©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Association between the Human Myeloid Immunoglobulin A Fc Receptor (CD89) and FcR Chain
MOLECULAR BASIS FOR CD89/FcR CHAIN ASSOCIATION (*)

(Received for publication, July 17, 1995; and in revised form, September 25, 1995)

H. Craig Morton Ingrid E. van den Herik-Oudijk Paula Vossebeld (1) Alies Snijders (§) Arthur J. Verhoeven (1) Peter J. A. Capel Jan G. J. van de Winkel (¶)

From the Department of Immunology, University Hospital Utrecht, Heidelberglaan 100, 3584 CX, Utrecht Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

FcR chain has previously been shown to interact with the TCR-CD3 complex, the IgE Fc receptor I (FcRI), and the class I and IIIA IgG receptors (FcRI and FcRIIIa). Here, we demonstrate that the Fc receptor chain associates with FcalphaR in transfected IIA1.6 B lymphocytes. FcalphaR could be expressed at the surface of IIA1.6 B cells by itself, but was devoid of signaling capacity. Upon co-expression of FcR chain, a physical interaction with FcalphaR could be demonstrated. This association proved crucial for the triggering of both proximal (intracellular calcium increase and tyrosine phosphorylation), as well as distal (IL-2 release), signal transduction responses. We next tested the hypothesis that a positively charged arginine residue (Arg) within the transmembrane domain of FcalphaR promotes association with FcR chain. We therefore constructed FcalphaR molecules where Arg was mutated to either a positively charged histidine, a negatively charged aspartic acid, or an uncharged leucine. A functional association between FcalphaR and FcR chain was observed only with a positively charged residue (Arg or His) present within the FcalphaR transmembrane domain. These data show that transmembrane signal transduction by the FcalphaR is mediated via FcR chain, and that FcalphaR requires a positively charged residue within the transmembrane domain to promote functional association.


INTRODUCTION

IgA is the primary immunoglobulin in bodily secretions and plays a critical role in protection against the constant environmental challenges at mucosal sites. Although the protective mechanisms are incompletely defined, a significant role in IgA-mediated immune defense has been proposed for IgA Fc receptors. These molecules have been detected on most populations of phagocytic cells in blood and mucosal tissues. Engagement of these molecules can trigger phagocytosis, degranulation, oxidative burst, inflammatory mediator release, and antibody-dependent cellular cytotoxicity(1, 2) . FcalphaR on monocytes/macrophages and neutrophils has been defined as a 55-75-kDa glycoprotein(3, 4) , whereas the eosinophil FcalphaR is more heavily glycosylated (70-100 kDa)(5) . Both types of myeloid receptors are recognized by the CD89 mAb (^1)panel (4, 5) and bind both IgA1 and IgA2 via their Fc regions(1) . The cDNA encoding the myeloid FcalphaR has been characterized and was found to encode a 30-kDa peptide, with two extracellular Ig-like domains, a hydrophobic transmembrane region and a cytoplasmic tail devoid of recognized signaling motifs(2, 6) . Additionally, we have recently isolated and characterized the human gene encoding the CD89 molecule. The gene structure indicates FcalphaR to represent a more distantly related member of the immunoglobulin receptor gene family (7) .

To explore the capacity of FcalphaR to trigger biological functions, we have now generated different transfectants in the mouse IIA1.6 B cell line. This line, derived from the A20 B cell lymphoma, lacks the 5`-end of the FcRII gene and, consequently, is Fc receptor-negative(8) . Previous work showed this line to represent an excellent model for assaying FcR-mediated functioning(9, 10, 11) . Following transfection, FcalphaR was expressed at the surface of IIA1.6 cells by itself, but lacked signaling capacity. We therefore hypothesized that FcalphaR may associate with a specialized signaling molecule. The FcR chain was known previously to associate with all three classes of FcR, FcRI, and the TCR-CD3 complex(12, 13, 14, 15) . FcR chain is responsible for coupling these receptors to intracellular signaling pathways(16) . By co-transfection experiments, we tested whether FcR chain could mediate signal transduction via FcalphaR. Our results show that co-expression of FcalphaR and FcR chain in IIA1.6 B cells conferred both proximal and distal signaling capacity to FcalphaR. During the preparation of this manuscript, it was shown that, in the U937 cell line, FcalphaR was associated with FcR chain and that chain was phosphorylated on tyrosine residues following FcalphaR cross-linking (17) . The data presented here indeed confirm that FcalphaR associates with FcR chain in transfected IIA1.6 B cells and, furthermore, suggest that FcalphaR and FcR chain can associate in normal blood PMN. The present experiments demonstrate FcR chain to be critical for FcalphaR-mediated transmembrane signal transduction.

