16 amino acids) revealed, that these short amino acid stretches are sufficient to allow reduced receptor-mediated endocytosis of bound ligand. Furthermore, using FcRIIa deletion mutants with a cytoplasmic domain consisting of 62, 46, and 28 amino acids, respectively, we found that the capability of these mutants to mediate immune complex uptake decreased gradually with the truncation of the cytoplasmic tails. Thus, only short amino acid sequences of the cytoplasmic domain are sufficient to enable an, albeit reduced, receptor-mediated endocytosis.

Microbial Immune Suppression Mediated by Direct Engagement of Inhibitory Fc Receptor

“…In this study, we demonstrate that two isoforms, Fc␥RIIA and Fc␥RIIB, are involved in recognition of immune complexes and of GXM, respectively. Fc␥RIIA is regarded as an activator of multiple intracellular pathways (45); in contrast Fc␥RIIB has inhibitory functions because of its ITIM, that activates the SHIP (46). Indeed, engagement of Fc␥RIIB by GXM leads to recruitment on macrophages of SHIP, a molecule considered a mediator of cellular activation/inhibition.…”

Phosphorylation of FcγRIIA is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts

“…The baby hamster kidney BHK-21 cells stably transfected with either wild-type FcγRIIA or Y298F mutant of FcγRIIA (C-terminal tyrosine 298 of ITAM was replaced by phenylalanine) were generated and cloned in the eukaryotic expression vector pBEH pac18 containing the puromycin resistance gene as described previously (Engelhardt et al, 1991;Bewarder et al, 1996). BHK cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml puromycin, 1 mM sodium pyruvate and 2 mM glutamine (all Gibco BRL).…”

In Vivo and In Vitro Specificity of Protein Tyrosine Kinases for Immunoglobulin G Receptor (FcγRII) Phosphorylation

“…Seed (Department of Molecular Biology, Massachusetts General Hospital, Boston). Cytoplasmic deletion mutants of Fc␥RIIa were constructed as described previously (16). The tyrosine exchange mutants of Fc␥RIIa were generated with the U.S.E.…”

“…MluI-ScaI selection primer and different mutagenesis primers which were designed to change one of the three cytoplasmic tyrosine residues to a phenylalanine residue: Fc␥RIIa F-298, 5Ј-GATAAAAAT ATTTTCCTGACTC-3Ј; Fc␥RIIa F-282, 5Ј-CAGCAGACGGCGGCTTCATG-3Ј; and Fc␥RIIa F-275, 5Ј-AATGACTTTGAAACAGCAGAC-3Ј. The isolation and modifications of the cDNAs of Fc␥RIIb1 and Fc␥RIIb2 were described recently (5,15,16).…”