Fine Structure Analysis of Interaction of FcεRI with IgE*

The high affinity receptor for IgE (FcεRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcεRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the α-subunit (FcεRIα). In this study, the IgE binding site of human FcεRIα has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcεRIα and the functionally distinct but structurally homologous low affinity receptor for IgG (FcγRIIa) have been used to localize two IgE binding regions of FcεRIα to amino acid segments Tyr129–His134 and Lys154–Glu161. Both regions were capable of independently binding IgE upon placement into FcγRIIa. Molecular modeling of the three-dimensional structure of FcεRIα-D2 has suggested that these binding regions correspond to the “exposed” C′-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129–His134 and Lys154–Glu161 regions of FcεRIα was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116in the B-C loop region, which lies adjacent to the C′-E and F-G loops, has suggested Trp113 also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcεRIα-IgE interaction for the possible treatment of IgE-mediated allergic disease.

The binding of IgE by Fc⑀RI on mast cells and basophils is a fundamental step in the cascade of events that lead to allergic disease. The interaction of multivalent allergen with Fc⑀RI-bound IgE results in cross-linking of the receptor, which triggers a range of biological sequelae that ultimately leads to the release of inflammatory mediators and the onset of the type I hypersensitivity response (1,2). Approaches that intervene in the binding of IgE by Fc⑀RI may prove useful in the treatment of allergic disease. Clearly, understanding the molecular basis of the interaction of Fc⑀RI with IgE would provide valuable information for such a therapeutic strategy.
Studies from our group and others using chimeric receptors together with the epitope mapping of anti-Fc⑀RI␣ monoclonal antibodies have identified the second extracellular domain of Fc⑀RI␣ as the principle IgE interactive domain (9 -12). The first extracellular domain has not been demonstrated to have a direct IgE binding role; however, it does appear to make an important structural contribution in the maintenance of the high affinity IgE binding of the receptor (9,11). Multiple regions of Fc⑀RI␣-D2 have been implicated in the binding of IgE. In a series of "gain of function" experiments using chimeric Fc⑀RI␣/Fc␥RIIa receptors, we identified three relatively large regions of Fc⑀RI␣-D2, each capable of independently binding IgE (9). The Fc⑀RI␣ regions encompassed by residues Trp 87 -Lys 128 , Tyr 129 -Asp 145 , or Ser 146 -Val 169 when inserted into Fc␥RIIa were each able to impart IgE binding to the receptor. Mallamaci et al. (10) have used a similar approach with chimeric Fc⑀RI␣/Fc␥RIII receptors, however, in "loss of function" experiments and identified four regions of Fc⑀RI␣-D2 that putatively contribute to IgE binding. The replacement of each of the Fc⑀RI␣ regions encompassed by residues Ser 93 -Phe 104 , Arg 111 -Glu 125 , Asp 123 -Ser 137 , and Lys 154 -Ile 167 with the corresponding regions of Fc␥RIII, was found to result in reduced IgE binding. In addition, a recent study by McDonnell et al. (13) has demonstrated that residues Ile 119 -Tyr 129 of Fc⑀RI␣-D2, when synthesized as a conformationally constrained peptide, can inhibit the binding of IgE to Fc⑀RI.
Despite the localization of multiple binding regions in Fc⑀RI␣-D2, the interaction of Fc⑀RI with IgE at the level of individual residues has not been defined. In this study, we have identified small IgE binding subregions of Fc⑀RI␣-D2, which have been analyzed by site-directed mutagenesis, and residues putatively involved in the interaction with IgE have been determined. These findings have enabled the development of a model of how Fc⑀RI␣ binds IgE and contribute to our understanding of the interaction of the leukocyte FcR family with their Ig ligands.

Generation of Chimeric Fc⑀RI␣/Fc␥RIIa and Mutant Fc⑀RI␣ Receptor cDNA Expression Constructs
Chimeric Fc⑀RI␣/Fc␥RIIa or mutant Fc⑀RI␣ cDNAs were constructed by splice overlap extension PCR 1 (14) using an expressible form of the Fc⑀RI␣ chain (15) or Fc␥RIIa NR cDNA (16) as templates. The expressible form of the Fc⑀RI␣ chain consists of the extracellular region of Fc⑀RI␣ linked to the transmembrane and cytoplasmic tails of Fc␥RIIa and is expressed on the cell surface and binds monomeric hIgE with an affinity comparable with that of the wild-type Fc⑀RI␣ chain, as described previously (15). Splice overlap extension PCR was performed as follows. Two PCR reactions were used to amplify the Fc⑀RI␣-Fc␥RIIa or Fc⑀RI␣ fragments to be spliced together. The reactions were performed on 100 ng of the Fc⑀RI␣ cDNA in the presence of 500 ng of each oligonucleotide primer, 1.25 mM dNTPs, 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.25 mM dNTPs, 1.5 mM MgCl 2 using 2.5 units of Taq polymerase (Amplitaq; Cetus) for 25 amplification cycles. A third PCR was performed to splice the two fragments and amplify the spliced product. 100 ng of each purified fragment was used with the appropriate oligonucleotide primers under the above PCR conditions. Chimeric and mutant receptor cDNA expression constructs were produced by subcloning the cDNAs into the eukaryotic expression vector pKC3 (17). Each cDNA was engineered in the PCRs to have an EcoRI site at their 5Ј-end (the 5Ј-flanking oligonucleotide primer containing an EcoRI recognition site) and a SalI site at their 3Ј end (the 3Ј-flanking oligonucleotide primer 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 (18) using Sequenase TM (U.S. Biochemical Corp.) as described (19).