We, furthermore, explored the molecular basis for FcalphaR/FcR chain association. The transmembrane (TM) domain of FcalphaR is unusual as it contains a single positively charged arginine (Arg) residue. Since the TM domain of the FcR chain contains a single negatively charged aspartic acid residue, we hypothesized that these oppositely charged residues may promote association. A similar mechanism involving charged TM amino acids has previously been shown to be involved in the assembly of the TCR-CD3 complex(18, 19, 20, 21) . Using PCR, we mutated the Arg found in the wild type FcalphaR (R209) to either a positively charged histidine (R209H), a negatively charged aspartic acid (R209D), or an uncharged leucine (R209L). Mutated FcalphaR cDNAs were transfected together with FcR chain to IIA1.6 cells and assessed for their ability to trigger an increase in [Ca] following FcalphaR cross-linking. Our data show a positively charged residue within the TM domain of FcalphaR to be required for functional association with FcR chain.


MATERIALS AND METHODS

Cells

The murine B cell line IIA1.6 (8) and the human cell line U937 were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. FcalphaR and FcalphaR/ transfectants were maintained in the same medium supplemented with either Geneticin (G418, 0.8 mg/ml; Life Technologies, Inc.) alone or Geneticin and methotrexate (10 µM; Pharmachemie, Haarlem, The Netherlands), respectively. PMN were purified as described previously(22) . Murine macrophages were isolated from the peritoneal cavity by rinsing with RPMI 1640, 10% fetal calf serum.

Expression Vectors and Transfections

The human FcalphaR cDNA contained within the pCAV vector was a kind gift from Dr. C. Maliszewski(6) . The murine FcR chain cDNA (23) was cloned into the pNUT expression vector (pNUT-), allowing selection of transfectants with methotrexate(24) . Transfection of IIA1.6 B cells was performed by electroporation as described previously(10) . IIA1.6 B cells were stably transfected with FcalphaR alone (FcalphaR) or with FcalphaR and chain (FcalphaR/) as follows. FcalphaR cells were generated by co-transfection of FcalphaR cDNA in pCAV, using pRC-CMV.neo as selection marker(10) . Two days following transfection, cells were transferred to medium containing G418 and seeded in 24-well plates. After 14 days, each well was tested for FcalphaR expression by FACS analysis using anti-FcalphaR mAb A77 (murine IgG1; (4) ) or My43 (murine IgM; (25) ). FcalphaR cells were isolated (two rounds of selection) using mAb A77 and Dynabeads coated with rat anti-mouse IgG1 (Dynal, Oslo, Norway)(10) . FcalphaR/ cells were generated by co-transfection of pCAV, pNUT-, and pRC-CMV.neo. Two days following transfection, cells were transferred to medium containing G418 and 1 µM methotrexate. After 2 weeks, the concentration of methotrexate was increased to 10 µM. All cells surviving this selection procedure were found to be FcalphaR-positive. Expression of FcR chain was analyzed by RT-PCR, immunoprecipitation, and Western blotting.

Construction of Mutant FcalphaR cDNAs

Overlap extension PCR (26) was used to construct mutant FcalphaR cDNAs where the codon CGC (encoding Arg in the wild type FcalphaR) was mutated to either CAC (histidine), GAC (aspartic acid), or CTA (leucine). For each mutant, a first round of PCR was performed, as follows, generating two DNA fragments with overlapping ends. 5` fragments were generated using a sense oligonucleotide primer FCAL (5`-ATGGACCCCAAACAGACC-3`) and either antisense primer H2 (5`-TGCCACGGCCATGTGGATCAAGTT-3`), D2 (5`-TGCCACGGCCATGTCGATCAAGTT-3`), or L2 (5`-TGCCACGGCCATTAGGATCAAGAT-3`). Similarly, 3` DNA fragments were generated using the antisense primer AS1 (5`-TCCAGGTGTTTACTTGCAGACAC-3`) and either the sense primer H1 (ACCTTGATCCACATGGCCGTGGCA-3`), D1 (5`-ACCTTGATCGACATGGCCGTGGCA-3`), or L1 (5`-ACCTTGATCCTAATGGCCGTGGCA-3`). Mismatch nucleotides responsible for the introduction of the desired mutation are underlined. First round PCR amplification was carried out by using 50 ng of FcalphaR cDNA as template, in a reaction mixture containing 50 mM KCl, 20 mM TrisbulletCl, pH 8.3, 2.5 mM each dNTP, 2.5 mM MgCl(2), 0.1 mg/ml bovine serum albumin, 10 pmol of each primer, and 2 units of Vent DNA polymerase (New England Biolabs) in a final volume of 100 µl. Samples were overlaid with light mineral oil, denatured at 94 °C for 5 min, before 30 cycles of amplification in a DNA thermal cycler 480 (Perkin Elmer) using a step program (94 °C, 1 min; 55 °C, 1 min; 72 °C, 2 min) followed by a final extension at 72 °C for 7 min. A second round of PCR was subsequently used to generate mutated cDNAs encoding the entire FcalphaR coding region. PCR conditions were as above, except that 25 ng of each overlapping DNA fragment was added as template along with 10 pmol of primers FCAL and AS1, and 1 unit of AmpliTaq DNA polymerase (Perkin Elmer) was used to amplify mutated cDNA fragments. PCR products were subcloned into pGEM-T (Promega) vectors, and the integrity of all mutants was confirmed by sequence analysis. Mutant cDNAs were cloned into the eukaryotic expression vector pSG5 (27) prior to transfection as described above.

