Cell surface receptors for the Fc portion of IgG (FcR) are expressed on most hematopoietic cells, and through the binding of IgG they play a key role in homeostasis of the immune system and host protection against infection. Three structurally related but functionally distinct classes of FcR have been defined: FcRI, FcRII, and FcRIII(1, 2, 3) . FcRII is a low affinity receptor for IgG that binds only IgG immune complexes and is expressed on a diverse range of cells such as monocytes, macrophages, neutrophils, eosinophils, platelets, and B cells(1, 2, 3) . FcRII is involved in a number of immune responses including antibody-dependent cell-mediated cytotoxicity, clearance of immune complexes, release of inflammatory mediators, and regulation of antibody production(1, 2, 3, 4, 5, 6) .

The extracellular region of FcRII comprises two Ig-like disulfide-bonded extracellular domains that are related to the Ig superfamily proteins and are most closely related to the C2 set of Ig domains(7, 8, 9, 10, 11, 12) . The two Ig-like domain extracellular region of FcRII is structurally conserved in all of the Ig superfamily leukocyte FcRs (including FcRI, FcRIII, FcRI, and FcRI) and presumably represents an Ig-interactive motif(13, 14, 15, 16, 17) . The elucidation of the molecular basis of FcR-Ig interactions is fundamental for understanding the mechanisms by which these receptors mediate biological functions such as activation of inflammatory cells, induction of cytokine release, and destruction of pathogens. In previous studies we utilized chimeric Fc receptors to identify the IgG binding region of human FcRII(18, 19) . Chimeric FcRII/FcRI chain receptors were used to demonstrate that the second extracellular domain of FcRII was responsible for the binding of IgG, with a single direct binding region located between residues Asn and Ser. Site-directed mutagenesis of the Asn-Ser region identified 5 residues as playing crucial roles in the binding of human and mouse IgG1 by FcRII: Ile, Gly, Leu, Phe, and Ser(20) .

However, despite the direct demonstration of only a single region involved in the binding of IgG, there is compelling evidence to suggest that other regions of FcRII contribute to binding. A genetic polymorphism of human FcRIIa, the so called ``responder/non-responder'' system, results in an amino acid substitution in domain 2 at residue 131 (Arg His), which has been shown to influence the binding of mouse IgG1 and human IgG2(21, 22, 23) . Similarly, in the mouse a genetic polymorphism of FcRII, identified as differences at residues 116 and 161, defines the epitope of the anti-Ly17.2 mAb ()that blocks the binding of IgG to this receptor(24, 25) . Our previous molecular modeling studies of FcRII domain 2 (wherein the Asn-Ser binding region was located to an exposed loop region; the F/G loop) suggest that these functionally important amino acid changes are situated in the B/C and C`/E loops (containing residues 116 and 131, respectively), which are juxtaposed to the F/G loop (contains residue 161) at or near the interface with domain 1(20) . Furthermore, the studies using chimeric FcRII/FcRI receptors have identified three regions in the structurally homologous receptor, FcRI, capable of directly binding IgE: residues 87-128, 130-135, and 154-161, which encompass the B/C, C`/E, and F/G loops respectively(1, 18, 19) . Taken together, these findings suggest that the B/C and C`/E loops of FcRII may in addition to the F/G loop also play a role in the binding of IgG by FcRII. Also of interest is that while the role of domain 2 of FcRII in Ig binding has been clearly defined, a role for domain 1 of FcRII has not been determined. However, domain 1 of FcRI, although demonstrated to not have a direct role in IgE binding, has been shown to play an important role in high affinity binding (18, 26) possibly by maintaining the structural integrity of the receptor or by providing additional contact sites. Since FcRII is structurally related to FcRI, domain 1 of FcRII may also play a similar role.

The possibility that domain 1 and the B/C or C`/E loop regions of domain 2 also contribute to the binding of IgG1 by FcRII is addressed herein, using both chimeric receptor and site-directed mutagenesis strategies.

