Immunoglobulin-binding Sites of Human Fcalpha RI (CD89) and Bovine Fcgamma 2R Are Located in their Membrane-distal Extracellular Domains

By H. Craig Morton,* Ger van Zandbergen,Dagger Cees van Kooten,Dagger Chris J. Howard,§ Jan G. J. van de Winkel,parallel and Per Brandtzaeg*

From the * Laboratory of Immunohistochemistry and Immunopathology (LIIPAT), The National Hospital, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway; the Dagger  Department of Nephrology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands; the § Division of Immunology and Pathology, The Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, United Kingdom; and the parallel  Department of Immunology and Medarex Europe, University Hospital Utrecht, 3584 CX Utrecht, The Netherlands

    Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

To localize the immunoglobulin (Ig)-binding regions of the human Fcalpha receptor (Fcalpha RI, CD89) and the bovine Fcgamma 2 receptor (bFcgamma 2R), chimeric receptors were generated by exchanging comparable regions between these two proteins. Fcalpha RI and bFcgamma 2R are highly homologous and are more closely related to each other than to other human and bovine FcRs. Nevertheless, they are functionally distinct in that Fcalpha RI binds human IgA (hIgA) but not bovine IgG2 (bIgG2), whereas bFcgamma 2R binds bIgG2 but not hIgA. Fcalpha RI and bFcgamma 2R possess extracellular regions consisting of two Ig-like domains, a membrane-distal extracellular domain (EC1), a membrane-proximal EC domain (EC2), a transmembrane region, and a short cytoplasmic tail. Chimeras constructed by exchanging complete domains between these two receptors were transfected to COS-1 cells and assayed for their ability to bind hIgA- or bIgG2-coated beads. The results showed that the Ig-binding site of both Fcalpha RI and bFcgamma 2R is located within EC1. Supporting this observation, monoclonal antibodies that blocked IgA binding to Fcalpha RI were found to recognize epitopes located in this domain. In terms of FcR-Ig interactions characterized thus far, this location is unique and surprising because it has been shown previously that leukocyte Fcgamma Rs and Fcepsilon RI bind Ig via sites principally located in their EC2 domains.

Key words: Fc receptor;  CD89;  bovine Fcgamma 2 receptor;  immunoglobulin A;  myeloid
    Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

Immunoglobulin (Ig) Fc receptors (FcRs) expressed on phagocytic cells provide a crucial link between the humoral and cellular branches of the immune system. Ligation of FcRs by antigen-bound Ig leads to cellular activation and triggering of powerful effector mechanisms (1, 2). In humans and other mammals, IgA predominates in mucosae and, furthermore, comprises a substantial proportion of the circulating Ig pool. At mucosal surfaces IgA provides a first-line protective function, termed immune exclusion, whereby it inhibits microbial colonization on epithelial cells and penetration of harmful antigens. In addition, the protective function of IgA both in mucosa and in the circulation may be reinforced by interaction of IgA-complexed antigens with the myeloid Fcalpha RI (CD89)(3, 4). Fcalpha RI is expressed on monocytes, macrophages, polymorphonuclear granulocytes, and eosinophils, and its cross-linking triggers a variety of immunological effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators and cytokines (3).

Among FcRs characterized until now, Fcalpha RI is most closely related to the bovine Fc receptor for IgG2 (bFcgamma 2R)1 expressed on monocytes and granulocytes. In fact, these two FcRs are more closely related to each other than to any known human or bovine FcRs (7). More recently, it has been shown that Fcalpha RI and bFcgamma 2R are members of a new gene family that apparently evolved from a common ancestral gene. Other human genes belonging to this family include the natural killer cell inhibitory receptors (KIRs), the Ig-like transcripts (ILTs), the leukocyte and monocyte/ macrophage Ig-like receptors (LIRs, MIRs), LAIR-1, and HM18 (8). These genes are located close to the Fcalpha RI gene within the so-called leukocyte receptor complex on chromosome 19q13.4 (13). Several murine members of the same gene family, gp49B1 (a structural homologue of human KIRs), and the paired Ig-like receptors A and B (PIR-A and PIR-B), have also been described (16).