Immunofluorescence

Cells were incubated with either mAb A77 or My43 culture supernatant for 30 min at 4 °C. Cells were washed twice with PBS, 1% bovine serum albumin, 0.1% NaN(3), and subsequently incubated with either FITC-conjugated goat anti-mouse (GAM) IgG1 or FITC-conjugated GAM IgM, respectively (Southern Biotechnology). Following 30 min incubation at 4 °C, cells were washed twice and analyzed on a FACScan flow cytometer (Becton Dickinson). For intracellular staining, cells were permeabilized by incubation in FACS Lysing Solution (Becton Dickinson) for 10 min at room temperature. After washing, FcR chain expression was detected by incubation with a rabbit antiserum against FcR chain (generously provided by Dr J.-P. Kinet; (28) ) followed by FITC-conjugated goat anti-rabbit F(ab`)(2) fragments (Southern Biotechnology).

Calcium Mobilization Assays

Intracellular free calcium levels were analyzed using a FACScan(10) . Briefly, cells were loaded simultaneously with SNARF-1 (2.8 µM) and Fluo-3 (1.4 µM) (Molecular Probes) by incubation for 30 min at 37 °C. After washing, cells were resuspended in calcium mobilization buffer at a concentration of 1 times 10^7 cells/ml. A flow rate of 140 cells/s was used for calcium mobilization studies with the initial 24 s of each run used to establish a baseline value for the intracellular free calcium concentration, [Ca](i). The ability of FcalphaR (with or without FcR chain) to trigger [Ca](i) increases was determined by incubating SNARF-1/Fluo-3-loaded cells with either mAb A77 or My43 for 20 min at room temperature (in the dark). After washing, cells were run on a flow cytometer for 24 s, and GAM IgG1 or GAM IgM Ab was added to cross-link FcalphaR.

RNA Isolation, Reverse Transcription, and RT-PCR

Total cellular RNA was extracted from 1 times 10^7 cells using the RNAzol B isolation method followed by cDNA synthesis (10) . FcR chain transcripts were detected via PCR using two chain-specific primers, MG1 (sense; 5`-ATCTCAGCCGTGATCTTG-3`) and MG2 (antisense; 5`-TCAGGTCTCTGGCAGCTT-3`) (Isogen Bioscience, Amsterdam) encompassing nucleotides 46-578 of murine chain. FcalphaR transcripts were amplified using primers FCAL and AS1 (see above). As a control, we used a sense and antisense primer set for the hypoxanthine-guanine phosphoribosyltransferase housekeeping gene ((29) ; a gift from Dr. H. Savelkoul, Erasmus University, Rotterdam).

Immunoprecipitations

Cells (1 times 10^7 per precipitation) were washed three times in PBS before surface radioiodination by the lactoperoxidase method. Cells were lysed in 1% digitonin, 150 mM NaCl, 10 mM triethanolamine, pH 7.4, containing the protease inhibitors phenylmethylsulfonyl fluoride, N-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor, and leupeptin (15) for 45 min at 4 °C. Insoluble material was removed by centrifugation at 13,000 times g for 30 min. Lysates were then precleared four times with Protein G-coated Sepharose CL-4B beads (Pharmacia) and once with beads coated with either an irrelevant mouse IgG1 mAb directed against plant allergens (CLB, Amsterdam) (A77 control) or rabbit serum (FcR chain antiserum control). Specific precipitations were then performed with beads coated with either mAb A77 or a rabbit antiserum against FcR chain. The beads were washed four times with digitonin lysis buffer, and precipitates were analyzed by reducing SDS-PAGE on 10% polyacrylamide gels followed by autoradiography.