MATERIALS AND METHODS

Generation of Chimeric FcRII/FcRI and Mutant FcRII Receptor cDNA Expression Constructs

The chimeric FcRII/FcRI chain receptors were generated as follows. For chimera 109-116, oligonucleotide pairs NR1 + CHM10 and CHM09 + EG5 were used to produce two fragments, which were spliced together using oligonucleotides NR1 and EG5. For chimera 130-135, oligonucleotide pairs NR1 + PM12 and PM11 + EG5 followed by NR1 and EG5. The sequences of the oligonucleotides used and their positions of hybridization with the FcRIIa cDNA are as follows: NR1, 5`-TACGAATTCCTATGGAGACCCAAATGTCTC-3` (nucleotide positions 10-30); EG5, 5`-TTTGTCGACCACATGGCATAACG-3`(967-981); CHMO9, 5`-CACATCCCAGTTCCTCCAACCGTGGCACCTCAGCATG-3` (419-437 with nucleotides 442-462 of FcRI chain); CHM10, 5`-AGGAACTGGGATGTGTACAAGGTCACATTCTTCCAG-3` (462-487 with 446-462 of FcRI chain), PM11, 5`-GTGGTTCTCATACCAGAATTTCTGGGGATTTTCC-3` (473-490 with 492-506 of FcRI chain); PM12, 5`-CTGGTATGAGAACCACACCTTCTCCATCCCAC-3` (516-531 with 491-506 of FcRI chain).

Sequences derived from FcRI chain are underlined, FcRII is not underlined, and nonhomologous sequences including restriction enzyme sites used in cloning of the PCR products are in boldface type. Nucleotide positions refer to the previously published FcRIIa and FcRI chain cDNA sequences(8, 16) .

The FcRII alanine point mutant cDNAs were generated using the following oligonucleotide combinations: Lys-Ala, GBCO3 + EG5 and GBCO4 + NR1; Pro-Ala, GBCO1 + EG5 and GBCO2 + NR1; Leu-Ala, GBCO5 + EG5 and GBCO6 + NR1; Val-Ala, GBCO7 + EG5 and GBCO8 + NR1; Phe-Ala, GCEO1 + EG5 and GCEO2 + NR1; Ser-Ala, GCEO3 + EG5 and GCEO4 + NR1; Arg-Ala GCEO5 + EG5 and GCEO6 + NR1; Leu-Ala, GCEO7 + EG5 and GCEO8 + NR1; Asp-Ala, GCEO9 + EG5 and GCE10 + NR1; Pro-Ala, GCE11 + EG5 and GCE12 + NR1. Oligonucleotides NR1 and EG5 were used to splice together the two component fragments of each mutant to produce the point-substituted cDNAs. The sequences of the oligonucleotides used and their positions of hybridization with the FcRIIa cDNA are as follows: GBCO1, 5`-GAAGGACAAGGCTCTGGTCAAG-3` (nucleotide positions 443-464); GBCO2, 5`-CTTGACCAGAGCCTTGTCCTTC-3`(443-464); GBCO3, 5`-CTGGAAGGACGCTCCTCTGGTC-3`(440- 461); GBCO4, 5`-GACCAGAGGAGCGTCCTTCCAG-3`(440-461); GBCO5, 5`-GGACAAGCCTGCTGTCAAGGTC-3`(446-467); GBCO6, 5`-GACCTTGACAGCAGGCTTGTCC-3`(446-467); GBCO7, 5`-GACAAGCCTCTGGCTAAGGTCAC-3`(447-469); GBCO8, 5`-GTGACCTTAGCCAGAGGCTTGTC-3`(447-469); GCEO1, 5`-CCCAGAAAGCTTCCCGTTTGG-3`(490-511); GCEO2, 5`-CCAAACGGGAAGCTTTCTGGG-3`(490-511); GCEO3, 5`-CAGAAATTCGCTCGTTTGGATC-3`(492-514); GCEO4, 5`-GATCCAAACGAGCGAATTTCTG-3`(492-514); GCEO5, 5`-GAAATTCTCCGCTTTGGATCCC-3`(494-516); GCEO6, 5`-GGGATCCAAAGCGGAGAATTTC-3`(494-516); GCEO7, 5`-ATTCTCCCGTGCTGATCCCACC-3`(497-519); GCEO8, 5`-GGTGGGATCAGCACGGGAGAAT-3`(497-519); GCEO9, 5`-CTCCCGTTTGGCTCCCACCTTC-3`(500-522); GCE10, 5`-GAAGGTGGGAGCCAAACGGGAG-3`(500-522); GCE11, 5`-CCGTTTGGATGCTACCTTCTCC-3`(503-525); GCE12, 5`-GGAGAAGGTAGCATCCAAACGG-3`(503-525). The alanine codon GCT or its complement AGC are underlined. Oligonucleotides NR1 and EG5 are described above.