Fcalpha RI and bFcgamma 2R are both transmembrane glycoproteins composed of two extracellular (EC) Ig-like domains (EC1 and EC2), a transmembrane region containing a charged arginine residue, and a short cytoplasmic tail devoid of signaling motifs (7, 19). Signal transduction via Fcalpha RI is mediated via the FcR gamma  chain, which associates with Fcalpha RI through the charged arginine residue within its transmembrane domain but does not affect its affinity for IgA (20). Despite the high level of amino acid identity (41%) within the EC and transmembrane regions of Fcalpha RI and bFcgamma 2R, these two receptors are functionally quite distinct in that Fcalpha RI binds IgA but not bIgG2, whereas bFcgamma 2R binds bIgG2 but not IgA. Therefore, to map the ligand-binding domains of these two FcRs we utilized their high degree of identity and exchanged homologous regions between them. Based on knowledge of interactions between other two-domain FcRs (Fcgamma RII, Fcgamma RIII, and Fcepsilon RI) with their respective ligands (IgG or IgE), we expected the Ig-binding sites to be located within the membrane-proximal EC2 domain (24- 30). Surprisingly, however, our results demonstrated that the ligand-binding region of both Fcalpha RI and bFcgamma 2R is located in their membrane-distal EC1 domain. In part, this finding probably reflects the evolutionary development of Fcalpha RI and bFcgamma 2R from an ancestral gene distinct from the putative Fcgamma R/Fcepsilon R precursor.

    Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cell Culture.

COS-1 cells were maintained in DMEM (BioWhittaker) supplemented with 10% FCS, 1 mM L-glutamine, and 50 µg/ml gentamycin (Life Technologies, UK). The murine IIA1.6 B cell line that coexpresses Fcalpha RI and the FcR gamma  chain has been described previously (21).

cDNAs and Construction of Chimeric FcRs.

cDNAs encoding the complete Fcalpha RI coding region and a mutant cDNA encoding a soluble form of Fcalpha RI were gifts from Dr. C. Maliszewski (Immunex Corp., Seattle, WA) (19, 31). cDNA for bFcgamma 2R has been described previously (7). Chimeric cDNAs were constructed by overlap extension PCR (21). Although the genomic structure of bFcgamma 2R is unknown, the high homology to Fcalpha RI allowed us to infer intron-exon boundaries because amino acid residues that link the Fcalpha RI exons are identical to those at comparable positions in bFcgamma 2R (Fig. 1). Primers were thus designed to allow the fusion of exons at these residues. To construct the bEC1(1-50)-Fcalpha RI mutant, primers were designed to allow the fusion of the first 50 amino acids of bFcgamma 2R to Fcalpha RI at isoleucine 50; both Fcalpha RI and bFcgamma 2R have isoleucine residues at this position, which lies almost exactly in the middle of the EC1 domains. The integrity of all chimeric cDNAs was confirmed by sequence analysis. Chimeric FcR cDNAs were cloned into the pCDNA3 mammalian expression vector (Invitrogen, The Netherlands) before transfection. The pCMV-GFP plasmid, which directed the expression of green fluorescent protein (GFP), was constructed by inserting the CMV promoter region from pCDNA3 into the multiple cloning site of the pEGFP-1 vector (Clontech).


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Fig. 1.   Alignment of amino acid sequences of Fcalpha RI (X54150) and bFcgamma 2R (Z37506). S1 exons are shown from the methionine initiation codons. Because the gene structure of bFcgamma 2R is unknown, intron-exon boundaries are shown only for Fcalpha RI. Amino acids that link two Fcalpha RI exons are underlined, and the exon designation is shown above the sequence. Note that exon-linking amino acids are conserved between Fcalpha RI and bFcgamma 2R. The first 19 amino acids of both sequences are considered to represent NH2-terminal signal peptides, which are removed before cell surface expression. Thus, glutamine (Q) 22 is proposed to be the first amino acid of both mature proteins and therefore is designated +1. Isoleucine (I) 50 is also underlined. Residues of bFcgamma 2R identical to those of Fcalpha RI are designated (*), and gaps that have been inserted to line up the sequences are designated (-) (also see reference 7).