Western Blotting

Proteins were immunoprecipitated as described above, separated by nonreducing SDS-PAGE on 15% polyacrylamide gels, and electrotransferred to nitrocellulose membranes (14) . The blots were incubated in blocking buffer (PBS containing 5% nonfat dry milk) for 1 h at room temperature and then with rabbit anti- chain serum (1:2000) for 2 h (room temperature). After several washes, blots were incubated with horseradish peroxidase-conjugated horse anti-rabbit Ig (1:2000; CLB, Amsterdam) in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 8.0) containing 1.5% nonfat dry milk. After extensive washing, bound antibody was detected by chemiluminescence (Boehringer Mannheim, Mannheim, Germany).

Tyrosine Phosphorylation Assays

Transfectants were incubated with mAb A77 for 30 min at room temperature, washed twice with RPMI 1640 medium, and resuspended in separate tubes (2 times 10^5 cells/30 µl). GAM IgG1 (final concentration 20 µg/ml) was added for the indicated time periods at 37 °C to cross-link FcalphaR. Reactions were stopped by the addition of 70 µl of reducing SDS-PAGE sample buffer (50 mM Tris/HCl (pH 6.8), 10% glycerol, 4.3% SDS, 0.05% bromphenol blue, 4% beta-mercaptoethanol). Tyrosine phosphorylation signals resulting from cross-linking sIgG2a by addition of GAM F(ab`)(2) fragments for 2 min at 37 °C served as a positive control. Background levels of tyrosine phosphorylation were determined by omitting the cross-linking GAM IgG1. After boiling for 3 min, 15-µl aliquots were separated on 7.5% SDS-PAGE gels and electrotransferred to nitrocellulose membranes (0.45 µm; Schleicher & Schuell, Dassel, Germany) using a Transblot cell system (Bio-Rad). Membranes were blocked in PT buffer (PBS, 0.1% Tween 20) containing 5% nonfat dry milk (Nutricia, Zoetermeer, The Netherlands) and probed with anti-phosphotyrosine mAb (4G10; UBI, Lake Placid, NY) in PT, 0.05% nonfat dry milk for 1.5 h at room temperature. Following washing three times in PT and once in PT, 0.5% nonfat dry milk for 20 min, membranes were incubated with peroxidase-conjugated GAM IgG (Dako, Glostrup, Denmark) for 1.5 h at room temperature. After washing twice with PT and once with PT, 0.5% NaCl, bound antibodies were detected using the ECL detection system (Amersham, Buckinghamshire, UK).

IL-2 Production

Transfectants were incubated with mAb A77 for 30 min at room temperature. Following washing, cells were seeded into 96-well plates and GAM IgG1 (30 µg/ml) added to the culture supernatant for 24 h at 37 °C. Alternatively, transfectants were added to wells pre-coated with either human serum IgA (Organon Teknika, Belgium) or human polymeric IgA, isolated from the serum of myeloma patients (a gift from Dr. M. Daha), and incubated for 24 h at 37 °C. As control, IL-2 production was triggered via surface IgG2a cross-linking by addition of GAM Ab to culture supernatants for 24 h at 37 °C. IL-2 production was assessed by culturing 1 times 10^4 CTLL-2 IL-2-dependent cells with the transfectant culture supernatants in 96-well plates. After a 24-h incubation at 37 °C, 1 µCi of [^3H]thymidine was added to each well and cultured for 4 h, and cells were harvested onto glass fiber filters (Wallac, Turku, Finland) for liquid scintillation counting.


RESULTS AND DISCUSSION

Expression of FcalphaR and Chain in Transfected IIA1.6 B Cells

We transfected IIA1.6 B lymphoma cells with cDNAs encoding either FcalphaR alone, or FcalphaR and FcR chain. The FcalphaR was surface-expressed at a high level in both FcalphaR and FcalphaR/ transfectants (Fig. 1A). In FcalphaR/ transfectants, the presence of a chain message was detected by specific RT-PCR (Fig. 1B). Expression of chain at the protein level was confirmed by FACS analysis of permeabilized cells using an anti- chain serum (Fig. 1C). FcalphaR expressed on IIA1.6 cells were reactive with previously described CD89 mAb A77 (Fig. 1A), My43, and A59 and were capable of binding human IgA (data not shown). The increased level of FcalphaR expression seen in FcalphaR/ cells was presumably not due to co-expression of the FcR chain since no increase in FcalphaR expression was observed following transfection of FcR chain cDNA to FcalphaR IIA1.6 cells. Furthermore, these latter cells were capable of all signaling processes attributable to FcalphaR/ cells (data not shown).