Chimeric and mutant receptor cDNA expression constructs were produced by subcloning the cDNAs into the eukaryotic expression vector pKC3(29) . Each cDNA was engineered in the PCRs to have an EcoRI site at its 5` end (the 5`-flanking oligonucleotide primer NR1 containing an EcoRI recognition site) and a SalI site at the 3` end (the 3`-flanking oligonucleotide primer EG5, containing a SalI recognition site), which enabled the cDNAs to be cloned into the EcoRI and SalI sites of pKC3. The nucleotide sequence integrities of the chimeric cDNAs were determined by dideoxynucleotide chain termination sequencing (30) using Sequenase (U.S. Biochemical Corp.) as described(31) .

Monoclonal Antibodies and Ig Reagents

Transfection

Immune Complex Binding

Generation of FcRII Domain 1-Domain 2 Model Structure

RESULTS

Chimeric Receptors Identify Multiple Regions of FcRII Involved in IgG Binding

The substitution of the FcRII domain 1 with that of the FcRI chain produced a receptor (designated D1D2), which as expected retained the capacity to bind the multivalent IgG-EA complexes, as did the wild-type FcRII (Fig. 1a). However, in contrast to the wild-type receptor the D1D2 chimeric FcR did not bind dimeric-hIgG1 at any concentration (Fig. 2). This suggests that domain 1 is necessary for optimal Ig binding as demonstrated by the binding of highly substituted but not small dimeric complexes.

Figure 1: IgG complex binding of chimeric Fc receptors. COS-7 cell monolayers were transfected with the following chimeric cDNA constructs: D1D2 (a), 109-116 (b), 130-135 (c), or an expressible form of the FcRI chain (d). The binding of IgG immune complexes was assessed directly on the monolayers by EA rosetting using mouse IgG1-sensitized erythrocytes. The transfections were performed using a transient expression system, resulting typically in 30-50% of cells expressing the chimeric FcR. IgG binding of the chimeric FcR is evident by COS-7 cells binding IgG-sensitized eythrocytes, i.e. forming ``rosettes,'' which appear as cell outlines covered in eythrocytes. Cells not expressing FcR or expressing FcR incapable of binding IgG do not bind the sensitized erythrocytes.

Figure 2: Human IgG1 dimer binding of chimeric Fc receptors. Radiolabeled dimeric human IgG1 was titrated on COS-7 cells transfected with wild-type FcRII (), an expressible form of the FcRI chain (), or the following chimeric receptor cDNAs: D1D2 (), 109-116 (), 130-135 (). All of the chimeras were expressed on the cell surface as determined by EA rosetting, outlined in the Fig. 1legend.