Transfections.

COS-1 cells were transiently transfected with 2 µg chimeric FcR cDNA constructs by means of Fugene 6 transfection reagent (Boehringer Mannheim, Germany) according to the manufacturer's instructions. In some experiments, 1 µg pCMV-GFP was cotransfected together with the FcR constructs. Cells were incubated at 37°C in a humidified CO2 atmosphere for 48 h before harvesting.

Ig-binding Assays.

Uncoated magnetic M-450 Dynabeads (Dynal, Norway) were coated, according to the manufacturer's instructions, with either human serum IgA (hIgA) or bovine IgG2 (bIgG2), which were purified as previously described (7, 32). Due to low transfection efficiency of some DNA constructs (see Results), transfected COS-1 cells were first enriched for those becoming positive for gene expression by cotransfection of the FcR and pCMV-GFP constructs. Experiments showed that most fluorescent (GFP+) cells had also taken up both plasmids, thus expressing the chimeric FcR together with GFP (see Results). Therefore, binding assays were performed as follows: 5 × 104 GFP+ COS-1 cells (which had also been cotransfected with an FcR construct) were purified in a FACSVantage® cell sorter (Becton Dickinson) and mixed with Ig-coated Dynabeads in a final volume of 50 µl per well in V-bottomed microtiter plates. After a 15-min incubation at room temperature, the plate was spun at 50 g for 1 min and incubated for an additional 45 min at room temperature. Cells and beads were resuspended and examined for the presence of rosettes, using a combination of light and fluorescent microscopy, in a Nikon Eclipse E800 microscope. Rosettes were defined as GFP+ cells binding four or more Ig-coated beads and at least 200 GFP+ COS-1 cells were counted for each determination. For blocking studies, cells were incubated with either mAb My43 (50 µl culture supernatant) or CC-G24 (50 µl ascites fluid diluted 1:4) for 30 min at room temperature before the addition of Ig-coated beads.

Production and Purification of Recombinant Soluble Fcalpha RI.

A cDNA that encodes a soluble form of Fcalpha RI was expressed in Chinese hamster ovary cells by means of the pEE14 expression system (Lonza, UK), and the protein was isolated from the culture supernatant by affinity chromatography with Sepharose-bound human IgA (van Zandbergen, G., and C. van Kooten, unpublished data).

Monoclonal Antibodies.

The previously described Fcalpha RI mAbs My43 (murine IgM), A3, A59, A62, and A77 (all murine IgG1) were used in this study (33, 34). My43 and A62 were gifts from Dr. Li Shen (Dartmouth Medical School, Lebanon, NH) and Dr. Max Cooper (University of Alabama, Birmingham, AL), respectively. A77 was supplied by Medarex Europe (The Netherlands), and A3 and A59 were purchased from Immunotech (France) and Research Diagnostics Inc. respectively. The bFcgamma 2R mAb CC-G24 (murine IgM) was generated by immunizing mice with bFcgamma 2R protein purified from cattle leukocytes, and the specificity was confirmed by staining COS-7 cells transfected with cDNA encoding the bFcgamma 2R or bovine Fcgamma RII (Howard, C.J., unpublished data). To obtain new Fcalpha RI mAbs, female BALB/C mice were immunized with purified soluble Fcalpha RI. Splenocytes isolated from immunized animals were fused with myeloma cells (SP20) in the presence of 50% polyethylene glycol. The cell suspension was diluted in IMDM supplemented with 10% FCS, hypoxanthine (100 µmol), aminopterin (0.4 µM), thymidine (16 µM), 500 pg/ml IL-6, 100 U/liter penicillin, and 100 µg/ml streptomycin. Cells producing antibodies to Fcalpha RI were subcloned by limiting dilution. Five clones producing mAb to Fcalpha RI were expanded and the specificity was determined by FACS® analysis (see below). To define the capacity of these new mAbs to inhibit binding of IgA to Fcalpha RI, blocking studies were performed as follows: Fcalpha RI mAbs or control mAbs of the same isotype were diluted in FACS buffer and incubated together with Fcalpha RI-transfected IIA1.6 cells for 15 min at 4°C. Purified human serum IgA, which had previously been heat-aggregated for 1 h at 63°C (aIgA), was then added for 1 h at 4°C. Cells were washed and bound aIgA was detected by incubation with a goat anti- human IgA F(ab)2-PE polyclonal antibody conjugate (Southern Biotechnology Associates, Inc.) in FACS® analysis. Isotype control antibodies for murine IgG1 were purchased from Becton Dickinson, and those for murine IgM were provided by Dr. Robert Burns (Scottish Agricultural Science Agency, Edinburgh, UK).