Figure 1: Expression of FcalphaR and FcR chain in transfected IIA1.6 cells. A, surface expression of FcalphaR in IIA1.6 B cells. Cells transfected with FcalphaR alone (FcalphaR) or with both FcalphaR and chain (FcalphaR/) were incubated with CD89 mAb A77 (solid line) or with immunofluorescence buffer alone (dotted line) followed by FITC-conjugated GAM IgG1 Ab. B, detection of FcR chain message. Total cellular RNA was isolated, reverse-transcribed to cDNA, and FcR chain and hypoxanthine-guanine phosphoribosyltransferase (HPRT) transcripts were detected via PCR. C, expression of FcR chain. Permeabilized cells were incubated with anti- chain serum followed by FITC-conjugated anti-rabbit serum and analyzed by flow cytometry. Fluorescence intensity of untransfected IIA1.6 cells was identical with that of FcalphaR transfectants (data not shown).



We found untransfected IIA1.6 cells not to express endogenous FcR chain (Fig. 1B). This observation suggests that, in contrast to FcRIIIa and FcRI(12, 16) , mere cell surface expression of FcalphaR in IIA1.6 cells is not dependent on the presence of FcR chain. Previously, it was also shown that FcalphaR could be expressed on the surface of COS cells in the absence of FcR chain(6) . Similarly, the high affinity IgG receptor FcRI (CD64), which also associates with FcR chain(13, 14) , can be expressed by itself in COS (13) and 3T3 transfectants(30) .

Physical Association between FcalphaR and Chain in Transfected IIA1.6 B Cells and in PMN

We next performed experiments to determine whether there is physical association between FcalphaR and FcR chain. Cells were surface-labeled with I and lysed in 1% digitonin prior to immunoprecipitation with anti-FcalphaR mAb A77 or anti-FcR chain antiserum (Fig. 2A). Immunoprecipitation with CD89 mAb A77 resulted in the isolation of one major band of 60 kDa (arrowheads) from both FcalphaR (lane 2) and FcalphaR/ (lane 3) transfectants, but not from untransfected IIA1.6 cells (lane 1). The molecular weight of the observed band is consistent with the predicted size of FcalphaR(3, 4, 5) . Immunoprecipitation of radiolabeled cell lysates with an anti- chain-specific antibody precipitated one band of 60 kDa only from the FcalphaR/ transfectants (Fig. 2A).


Figure 2: Physical association between FcalphaR and FcR chain in transfected IIA1.6 B cells and PMN. Surface radioiodinated cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with either mAb A77 or anti-FcR chain antiserum. Immunoprecipitates were separated by gel electrophoresis under reducing conditions, followed by autoradiography. A, radiolabeled cell lysates from IIA1.6 cells (lanes 1 and 4), FcalphaR (lanes 2 and 5), and FcalphaR/ (lanes 3 and 6) transfectants were immunoprecipitated with either A77 (lanes 1-3) or anti- chain serum (lanes 4-6). Positions of FcalphaR are marked by arrowheads. B, 1% digitonin cell lysates (as indicated) were immunoprecipitated with either A77 mAb (lanes 1-4) or anti- chain serum (lane 5). Precipitates were separated by nonreducing SDS-PAGE, transferred to nitrocellulose, and probed with anti- chain serum. C, control precipitations were also performed using beads coated with an irrelevant murine IgG1 Ab (mIgG1), as isotype control for A77 (lanes 1-4), or normal rabbit serum (Rbt serum) as a control for anti- chain serum (lane 5). D, mouse and human FcR chains were precipitated from lysates of different mouse (lanes 1 and 2) or human (lanes 3 and 4) cells as detailed under ``Materials and Methods.'' The position of molecular mass standards are marked on the left.



Western blotting analysis of immunoprecipitated proteins further supported the presence of a physical interaction between FcalphaR and FcR chain in transfectants and suggest that FcalphaR and FcR chain can also associate in peripheral blood PMN. Proteins were precipitated from digitonin-solubilized cells with beads coated with either A77, anti-FcR chain serum, or control serum (see ``Materials and Methods''), transferred to nitrocellulose, and probed with anti-FcR chain serum. In IIA1.6 cells, a specific band of 20 kDa, corresponding to the expected size of FcR chain homodimers(12) , was co-precipitated by anti-FcalphaR mAb A77 from FcalphaR/ cells only (Fig. 2B, lane 3). No bands of this size were precipitated by A77 from either untransfected IIA1.6 or FcalphaR transfectant cell lysates (lanes 1 and 2). Similarly sized bands were detected in PMN cell lysates following immunoprecipitation with either A77 (lane 4) or anti- chain serum (lane 5). No specific bands of this size were seen following immunoprecipitation with either an irrelevant mouse IgG1 antibody (Fig. 2C, lanes 1-4; A77 control) or with normal rabbit serum (Fig. 2C, lane 5; anti- chain control). The 28-kDa bands seen in Fig. 2B, lanes 1-5, are considered to be nonspecific since these bands were also seen in the control immunoprecipitations (Fig. 2C, lanes 1-5). We, furthermore, observed a slight difference in mobility of the FcR chain between transfectants (murine chain) and PMN (human chain) (Fig. 2B, lane 3 versus lanes 4 and 5). This different migration profile was surprising in view of the high homology between murine and human chains(12) . Therefore, we next precipitated murine chain from FcalphaR/ transfectants and murine peritoneal macrophages, and human chain from PMN and U937 cells. Following SDS-PAGE and transfer to nitrocellulose, the blots were probed with anti- chain serum. Results presented in Fig. 2D, clearly showed a slight, albeit significant, difference in mobility between murine (lanes 1 and 2) and human (lanes 3 and 4) FcR chains. The observed difference in the mobilities of human and mouse chains may be explained by the slightly different amino acid sequences of these two chains. However, the phosphorylation state of FcR chain has also been shown to affect its mobility in SDS-PAGE(31) ; therefore, differential phosphorylation between human and murine cells may also explain the observed size difference.

