Published January 19, 2015, doi:10.4049/jimmunol.1401866 The Journal of Immunology Unique Ligand-Binding Property of the Human IgM Fc Receptor Kazuhito Honjo,* Yoshiki Kubagawa,* John F. Kearney,† and Hiromi Kubagawa*,1 The IgM Fc receptor (FcmR) is the newest FcR, and coligation of FcmR and Fas/CD95 on Jurkat cells with agonistic IgM anti-Fas mAb was shown to inhibit Fas-induced apoptosis. The ligand-binding activity of human FcmR was further examined. FcmR- mediated protection from apoptosis was partially blocked by addition of 104 molar excess of IgM or its soluble immune complexes, but it could be inhibited by addition of 10-fold excess of IgM anti-CD2 mAb. This suggests that FcmR binds more efficiently to the Fc portion of IgM reactive with plasma-membrane proteins than to the Fc portion of IgM in solution. The former interaction occurred in cis on the same cell surface, but not in trans between neighboring cells. This cis engagement of FcmR resulted in modulation of Ca2+ mobilization via CD2 on Jurkat cells or BCRs on blood B cells upon cross-linkage with the corresponding IgM mAbs. Several functional changes were observed with FcmR mutants: 1) significant increase in IgM ligand binding in the cytoplasmic tail-deletion mutant, 2) enhanced cap formation in FcmR upon IgM binding at 4˚C with a point mutation of the transmembrane His to Phe, and 3) less protective activity of FcmR in IgM anti-Fas mAb-mediated apoptosis assays with a point mutation of the membrane-proximal Tyr to Phe. These findings show the importance of the cis engagement of FcmR and its critical role in receptor function. Hence, FcmR on B, T, and NK cells may modulate the function of surface proteins recognized by natural or immune IgM Abs on the shared membrane cell surface. The Journal of Immunology, 2015, 194: 000–000. ntibodies have dual binding activity: to Ag via their response, has defied identification, despite extensive biochemical N-terminal variable regions, and to effector molecules evidence of IgM Fc–binding proteins accumulated over decades A such as FcRs via their C-terminal constant regions. FcRs (11–13). We previously successfully identified a cDNA encoding are expressed by many different cell types, and their interaction an authentic FcmR from cDNA libraries of human B-lineage cells with Abs can initiate a broad spectrum of effector functions es- using a functional cloning strategy (14). FCMR is a single-copy sential in host defense. These functions include phagocytosis gene located on chromosome 1q32.2, adjacent to two other IgM- of Ab-coated microbes, lysosomal degradation of endocytosed binding receptor genes, Fca/mR and polymeric Ig receptor. The immune complexes, Ab-dependent cell-mediated cytotoxicity, predicted FcmR is a transmembrane protein that consists of secretion of cytokines and chemokines, release of potent inflam- a single V-set Ig-like domain responsible for Fcm binding, an matory mediators, regulation of Ab production by B cells, survival additional extracellular region with no known domain structure, of plasma cells, and presentation of degradable as well as non- a transmembrane segment containing a charged His residue, and degradable Ags (1–7). These diverse functions depend on the Ab a relatively long cytoplasmic tail (118 aa) containing three con- isotype and the cell type expressing the FcR. Structurally and served Tyr and five conserved Ser residues. FcmR binds pentameric functionally diverse FcRs, namely FcR for IgG (FcgRI/CD64, IgM with a surprisingly high avidity of ∼10 nM as determined by FcgRII/CD32, FcgRIII/CD16, FcgRIV, FcRn), IgE (FcεRI, Scatchard plot analysis, with the assumption of a 1:1 stoichiometry FcεRII/CD23), IgA (FcaR/CD89), and both polymeric IgA and of FcmR to IgM ligand. Upon ligation of FcmR with IgM IgM (Fca/mR/CD351), have been characterized extensively at ligands, both Tyr and Ser residues in the cytoplasmic tail are both the protein and genetic levels (1–5, 8–10). phosphorylated (14), and receptors are rapidly internalized into It has long been a puzzle why an FcR for IgM (FcmR), the first Ig lysosomal compartments (15). Unlike other FcRs, the expres- isotype to appear during phylogeny, ontogeny, and the immune sion of FcmR is restricted to lymphocytes: B, T, and NK cells (14, 16). This suggests potentially distinct functions of FcmRas compared with other FcRs that are mainly expressed by myeloid *Division of Laboratory Medicine, Department of Pathology, University of Alabama cells. at Birmingham, Birmingham, AL 35294; and †Department of Microbiology, Univer- Alternatively, the FcmR was initially designated as Fas apo- sity of Alabama at Birmingham, Birmingham, AL 35294 ptotic inhibitory molecule 3 (FAIM3), because coligation of Fas 1 Current address: Deutsches Rheuma-Forschungszentrum Berlin, Berlin, Germany. and FcmR/FAIM3 with an agonistic IgM anti-Fas mAb prevented Received for publication July 22, 2014. Accepted for publication December 16, 2014. Fas-mediated apoptosis (17). Unlike the