FACS® Analysis.

Cells (5 × 105) were washed twice with FACS buffer (PBS/0.5% BSA/0.02% azide) and incubated with either Fcalpha RI mAb (murine IgM or IgG1) culture supernatant or the appropriate isotype control supernatant for 1 h at 4°C. Cells were then washed twice with FACS buffer and incubated for 1 h at 4°C with either goat anti-mouse (GAM) IgM-FITC conjugate (1:150 final dilution), or a GAM IgG1-PE conjugate (1:150 final dilution) (both from Southern Biotechnology Associates, Inc.). In experiments where GFP+ cells were analyzed for chimeric FcR expression, a GAM IgG1 Tricolor secondary reagent (1:200 final dilution) (Caltag Labs.) was used. After washing twice with FACS buffer, cells were fixed in PBS-buffered 1% (wt/vol) paraformaldehyde at 4°C and analyzed on a FACScan®. Data acquisition was conducted with Lysis II software (Becton Dickinson), and data analysis was performed using WinMDI software (available from The Scripps Research Institute, La Jolla, CA).

    Results
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
The EC1 Domains of Fcalpha RI and bFcgamma 2R Mediate Ligand Binding.

To map the ligand-binding domains of Fcalpha RI and bFcgamma 2R, we generated five chimeric receptors as follows: hEC-bFcgamma 2R, consisting of the two EC domains of Fcalpha RI fused to the transmembrane/cytoplasmic (TM/C) domain of bFcgamma 2R; bEC-Fcalpha RI, the two bovine EC domains fused to the TM/C domain of Fcalpha RI; hEC1-bFcgamma 2R, the EC1 domain of Fcalpha RI fused to the EC2 TM/C region of bFcgamma 2R; bEC1-Fcalpha RI, the EC1 domain of bFcgamma 2R joined to the EC2 TM/C region of Fcalpha RI; and bEC1(1-50)-Fcalpha RI, the first 50 amino acids of bFcgamma 2R fused at isoleucine 50 to Fcalpha RI (Fig. 1). Together with wild-type Fcalpha RI and bFcgamma 2R cDNAs, individual chimeric FcR constructs were transfected to COS-1 cells, and their cell surface expression was assessed by FACS® analysis and by a specific binding assay using Ig-coated beads (see Materials and Methods). Initial experiments revealed the transfection efficiency of individual constructs to be quite variable. The most efficient construct directed expression of Fcalpha RI on the surface of ~30% of COS-1 cells, whereas the least efficient (bEC1-Fcalpha RI) was expressed by only 3% of the transfectants (Fig. 2 A). Therefore, we developed a method to selectively enrich for transiently transfected cells by cotransfecting a plasmid that directed expression of GFP (visualized by green fluorescence) together with the chimeric FcR constructs. Most GFP+ COS-1 cells cotransfected with Fcalpha RI were recognized by mAb A62 (Fig. 2 B), and formed rosettes with hIgA-coated beads (Fig. 2 C). By this procedure we obtained an approximately twofold enrichment of chimeric FcR-expressing cells, which in the case of Fcalpha RI resulted in >60% of cells being reactive with Fcalpha RI mAb and further able to form rosettes with hIgA coated beads (Fig. 2, B and C). It should be noted that GFP- COS-1 almost never formed rosettes, most likely because transfectants expressing Fcalpha RI alone accounted for only ~1% of the total cells (Fig. 2 B). We, furthermore, demonstrated the specificity of our binding assay by using blocking mAbs specific for Fcalpha RI or bFcgamma 2R to inhibit rosette formation (Fig. 3). The finding that the inhibition obtained with My43 was only partial (~50%) was most likely explained by the use of culture supernatant other than a higher concentration of purified antibody which was not available.