The previous analysis of genetic polymorphisms of FcRII (21, 22, 23, 24, 25) in conjunction with our molecular modeling studies described above(20) , suggest that the region around residue 114 (human equivalent of polymorphic residue 116 in mouse FcRII) in the predicted B/C loop may be important in Ig binding. To investigate this possibility a chimeric FcR (109-116) was constructed wherein the B/C loop of FcRII (residues Ser, Trp, Lys, Asp, Lys, Pro, Leu, Val) was replaced with the homologous region of the FcRI chain (Gly, Trp, Arg, Asn, Trp, Asp, Val, Tyr). After transfection into COS-7 cells, this receptor was clearly able to bind Ig in the form of multivalent immune complexes, i.e. erythrocytes highly sensitized with IgG (IgG-EA) (Fig. 1b). By contrast, this receptor was unable to bind dimeric hIgG1 at any concentration, implying that the B/C loop is essential for optimal Ig binding (Fig. 2). Similarly, the region surrounding residue 131 responsible for the responder/nonresponder phenotype of FcRIIa, i.e. the C`/E loop (Ser, Arg/His, Leu, Asp, Pro, Thr) was replaced with the equivalent FcRI chain sequence (Trp, Tyr, Glu, Asn, His, Asn), generating a chimeric receptor (130-135) that upon transfection into COS-7 cells was able to bind IgG-EA (Fig. 1c) but not dimeric IgG1 (Fig. 2). As expected COS-7 cells transfected with an expressible form of the FcRI chain (18) did not bind hIgG1 dimers or IgG-EA (Fig. 1d and 2). Thus the ability of the chimeric FcRII containing B/C or C`/E domain 2 substitutions to bind the highly sensitized EA complexes but not dimeric hIgG1 suggests that these receptors bind IgG less avidly than wild-type FcRII and clearly indicates that the B/C and C`/E regions also make a contribution to the binding of IgG by FcRII.

Fine Structure Analysis of the B/C and C`/E loops of FcRII Domain 2

Figure 3: Human IgG1 dimer binding by FcRIIa alanine point mutants. Radiolabeled dimeric human IgG1 was titrated on COS-7 cells transfected with wild-type FcRII or FcRII containing alanine point mutations. A, B/C loop mutants: wild-type FcRII (), Lys Ala (), Pro Ala (), Leu Ala (), Val Ala (). B, C`/E loop mutants: wild-type FcRII (), Phe Ala (+), Ser Ala (), Arg/His Ala (), Leu Ala (), Asp Ala (⊞), Pro Ala (). A comparison of the levels of human IgG1 dimer binding to FcRII mutants relative to wild-type FcRIIa is shown. C, B/C loop mutants; D, C`/E loop mutants. The binding of wild-type FcRIIa was taken as 100% and mock-transfected cells as 0% binding. Results are expressed as ±S.E. To control for variable receptor expression between the mutant FcRII COS-7 cell transfectants, levels of expression were determined using a radiolabeled monoclonal anti-FcRII antibody 8.2, and dimer binding was normalized to that seen for wild-type FcRII. Typical levels of 8.2 binding in cpm ±S.E.: WT FcRII, 95,279; Lys Ala, 71,660; Pro Ala, 61,636; Leu Ala, 44,696; Val Ala, 110,722; Phe Ala, 74,707; Ser Ala, 139,802; Arg/His Ala, 140,475; Leu Ala, 121,096; Asp Ala, 100,149; Pro Ala, 172,047.

The replacement of the B/C loop residues (Lys, Pro, Leu, Val) in turn with Ala in each case resulted in diminished hIgG1 dimer binding (Fig. 3). The most dramatic effect was seen on substitution of Lys or Leu, which exhibited only 15.9 ± 3.4% (mean ± S.D.) and 20.6 ± 4.0% binding compared with wild-type FcRII. The replacement of Pro or Val with Ala had a lesser effect, these receptors displaying 53.5 ± 13.5% and 73.5 ± 7.9% wild-type binding respectively. It is interesting to note that the individual replacement of these amino acids did not result in the complete abolition of dimer binding seen in chimera 109-116. These results suggest that each of these residues in the B/C loop contribute to the binding of IgG by FcRII either as direct contact residues or indirectly by maintaining the correct conformation of the binding site. The same approach was used to analyze the role of individual amino acids within the C`/E loop (Phe, Ser, Arg/His, Leu, Asp, Pro). In contrast to that observed for residues of the B/C loop, mutation of individual residues of the C`/E loop resulted in both loss and enhancement of IgG binding. Substitution of Phe and Arg/His dramatically decreased hIgG1 dimer binding by approximately 90 and 80%, respectively, to 8.2 ± 4.4 and 21.9 ± 3.9 compared with that seen for wild-type FcRII (Fig. 3). Interestingly, replacement of residues Asp and Pro increased binding to 113.5 ± 8.8% and 133.5 ± 3.2% of the wild-type receptor. The substitution of Ser or Leu had no significant effect on the binding of hIgG1 dimers, since these mutants exhibited binding comparable with that seen for wild-type FcRII (Fig. 3). These findings suggest that Phe and Arg/His may play an important role in the binding of hIgG1, and the observation that the substitution of Asp and Pro increase binding also suggests an important role for these residues, which appears distinct from Phe and Arg/His. Again, a distinction between a possible direct binding role or contribution to structural integrity of the receptor cannot be made; however, these findings clearly identify both the B/C and C`/E loops as playing a role in the binding of IgG by FcRII.