Previously it has been demonstrated that FcR chain homodimers associate with TCR-CD3, FcRI, and all three classes of FcR (12, 13, 14, 15) . During the preparation of this manuscript, it was demonstrated that FcR chain may also be found in membrane complexes with FcalphaR in the U937 cell line(17) . Our results confirm this observation in a transfectant model system and, furthermore, provide evidence to suggest that FcalphaR may associate with FcR chain in normal peripheral blood PMN.

FcR Chain Is Critical for FcalphaR Signaling in IIA1.6 B Cells

We next assessed FcalphaR signal transduction by both types of IIA1.6 B cell transfectants. These FcR-negative B lymphoid cells have been used extensively as a model system for studying FcR functioning. IIA1.6 cells express endogenous surface IgG2a, which upon cross-linking triggers a calcium flux, protein tyrosine phosphorylation, and synthesis and release of IL-2(9, 10, 11) . Changes in [Ca](i) and tyrosine kinase activation are important proximal signaling events and have been correlated with the presence of immunoreceptor tyrosine-based activation motifs (ITAMs) (32) within receptor cytoplasmic domains. FcalphaR is devoid of ITAMs (6) while the FcR chain homodimer contains two such motifs(16) .

Our results show FcR chain to be required for generation of both proximal and distal signaling events via FcalphaR. Cross-linking of FcalphaR in FcalphaR/ transfectants leads to a rapid rise in [Ca](i) that was maintained for at least 2 min (Fig. 3A). In the absence of FcR chain, no [Ca](i) increase was observed. Following FcalphaR cross-linking, a rapid tyrosine phosphorylation of cellular proteins was also observed only in FcalphaR/ cells (Fig. 3B). Tyrosine phosphorylation was detected within 20 s and reached a maximum between 40 s and 2 min in all experiments. In all experiments, no detectable signaling responses were initiated when the cells were incubated with GAM IgG1 mAb alone, indicating that this antibody does not react with the surface IgG2a expressed by the IIA1.6 cells (as reported previously; (33) ).


Figure 3: Signalling events triggered by cross-linking FcalphaR in transfected IIA1.6 B cells. A, calcium mobilization triggered by FcalphaR in SNARF-1/Fluo-3-loaded transfectants. FcalphaR (dotted line) or FcalphaR/ (solid line) transfectants were incubated with CD89 mAb A77 for 20 min at room temperature, and FcalphaR was subsequently cross-linked with GAM IgG1 F(ab`)(2) (arrow). [Ca] levels were analyzed by flow cytometry as described under ``Materials and Methods.'' Data are representative of five individual experiments. B, tyrosine phosphorylation of cellular proteins upon cross-linking FcalphaR in FcalphaR (left panel) or FcalphaR/ (right panel) IIA1.6 B cells. Transfectants were incubated with mAb A77 for 30 min at room temperature and washed twice with RPMI 1640 medium. Cells were then incubated with GAM IgG1 Ab for the indicated time periods. As control, cells were also incubated either with A77 alone (A77) as negative control or with GAM IgG (GAM) to cross-link the surface IgG (positive control). Samples were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-phosphotyrosine mAb. C, induction of IL-2 production following cross-linking of FcalphaR in transfected IIA1.6 B cells. FcalphaR (open boxes) and FcalphaR/ (filled boxes) transfectants were incubated for 24 h in wells coated with either serum IgA (IgA) or polymeric IgA (pIgA). Alternatively, transfectants were incubated with CD89 mAb A77 for 30 min at room temperature, washed, and seeded into wells. A cross-linking GAM IgG1 Ab was then added to the culture supernatant for 24 h (A77+GAM1). Control cells were incubated with culture medium alone (medium) or had GAM Ab added to cross-link sIgG2a (GAM). Results shown are representative of data obtained for IL-2 release in three separate experiments.