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Fig. 2.   Expression of chimeric FcR by COS-1 cells. (A) Cos-1 cells were transfected with the indicated constructs 2 d before harvesting and FACS® analysis. Cells were stained with either Fcalpha RI EC2-specific mAb A62 (mouse IgG1) (bottom panels) or an appropriate isotype control (top panels), followed by a GAM IgG1 Tricolor reagent. Numbers in the top right corners of the plots refer to the percentage of positive cells. (B) Enrichment of Fcalpha RI expression in COS-1 cells cotransfected with GFP. Cells transfected with both Fcalpha RI and GFP were stained with Fcalpha RI EC2-specific mAb A62 followed by GAM IgG1 Tricolor reagent and analyzed by FACS®. More than 60% of the GFP+ cells also express Fcalpha RI. (C) Rosette formation by COS-1 cells cotransfected with both Fcalpha RI and GFP and exposed to hIgA-coated beads. Rosettes were quantified as specified in Materials and Methods. Black bar, total number of cells counted; white bar, total number of GFP+ cells assessed by fluorescent microscopy; gray bar, number of GFP+ cells forming rosettes. Results shown are representative of three separate experiments.


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Fig. 3.   Specificity of the bead rosetting assays. COS-1 cells were cotransfected with the indicated FcR construct together with pCMV-GFP. GFP+ cells were purified as described in Materials and Methods and incubated with the relevant murine IgM-blocking mAb or an irrelevant murine IgM control mAb for 30 min before addition of Ig-coated beads. Results are shown as percentage of inhibition of rosette formation when compared with transfectants that were not incubated with mAb before rosetting analysis. Results shown are representative of two experiments.

Fcalpha RI does not bind bIgG2, and bFcgamma 2R does not bind hIgA (reference 7 and this paper); accordingly, we neither observed rosettes when hIgA-coated beads were mixed with bFcgamma 2R transfectants, nor when bIgG2-coated beads were mixed with Fcalpha RI transfectants (Fig. 4). Binding studies with COS-1 cells enriched for FcR expression as described above, showed not unexpectedly that wild-type Fcalpha RI and bFcgamma R2 transfectants produced the highest levels of rosette formation with hIgA- and bIgG2-coated beads, respectively (Fig. 4). Transfectants expressing chimeras coding for the entire EC portions of the receptors (hEC-bFcgamma 2R and bEC-Fcalpha RI) bound their respective Ig-coated beads efficiently, although at a slightly lower level than their wild-type counterparts (Fig. 4). The fact that the hEC1-bFcgamma 2R chimera retained IgA-binding capacity demonstrated that the binding site of Fcalpha RI lies within the membrane-distal EC1 domain. Furthermore, because this chimera did not form rosettes with bIgG2-coated beads, our finding further suggested that the binding site for bIgG2 in bFcgamma 2R was not located within the EC2 domain of this receptor. Conversely, the bEC1-Fcalpha RI chimera did form rosettes with bIgG2-coated beads, but not with beads coated with hIgA.


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Fig. 4.   Rosette formation by FcR/GFP cotransfected COS-1 cells. Schematic representation of wild-type and chimeric FcRs. Unshaded regions are derived from Fcalpha RI, shaded regions from bFcgamma 2R. S, signal peptide; EC1, extracellular domain 1; EC2, extracellular domain 2; TM/C, transmembrane/cytoplasmic tail. GFP+ transfectants were purified from COS-1 cells cotransfected with GFP and FcR constructs as described. Ig-binding to FcRs coexpressed with GFP was assessed by rosetting with either hIgA- or bIgG2-coated beads. More than 200 cells were counted for each determination, and the number of cells binding four or more Ig-coated beads is expressed as percentage rosette formation. Results are representative of three separate experiments.