Site-directed mutagenesis was also performed on 3 residues of the C`/C loop, a region predicted to be distant from the putative binding region, i.e. the B/C, C`/E, and F/G loop regions. The substitution of residues Asn, Gly, and Lys had no effect on the binding of hIgG1 dimer, since each of these mutants exhibited similar binding to the wild-type receptor (data not shown).

Molecular Modeling of FcRII Extracellular Region

Figure 4: Molecular modeling of the extracellular region of human FcRII (domains 1 and 2) and location of residues putatively involved in the interaction with hIgG1. A, FcRII domain 1-domain 2 model structure. Domain 1 is shown in green and domain 2 in darkblue. The three regions of domain 2 putatively involved in IgG binding (B/C, C`/E, and F/G loops) are highlighted in paleblue. The side chains of amino acids implicated in hIgG1 binding as described under ``Results'' are indicated. Those side chains that when substituted result in decreased or increased binding are shown in paleyellow or red, respectively. The brightyellow regions represent the A/B and G strands of domain 1, predicted to be in close proximity to the domain 2 active binding region. B, location of residues putatively involved in the interaction of FcRII with hIgG1. Domain 2 and the domain 1 interface region of the FcRII domain 1-domain 2 model is shown to highlight the putative binding region. Residues implicated in IgG1 binding are indicated as described above. The computer model of FcRII domain 1domain 2 was generated by molecular modeling based on the structure of the related CD4 domains 1 and 2 as described under ``Materials and Methods.''

DISCUSSION

The studies described herein provide evidence to suggest that the interaction of IgG with human FcRII involves multiple regions juxtaposed in the receptor. Previously, we have described the localization of a single region of FcRII capable of directly binding IgG situated in the second extracellular domain between residues Asn and Ser(20) . Of the entire extracellular region, only the 154-161 segment was demonstrated to directly bind IgG, since placement of only this region in the corresponding region of the human FcRI chain, imparted IgG binding function to the IgE receptor FcRI. Moreover, replacement of this region in FcRII with that of FcRI resulted in the total loss of IgG binding including large complexes, implying that residues Asn-Ser comprise the key IgG1 interactive site of FcRII. However, the generation of further chimeric FcRII/FcRI receptors as described herein indicates that two additional regions of FcRII domain 2 also influence the binding of IgG by FcRII. The replacement of the regions encompassing Ser-Val (B/C loop) and Ser- Thr (C`/E loop) of FcRII with the equivalent regions of the FcRI chain, produced receptors that, despite containing the putative binding site (Asn-Ser) and retaining the ability to bind large complexes (IgG-EA), lost the capacity to bind small complexes (dimeric hIgG1). Indeed, site-directed mutagenesis performed on residues of the B/C and C`/E regions identified a number of amino acids that appear to play crucial roles in hIgG1 binding by FcRII. The replacement of Lys, Pro, Leu, and Val of the B/C loop and Phe and Arg/His of the C`/E loop with alanine all resulted in diminished hIgG1 binding. Furthermore, the substitution of Asp and Pro of the C`/E loop increased hIgG1 binding. Therefore, these findings provide strong evidence to suggest that the B/C and C`/E loops of FcRII, in addition to the F/G loop, also contribute to the binding of IgG.