effect of IgM anti-Fas This work was supported in part by National Institutes of Health/National Institute mAb, however, ligation of Fas with an agonistic IgG mAb in- of Allergy and Infectious Diseases Grants AI4782-36 (to J.F.K.) and R21AI094625 (to H.K.). duced apoptosis irrespective of the expression of FcmR/FAIM3 (14, 16, 18). Notably, coligation of Fas and FcmR/FAIM3 with Address correspondence and reprint requests to Dr. Hiromi Kubagawa, Department of Pathology, University of Alabama at Birmingham, 506 Shelby, 1825 University the corresponding mouse IgG mAbs plus a secondary reagent Boulevard, Birmingham, AL 35294-2182. E-mail address: [email protected] [e.g., F(ab9)2 fragments of anti-mouse g Ab] had no demonstrable The online version of this article contains supplemental material. effects on the IgG anti-Fas mAb-induced apoptosis (14). This 2+ Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; [Ca ]i, intracel- suggests that the antiapoptotic activity of FcmR/FAIM3 depends 2+ lular Ca concentration; FAIM3, Fas apoptosis inhibitory molecule 3; NP, nitro- on usage of the IgM anti-Fas mAb, and not on physical proximity phenyl; WT, wild-type. of two receptors induced by artificial coligation. However, be- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 cause the FcmR-mediated inhibition of the IgM anti-Fas mAb- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401866 2 THE CIS ENGAGEMENT OF FcmR induced apoptosis is a reproducible and well-defined function, we FcmR mutants employed this antiapoptotic assay in the present study as one of FcmR cDNA constructs with point mutations (H253F, Y315F, Y366F, or the readouts to explore the mode of action of FcmR. In this study, Y385F) were generated by a QuickChange site-directed mutagenesis kit we have defined the unique ligand-binding properties of FcmR and (Stratagene) using nonmutated FcmR cDNA as a template DNA and a set of the key residues involved in its receptor function. primers: 1) 59-GAAGGCCAAGGATTTTTCATCCTGATCCCGACCAT-39 and 59-ATGGTCGGGATCAGGATGAAAAATCCTTGCCCTTC-39for H253F, 2) 59-CTCCCAAAACAACATCTTCAGCGCCTGCC-39 and 59-GGCA- Materials and Methods GGCGCTGAAGATGTTGTTTTGGGAG-39 for Y315F, 3) 59-GACCA- Cells GCTGTGAATTTGTGAGCCTCTACCACCAG-39 and 59-CTGGTGGTA- 9 9 The human leukemic T cell (Jurkat) and mouse thymoma (BW5147) lines, GAGGCTCACAAATTCACAGCTGGTC-3 for Y366F, and 4) 5 -GG- 9 9 which stably expressed either only GFP as a control or both GFP and ACAGTGATTCAGATGACTTCATCAATGTTCCTG-3 and 5 -CAGGA- 9 human FcmR, were prepared by retrovirus-mediated transduction as ACATTGATGAAGTCATCTGAATCACTGTCC-3 for Y385F (underlining previously described (14) and were maintained in media containing indicates mutation sites), according to the manufacturer’s recommendation. For FcmR cytoplasmic tail deletion, an FcmR cDNA construct with a deletion puromycin at 1.5 and 0.75 mg/ml, respectively. BW5147 cells stably of most of the cytoplasmic tail (A281–A390; DCy) but with an inclusion of expressing human FcmR (without GFP) and wild-type (WT) Jurkat or BW5147 cells were maintained in media only as described (14). For Ca2+ the posttransmembrane basic aa-rich region (K273–K280) was generated by m mobilization, mononuclear cells isolated from healthy individuals by PCR using PrimeStar HS DNA polymerase (TaKaRa), Fc R cDNA as 9 GAATTC Ficoll-Hypaque density gradient centrifugation were enriched for B cells a template DNA, and a set of primers: 5 - TAGAAGGGACA- 9 9 GCGGCCGC 9 using a B cell isolation kit II (Miltenyi Biotec) with MACS. The fre- ATGGACT-3 and 5 - CTATTTCCTCCTTTCAACGGCC-3 + Eco Not quency of CD19 B cells in the resultant enriched fractions was .95% (italics indicate RI and I sites, and underlining indicates translation start and stop sites). The amplified PCR products of the expected size were as determined by flow cytometry. This study involving human subjects gel purified and subcloned into the ZeroBlunt TOPO vector (Invitrogen). All was approved by the institutional review board of the University of m Alabama at Birmingham. plasmid DNAs encoding Fc R mutants were digested with appropriate re- striction enzymes, gel purified, and ligated into the pMX-PIE retroviral ex- Immunofluorescence analysis pression vector as described (14). After confirming the correct sequences with the expected mutation of the resultant cDNAs at both strands of DNA, Flow cytometric analysis of cell surface FcmR on transductants was per- they were transfected into 293T-A packaging cell line before transducing formed by using biotin-labeled, receptor-specific mAb (HM14, g1k iso- Jurkat cells as described (14). GFP+ transductants were enriched by FACS type) and isotype-matched control mAb, along with PE-labeled streptavidin before IgM binding, apoptosis, and Ca2+ mobilization analyses. as described (14). For IgM binding, cells were incubated with biotin-labeled mouse myeloma IgM (TEPC183) at 1 mg/ml or PBS, washed, and then Epifluorescence microscopic analysis incubated with PE-labeled streptavidin.