Altogether these results showed that, in common with Fcalpha RI, the ligand-binding site of bFcgamma 2R appeared to lie within the EC1 domain. It should be noted, however, that the level of binding obtained with the two EC1 chimeras (hEC1-bFcgamma 2R and bEC1-Fcalpha RI) was reduced when compared with the wild-type receptors, and the EC chimeras (hEC-bFcgamma 2R and bEC-Fcalpha RI) (Fig. 4). Futhermore, to better localize the Ig-binding sites of these two receptors, a further chimera was constructed in which the first 50 amino acids of the EC1 domain were from bFcgamma 2R, while the remaining EC1 (49 amino acids) and the rest of the receptor were from Fcalpha RI [bEC1(1-50)-Fcalpha RI]. Although this chimera was expressed at the cell surface and could be recognized by the majority of Fcalpha RI mAb, it bound neither hIgA- nor bIgG2-coated beads.

EC1-specific Antibodies Block Fcalpha RI Binding of IgA.

To confirm that the IgA-binding site of Fcalpha RI lies within the EC1 domain, we mapped the specific epitopes for a number of blocking and nonblocking mAbs. Of the previously described Fcalpha RI mAbs, only My43 (murine IgM) was able to block the binding of hIgA to Fcalpha RI (33). Four others (A3, A59, A62, and A77, all murine IgG1) did not inhibit binding (34). We also included a number of new Fcalpha RI mAbs (2E6, 2D11, 7G4, 2H8, and 7D7, all murine IgG1), raised against a soluble form of Fcalpha RI. These mAbs were shown to be specific for Fcalpha RI by reaction with IIA1.6 cells expressing this receptor (Fig. 5 A). They were next assayed for ability to block binding of heat-aggregated hIgA to Fcalpha RI: mAbs 2E6, 2D11, 7G4, and 2H8 produced such inhibition while 7D7 did not (Fig. 5 B). Accordingly, we presumed that the blocking mAbs would prove to be EC1-specific, whereas the nonblocking ones would be EC2 specific.


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Fig. 5.   (A) FACS® analysis of newly produced mAbs to Fcalpha RI compared with mAb A77 of similar specificity. Fcalpha RI-expressing murine B cells (IIA1.6) were incubated with mAbs as indicated (white peaks) or with concentration and isotype-matched control mAbs (hatched peaks), followed by a GAM IgG1-PE conjugate. (B) Fcalpha RI-expressing IIA1.6 cells were incubated without (left panel) or with mAbs as indicated, followed by heat-aggregated hIgA (aIgA). After 1 h at 4°C, cells were washed and bound hIgA was detected by incubation with goat anti-human IgA F(ab)2-PE conjugate (white peaks). Cells incubated with only the secondary reagent served as negative controls (hatched peaks). (C) Reactivity of chimeras (left) with a panel of Fcalpha R and bFcgamma 2R mAb (top) as measured by FACS® analysis. Binding was graded as follows: +, strong binding; +/-, weak binding; -, no binding.

To test this hypothesis, we screened the reactivity of all mAbs against the panel of chimeric FcR expressed in COS-1 cells by FACS® analysis. Indeed, all mAbs capable of blocking the binding of heat-aggregated hIgA to Fcalpha RI (Fig. 5 B), mapped to the EC1 domain (Fig. 5 C). Also, all nonblocking mAbs were directed against the EC2 domain, except for mAb A3 that apparently recognized an epitope depending on parts of both domains. Unfortunately, because only one mAb against bFcgamma 2R was available, a similar detailed study could not be performed for this receptor. We showed that mAb CC-G24 only recognized wild-type bFcgamma 2R and the bEC-Fcalpha RI chimera (Fig. 5 C). Thus, like mAb A3, it is most likely directed against a conformational epitope depending on both EC1 and EC2.