A number of other studies have provided evidence to support the proposed IgG binding roles of the B/C and C`/E loop regions of FcRII. Studies of genetic polymorphisms of mouse and human FcRII have implicated residues 114, 131, and 159 in the binding of IgG by human FcRII. These residues are located in the B/C (residue 114), C`/E (131), and F/G (159) loops, respectively. The Ly-17 polymorphism of mouse FcRII has been described at the molecular level as two allelic variants (Ly17.1 and Ly17.2) that differ only at residues 116 and 161 (the equivalent of residues 114 and 159 in the human). Monoclonal antibodies specific for Ly17.2 inhibit the binding of IgG to the receptor, implying that residues 116 and/or 161 (and therefore their human equivalents) are involved in binding themselves or closely situated to residues crucial in the interaction of FcRII with IgG(24, 25) . Furthermore, the high responder/low responder polymorphism of hFcRIIa results in an amino acid substitution at residue 131, which has been shown to influence the binding of mIgG1 and hIgG2(21, 22, 23) . The findings described herein also indicate that the nature of the residue at 131 plays a role in the binding of hIgG1, since replacement with alanine results in almost complete loss in binding of this isotype to FcRII. Thus, although the F/G loop of FcRII is clearly a major region involved in the direct interaction with IgG, as demonstrated by the fact that only this region has been definitively shown to directly bind IgG(20) , residue 131 also appears to play a binding role. However, the question of whether residue 131 is directly participating in IgG binding or providing a secondary or indirect influence remains to be answered.

The mutagenesis data clearly implicate a number of distinct regions within FcRII in the interaction with IgG complexes as described above. The spatial relationship of these regions, i.e. residues 109-116 (B/C loop), 129-135 (C`/E loop), and 154-161 (F/G loop) is postulated in our model of FcRII (Fig. 4). This model suggests that these regions are juxtaposed to each other in domain 2 at the interface with domain 1 and form a hydrophobic pocket surrounded by a wall of additional residues. The data supporting this model include the following. 1) Mutagenesis of the hydrophobic residues Ile, Gly, Pro, Leu almost completely abolishes binding of dimeric hIgG1 complexes. 2) Substitution of residues that may contribute the wall (Lys in the B/C loop, Phe and Arg in the C`/E loop, and Leu and Phe in the F/G loop) also modify binding of immune complexes. 3) It may also be expected that such a wall would be accessible to anti-FcR antibodies. Indeed several anti-FcRII monoclonal antibodies detect epitopes in the B/C, C`/E, and F/G loops. For example, the epitope detected by the anti-human FcRII antibody 41H16 (39) is dependent on residue 131 of the C`/E loop, and the Ly-17 epitope of mouse FcRII is dependent on residues that equate to residues 114 and 159 in human FcRII (25) that are located in the B/C and F/G loops, respectively. 4) The studies described herein demonstrate that domain 1 of FcRII, although it does not appear to play a direct role in IgG binding, does play an important role in the affinity of IgG binding by FcRII. This is suggested since replacement of domain 1 of FcRII with domain 1 of FcRI reduced the capacity of FcRII to bind IgG, as shown by the failure of this receptor to bind dimeric hIgG1. These data imply that the IgG binding role of domain 1 is likely to be an influence on receptor conformation, stabilizing the structure of domain 2 to enable efficient IgG binding by FcRII. Again this proposal is consistent with the molecular modeling, which suggests the localization of the IgG binding site of FcRII to loop regions in domain 2 at the interface with domain 1. The binding site would therefore be in close proximity to domain 1 and as such predicted to be influenced in conformation, presumably by the loop and strand regions at the ``bottom'' of domain 1. These regions include the G strand and the A/B and E/F loops, which may therefore interact with the ``active'' binding region of domain 2.