Recently, it has been noted that FcalphaR cross-linking triggers phosphorylation of FcR chain in U937 cells(17) , supporting the hypothesis that FcR chain is critically involved in the generation of FcalphaR signal transduction responses.

Very little is known concerning signal transduction pathways associated with FcalphaR. Interestingly, it has recently been proposed that FcalphaR surface expression may be regulated by [Ca](i), since treatment of neutrophils with known calcium agonists leads to up-regulated FcalphaR expression (34) . This observation, taken together with our data that FcalphaR/ chain cross-linking mediates a rapid rise in [Ca](i), may explain the phenomena of IgA-induced FcalphaR expression-up-regulation, a function apparently unique to FcalphaR, at least among other FcRs(2) .

We, furthermore, examined the capacity of FcalphaR and FcalphaR/ transfectants to trigger the release of IL-2 from IIA1.6 B cells. Secretion of IL-2, triggered via FcalphaR cross-linking, also proved to be dependent on FcR chain co-expression (Fig. 3C). IL-2 was secreted following FcalphaR cross-linking by polymeric IgA, serum IgA, and CD89 mAb A77. The ability of polymeric IgA to trigger a higher level of IL-2 release than serum IgA may indicate FcalphaR to have higher affinity for this molecular species. IL-2 release represents a distal signaling event which can be triggered via cross-linking sIgG2a in IIA1.6 B cells(35) . IIA1.6 sIgG2a is part of the B cell antigen receptor signaling complex which includes at least four ITAMs(36) . In contrast, previous data from our laboratory utilizing a panel of FcR IIA1.6 transfectants showed that cross-linking of FcRIIa (CD32; with only one ITAM) was sufficient to induce a Ca response and tyrosine kinase activation but not IL-2 release(10, 32) . Taken together, these data suggest the number of ITAMs within signaling complexes to be of importance for determining the type of signals generated via receptor complexes in IIA1.6 B cells. This hypothesis is supported by recent work in Jurkat cells, where the number of ITAMs within the TCR chain was found to quantitatively affect T cell responses(37) .

Several reports in the literature suggest FcalphaR to be capable of synergizing with FcR in promoting ADCC, phagocytosis, and the respiratory burst(38, 39, 40) . Our results provide a model for such co-operation in which FcR chain mediates signal transduction via both types of receptors. The importance of FcR chain in triggering phagocytosis via FcR has recently been demonstrated in FcR chain knock-out mice (41) and transfection studies(42, 43) . Since our data suggest that FcalphaR functioning also depends on FcR chain association, we hypothesize that FcR chain-deficient mice may display aberrations in IgA-mediated mucosal immune responses.

Molecular Basis for FcalphaR/FcR Chain Association

Earlier reports proposed the interaction of FcR chain with FcRI and FcRIIIA to involve a conserved region (LFAVDTGL) within the TM domains of these receptors, containing a negatively charged aspartic acid residue (underlined)(12) . Human FcRI (which also associates with FcR chain) displays high homology with FcRI and FcRIIIA within this region of the TM domain (MFLVNTVL), but lacks such an aspartic acid residue(13, 14, 44) . The predicted TM domain of FcalphaR is 19 amino acids long and, unusually, contains a positively charged arginine residue at position 209 (Arg) (6; Fig. 4). Moreover, FcalphaR TM domain displays no obvious homology at the protein level to the TM domains of FcRI, FcRIIIA, or FcRI(13, 14) .


Figure 4: Protein sequence within the region of the transmembrane domains of FcalphaR, FcRI, and FcR chain. Predicted transmembrane spanning residues are underlined. Positively () and negatively () charged residues which may mediate association between FcalphaR, FcRI, and FcR chain are indicated.



Although, in general, charged residues are uncommon within the TM domains of integral membrane proteins, they are not unknown. For example, the TCR-alpha and TCR-beta chains have conserved positively charged residues within their TM domains while the invariant chains of the CD3 complex contain negatively charged TM residues. Elegant site-directed mutagenesis experiments have shown these charged residues to be of critical importance for surface expression of the TCR-CD3 complex(18, 19, 20, 21) .