    Discussion
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

By means of a panel of chimeric FcRs, we identified conclusively for the first time the ligand-binding sites of Fcalpha RI and bFcgamma 2R. Surprisingly, these sites were found to be located in the EC1 domains. Fcalpha RI and bFcgamma 2R are highly homologous both at the protein and nucleotide level (41 and 56% identity, respectively), but show much less homology with other human and bovine FcRs (7). This suggests that Fcalpha RI and bFcgamma 2R evolved from a common ancestral gene (also shared by KIR, ILT, MIR, LIR, LAIR-1, HM18, PIR, and gp49B1 genes) and not shared by other human or bovine FcRs (7). Mapping of the bFcgamma 2R gene to bovine chromosome 18, which corresponds to human chromosome 19, further supports this notion (35).

Our finding that the Ig-binding sites of both Fcalpha RI and bFcgamma 2R are located within their EC1 domains, was based on the fact that rosetting with Ig-coated beads was only seen for the corresponding Fcalpha RI or bFcgamma 2R EC1 chimera. In both cases, however, a reduction in binding activity was seen compared with that obtained for the wild-type receptors and for the comparable EC chimeras (Fig. 4). Although these differences in part may be attributed to the expression levels of individual constructs (especially for the bEC1-Fcalpha RI chimera, see above), it is also possible that the EC2 domains and membrane-proximal regions of the receptors contribute either directly (by forming "secondary" contact sites) or indirectly (by preserving three- dimensional structure) to the affinity and stability of the ligand interactions. This would be analogous to the activity of other two-domain FcRs, namely Fcgamma RII, Fcgamma RIII, and Fcepsilon RI in which the ligand-binding sites are located in the membrane-proximal EC2 domains, whereas structures within the EC1 domains contribute to the binding process (29, 30).

It should also be noted that the region of hIgA interacting with Fcalpha RI has recently been mapped to the Calpha 2/ Calpha 3 boundary (36). This is in contrast to the region of human IgG responsible for interaction with Fcgamma Rs, which is proposed to lie much closer to the hinge (30). Therefore, in terms of the evolution of Ig/FcR interactions, it should be interesting to map the region of bIgG2 that binds to bFcgamma 2R. Because recombinant bIgG2 is available, experiments can readily be designed to determine whether this region lies close to the hinge region as in human IgG or in a position analogous to that of hIgA (37).

To substantiate our observation that hIgA binds to the EC1 domain of Fcalpha RI, we mapped the epitopes for a panel of blocking and nonblocking Fcalpha RI mAbs that bound equally to the EC parts of wild-type Fcalpha RI and the hEC-Fcgamma 2R chimera. The nonblocking mAbs (A59, A62, A77, and 7D7) were shown to react with the membrane-proximal EC2 domain because they bound only to wild-type Fcalpha RI and the hEC-bFcgamma 2R, bEC1-Fcalpha RI, and bEC1(1-50)-Fcalpha RI chimeras. The only exception was the nonblocking mAb A3 that bound only to wild-type Fcalpha RI and the hEC-bFcgamma 2R and bEC1(1-50)-Fcalpha RI chimeras, suggesting that its epitope is conformational and depends on regions of both EC1 and EC2 (similar to the bFcgamma 2R mAb CC-G24; see Results section). In contrast, all blocking mAbs (My43, 2E6, 2D11, 7G4, and 2H8) were shown to react with the EC1 domain of Fcalpha RI because their binding activity was retained with the hEC1-bFcgamma 2R chimera. The epitopes recognized by My43 and 2D11 were further localized to the region of EC1 directly adjacent to EC2, because they were shown to bind the bEC1(1-50)-Fcalpha RI chimera. 2E6 and 2H8 on the other hand, bound only weakly to this chimera, whereas 7G4 did not bind at all. Similar mapping studies with blocking mAbs have previously been used to localize the IgG-binding sites of Fcgamma RII and Fcgamma RIII to their EC2 domains (25, 27).