Further support for the involvement of the B/C and C`/E loops of FcRII domain 2 in the binding of IgG has been provided in the cloning and subsequent Ig binding studies of rat FcRIII(40) , which is structurally and functionally homologous to FcRII. Two rat FcRIII isoforms, IIIA and IIIH, which have extensive amino acid differences in their second extracellular domains, have been shown to bind rat and mouse IgG subclasses differently. Both isoforms bind rtIgG1, rtIgG2a, and mIgG1; however, they differ in that only the IIIH form binds rtIgG2b and mIgG2b. Significantly, the amino acid differences between rat FcRIIIA and IIIH isoforms are situated predominantly in the predicted B/C and C`/E loops of domain 2. However, it should be noted that the F/G loop regions of rat FcRIIIA and IIIH are almost totally conserved, which together with the observation that both forms bind rtIgG1, rtIgG2a, and mIgG1, is consistent with the proposal that the F/G loop region is the major IgG interactive region and that the B/C and C`/E loop regions provide supporting binding roles. In addition, a recent mutagenesis study of human FcRIII has also implicated residues in the B/C and C`/E loops of this receptor in the binding of IgG(41) . It is also interesting to note that in this study the C/C` region of FcRIII was suggested to play a major role in IgG binding, which is in marked contrast to our findings with FcRII. Indeed, the substitution of 3 residues in the C/C` loop of FcRII with alanine, namely Asn, Gly, and Lys, did not have any effect on the binding of dimeric hIgG1. Therefore, these findings somewhat surprisingly suggest that FcRII and FcRIII, which exhibit substantial amino acid sequence conservation and similar IgG binding affinities and specificities, may interact differently with IgG.

It is interesting to note that a number of parallels are apparent in the molecular basis of the interaction of FcRII with IgG and that of FcRI with IgE. The Ig binding roles of the two extracellular domains of FcRI are similar to FcRII, with domain 2 responsible for the direct binding of IgE and domain 1 playing a supporting structural role (18, 26, 42) . Furthermore, as described for FcRII, we and others have also identified multiple IgE binding regions in domain 2 of FcRI. Using chimeric FcRII/FcRI receptors we have demonstrated that domain 2 of FcRI contains at least three regions, each capable of directly binding IgE, since the introduction of the FcRI regions encompassed by residues Trp-Lys, Tyr-Asp, and Lys-Glu into the corresponding regions of FcRII was found to impart IgE binding to FcRII(1, 18, 20) . A similar study using chimeric FcRIII/FcRI receptors has implicated 4 regions of FcRI domain 2 in IgE binding since the regions Ser-Phe, Arg-Glu, Asp-Ser, and Lys-Ile of FcRI when replaced with the corresponding regions of FcRIII resulted in the loss or reduction of IgE binding(42) . Taken together, these data suggest that at least four regions of FcRI domain 2 contribute to the binding of IgE, Ser-Phe, Arg-Glu, Tyr-Ser, and Lys-Glu. Three of these regions correspond to the three regions identified herein as important in the binding of IgG by FcRII, Arg-Glu, Tyr-Ser, and Lys-Glu, which encompass the B/C, C`/E, and F/G loops, respectively. In addition, studies with anti-FcRI chain mAb have indicated that the region encompassed by residues 100-115 contains an epitope detected by mAb 15A5, which can completely block the binding of IgE to FcRI(43) . Thus, these findings implicate the B/C, C`/E, and F/G loops juxtaposed in domain 2 at the domain 1 interface as the crucial IgE-interactive region of FcRI. Clearly, the findings described herein for FcRII together with those discussed for FcRI provide evidence to suggest that the Ig-interactive regions of FcRII and FcRI are conserved between the two receptors, with the domain 1-domain 2 interface forming the Ig binding site.

In conclusion, the results presented herein demonstrate that multiple regions of hFcRII are involved in the binding of IgG, with three putative loop regions juxtaposed in the second extracellular domain at the domain 1 interface comprising the IgG binding site. The proposition that the functionally distinct receptor FcRI also interacts with IgE in a structurally similar fashion, in conjunction with the conserved nature of the extracellular regions of the Ig superfamily FcR, strongly suggests that this region will also comprise the key Ig-interactive site of all members of this family.

FOOTNOTES

*

This work was supported with the assistance of the National Health and Medical Research Council and Harry Triguboff. 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.

§

Recipient of a National Health and Medical Research Council Australian Postdoctoral Research Award.

¶

To whom correspondence should be addressed: The Austin Research Inst., Austin Hospital, Studley Rd., Heidelberg Vic 3084, Australia. Tel.: 61-3-287-0666; Fax: 61-3-287-0600.

()

The abbreviations used are: mAb, monoclonal antibody; EA, antibody-sensitized erythrocyte; r.m.s., root mean square; SOE, splice overlap extension; PCR, polymerase chain reaction; SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate.

ACKNOWLEDGEMENTS

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