Therefore, based upon the predicted TM regions of FcalphaR and FcR chain (Fig. 4), we hypothesized the positively charged Arg to be important for association with FcR chain. To test this hypothesis, we constructed mutant FcalphaR molecules using overlap extension PCR (see ``Materials and Methods''), in which the wild type Arg residue was replaced by either a positively charged histidine (FcalphaR-R209H), a negatively charged aspartic acid (FcalphaR-R209D), or an uncharged leucine (FcalphaR-R209L). Mutant FcalphaR cDNAs were transfected together with FcR chain to IIA1.6 B cells. FcalphaR and FcR chain mRNA transcripts were readily detectable by RT-PCR, and the resulting mutated FcalphaR proteins were well expressed at the cell surface as shown by FACS analysis (Fig. 5, A and B). Surprisingly, however, FcR chain protein was only observed in transfectants co-expressing an FcalphaR molecule possessing a positively charged residue within the TM domain, i.e. wild type FcalphaR and FcalphaR-R209H (Fig. 5C). We next assayed mutant FcalphaRs for their ability to form functional FcalphaR/FcR chain signaling complexes, by measuring the increase in [Ca](i) triggered upon FcalphaR cross-linking (see ``Materials and Methods''). Predictably, only the FcalphaR-R209H mutant resulted in intact functional integrity of the FcalphaR/FcR chain complex comparable to the wild type FcalphaR (Fig. 5D). These data demonstrate that a positively charged residue within the FcalphaR TM domain promotes functional association with FcR chain. Our studies suggest furthermore that, in IIA1.6 cells, FcR chain molecules unable to associate with FcalphaR are degraded. This occurs possibly via a mechanism recognizing the charged aspartic acid residue within the TM domain (Fig. 4). The presence of charged residues within the TM domain of some proteins can result in their retention and degradation in the endoplasmic reticulum(44) .


Figure 5: A, cells transfected with either FcalphaR-R209D, -R209H, or -R209L and FcR chain were incubated with CD89 mAb My43 (curve 2) or with immunofluorescence buffer alone (curve 1) followed by FITC-conjugated GAM IgM Ab. B, detection of FcalphaR and FcR chain mRNA transcripts by RT-PCR as described under ``Materials and Methods.'' C, detection of FcR chain protein by Western analysis. Digitonin cell lysates (as indicated) were immunoprecipitated (IP Ab) with either anti- chain serum (upper panel) or normal rabbit serum (Rbt serum) as control (lower panel). Precipitates were separated by nonreducing SDS-PAGE, transferred to nitrocellulose, and probed with anti- chain serum (WB Ab). The position of molecular mass standards are marked on the left. D, calcium mobilization triggered by mutant FcalphaR/FcR chain in SNARF-1/Fluo-3-loaded transfectants. Transfectants were incubated with CD89 mAb My43 for 20 min at room temperature, and FcalphaRs were subsequently cross-linked with GAM IgM. [Ca] levels were analyzed by flow cytometry as described under ``Materials and Methods.'' The lines represent FcalphaR-R209H/ (box), FcalphaR-R209D/ (), and FcalphaR-R209L/ (). FcalphaR/ (bullet) and FcalphaR (circle) are also shown for comparison. Data are representative of three individual experiments.



A similar charge-based mechanism may also be operational for FcRI-FcR chain association, since a positively charged histidine residue is located directly preceding the predicted TM domain(45) . Charged residues located at or near the extracellular/TM boundary may also be able promote association between protein subunits as suggested for the alpha and beta subunits of the major histocompatibility complex class II molecule(46) . The fact that surface expression of both FcalphaR and FcRI appears independent of FcR chain (in contrast to FcRI and FcRIIIA) may, indeed, argue for a different type of FcR chain association between these two receptors and either FcRI or FcRIIIa.

In conclusion, our data demonstrate that FcalphaR is capable of associating with the FcR chain in a transfectant model system and also provides evidence that these two proteins can associate in peripheral blood PMN. We, further, show FcalphaR signal transduction responses to be critically dependent upon co-expression of FcR chain, and that a positively charged residue within the TM domain of FcalphaR is involved in the functional association between these two molecules. Implications of these observations in terms of cooperation (and competition) between immunoglobulin receptors in cellular activation processes remain to be elucidated.


FOOTNOTES

*
This work was supported by EC, The Netherlands Organization for Scientific Research, and Wellcome Trust (Reference 042417) Fellowships. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Laboratory of Cell Biology and Histology, Amsterdam Medical Center, Amsterdam, The Netherlands.

To whom correspondence should be addressed: Dept. of Immunology, University Hospital Utrecht, Rm G04.614, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands. Tel.: 31-30-2507-636; Fax: 31-30-2537-447.

(^1)
The abbreviations used are: mAb, monoclonal antibody; TCR, T cell receptor; PMN, polymorphonuclear cells; TM, transmembrane; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; GAM, goat anti-mouse; PAGE, polyacrylamide gel electrophoresis; IL, interleukin; ITAM, immunoreceptor tyrosine-based activation motif.


ACKNOWLEDGEMENTS

We thank Dr. Dirk Roos for valuable discussions, Drs. Thomas Reterink and Mohammed Daha for polymeric IgA, Susan Janssen for expert help with experiments, and Drs. Hans Clevers, Ton Logtenberg, and Sjef Verbeek for critical reading of the manuscript.


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