A number of Fcalpha RI mRNAs have been isolated and shown to encode splice variants of the receptor (38). One such report described cell surface expression of an Fcalpha RI variant that lacked the complete EC2 domain, and suggested the EC1 domain to be involved in hIgA binding (40). However, in contrast to our results, mAb My43 was proposed to react with EC2, and mAb A59 with EC1. We believe that observation to be spurious either due to incorrect cell surface expression of the splice variant and/or aberrant receptor structure caused by lack of the complete EC2 domain. This possibility was supported by our attempts to express various Fcalpha RI splice variants in COS cells with no success (38). Additionally, chimeras constructed between Fcalpha RI and Fcgamma RII were not expressed efficiently (Morton, H.C., and J.G.J. van de Winkel, unpublished observations), possibly reflecting a degree of structural incompatibility between these two FcRs. In fact, our unsuccessful experience with those approaches led us to construct chimeras between Fcalpha RI and bFcgamma 2R as reported here, because their levels of homology (and hence presumably their overall structure) are more similar than for other FcRs. Thus swapping of highly homologous regions should have minimal affect on the overall structural integrity of the resultant chimeras. Therefore, we feel that the present approach is more physiological than previous attempts to this end.

The surprising difference seen between the ligand-binding sites of Fcalpha RI and bFcgamma 2R versus those of other leukocyte Fcgamma Rs and Fcepsilon RI may have interesting implications in terms of Ig interactions. As mentioned above, this disparity could simply reflect the proposed evolution of Fcalpha RI and bFcgamma 2R from an ancestral gene distinct from that giving rise to other Fcgamma Rs and Fcepsilon RI. This notion is supported by the observation that residues within the membrane-distal domain of two KIR proteins determine their ability to bind to their respective ligands, the two groups of HLA-C allotypes (42). Moreover, due to their high levels of homology, the three-dimensional structure of Fcalpha RI and bFcgamma 2R might more closely resemble that of the KIR proteins than that of more distantly related FcRs (43). Indeed, more detailed mutational analysis, directed by modeling studies using the recently published three dimensional structure of the p58 KIR as a template for the protein backbones of Fcalpha RI and bFcgamma 2R, are currently underway in our laboratory to further localize the Ig-binding sites within these two receptors.

An alternative evolutionary explanation possibly applicable at least for Fcalpha RI might be that its ligand-binding site developed to ensure interaction with all molecular forms of IgA: monomeric IgA, dimeric IgA (including J chain), and secretory IgA (including J chain and secretory component). Fcalpha RI is reported to bind all these ligand variants (44, 45). Therefore, because the site of interaction with Fcalpha RI at the Calpha 2/Calpha 3 boundary appears to be accessible to the receptor in all these forms of IgA, Fcalpha RI could have evolved to accomplish this interaction via its EC1 domain to avoid potential problems of steric hindrance of a more membrane-proximal binding site in relation to large IgA polymers.

In conclusion, we have shown that the closely related Fcalpha RI and bFcgamma 2R bind their ligands via sites located in their membrane-proximal EC1 domains. The difference in the Ig-binding sites of these two receptors versus other leukocyte Fcgamma Rs and Fcepsilon RI, may reflect the proposed divergent evolutionary pathway from a distinct genetic precursor, or (at least in the case of Fcalpha RI) a specific adaptation for efficient interaction with large molecular forms of IgA.

    Footnotes

Address correspondence to H. Craig Morton, LIIPAT, Rikshospitalet, N-0027, Oslo, Norway. Phone: 47-22-86-86-31; Fax: 47-22-11-22-61; E-mail: craig.morton{at}labmed.uio.no

Received for publication 24 December 1998 and in revised form 29 March 1999.

   Note added in proof. A recent report by Wines et al. (J. Immunol. 1999. 162:2146-2153) likewise identified the EC1 domain of Fcalpha RI to be responsible for ligand binding.

We would like to thank Drs. Li Shen for My43, Max Cooper for A62, and Charles Maliszewski and Immunex for Fcalpha RI cDNAs. We also gratefully acknowledge the technical staff of LIIPAT, specifically Bjørg Simonsen, Marie Johannesen, and Inger Johanne Ryen, for expert laboratory assistance. We further thank Gøril Olsen for help with cell sorting, and Dr. Finn-Eirik Johansen (LIIPAT) for helpful discussions and provision of the pCMV-GFP plasmid.

Abbreviations used in this paper b, bovine; EC, extracellular; GAM, goat anti-mouse; GFP, green fluorescent protein; KIR, natural killer cell inhibitory receptor; TM/C, transmembrane/cytoplasmic.

    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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