Monoclonal Antibodies and Ig Reagents
The anti-Fc⑀RI␣ mAb 3B4 and the anti-Fc␥RIIa mAb 8.2 were produced in this laboratory (20). The anti-Fc⑀RI␣ mAb 15A5 was a gift of Dr. J. Kochan (12). The mouse IgE anti-2,4,6-trinitrophenyl mAb (TIB142) was produced from a hybridoma cell line obtained from the American Type Culture Collection (Rockville, MD); the mouse IgG 1 anti-2,4,6-trinitrophenyl mAb (A3) was produced from a hybridoma cell line that was a gift of Dr. A. Lopez (21). Human IgE myeloma protein was purified from the serum of a myeloma patient. IgE was precipitated with NH 4 SO 4 , and then IgG was removed by chromatography on protein A, and IgE was purified by size fractionation chromatography on Sephacryl S-300 HR ( Amersham Pharmacia Biotech). Purified IgE was analyzed by SDS-polyacrylamide gel electrophoresis and by enzymelinked immunosorbent assay, and contaminating IgG was estimated at Ͻ1%. Transfection COS-7 cells (30 -50% confluent per 5-cm 2 Petri dish) were transiently transfected with FcR cDNA expression constructs by the DEAE-dextran method (22). Cells were incubated with a transfection mixture (1 ml/5cm 2 dish) consisting of 5-10 mg/ml DNA, 0.4 mg/ml DEAE-dextran (Amersham Pharmacia Biotech), and 1 mM chloroquine (Sigma) in Dulbecco's modified Eagle's medium (Flow Laboratories, Australia) containing 10% (v/v) Nuserum (Flow Laboratories, Australia), for 4 h. The transfection mixture was then removed, and the cells were treated with 10% (v/v) dimethyl sulfoxide in phosphate-buffered saline (7.6 mM Na 2 HPO 4 , 3.25 mM NaH 2 PO 4 , 145 mM NaCl), pH 7.4, for 2 min, washed, and returned to fully supplemented culture medium for 48 -72 h before use in assays. COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM glutamine (Commonwealth Serum Laboratories, Melbourne, Australia), and 0.05 mM 2-mercaptoethanol (Koch Light Ltd., Birmingham, United Kingdom).

Ig Binding Assays
The binding of Ig by COS-7 cells following transfection with chimeric or mutant receptor cDNAs was determined using two approaches.
Erythrocyte-Antibody Rosetting-COS-7 cell monolayers transfected with FcR expression constructs were incubated with EA complexes, prepared by coating sheep red blood cells with trinitrobenzene sulfonate (Fluka Chemika, Switzerland) and then sensitizing these cells with mouse IgE or IgG 1 anti-2,4,6-trinitrophenyl mAb (23). Two ml of 2% EAs (v/v) were added per 5-cm 2 dish of transfected cells and incubated for 5 min at 37°C. Plates were then centrifuged at 500 ϫ g for 3 min and placed on ice for 30 min. Unbound EA were removed by washing with L-15 medium modified with glutamine (Flow Laboratories, Melbourne, Australia) and containing 0.5% bovine serum albumin.
Direct Binding of Monomeric Human IgE-COS-7 cells transfected with FcR expression constructs were harvested; washed in phosphatebuffered saline, 0.5% bovine serum albumin; and resuspended at 10 7 cells/ml in L-15 medium, 0.5% bovine serum albumin. 50 l of cells were incubated with 50-l serial dilutions of 125 I-hIgE for 120 min at 4°C. 125 I-hIgE was prepared by the chloramine-T method as described (24) and shown to compete equally with unlabeled hIgE in binding to Fc receptor expressing COS-7 cells. Cell-bound 125 I-hIgE was determined following centrifugation of cells through a 3:2 (v/v) mixture of dibutylphthalate and dioctylphthalate oils (Fluka Chemika, Buchs, Switzerland), and cell bound 125 I-hIgE was determined. Nonspecific hIgE binding was determined by assaying on mock-transfected cells and subtracted from total binding to give specific hIgE bound. Levels of COS-7 cell surface expression of the mutant Fc⑀RI␣ receptors were determined by assessing the binding of the anti-Fc⑀RI␣ mAb 22E7 (shown to bind distantly to the binding site; see Ref. 12) at 2 g/ml in a direct binding assay as described for the binding of hIgE. Any variation in cell surface receptor expression between the mutant Fc⑀RI␣ and wild-type Fc⑀RI␣ COS-7 cell transfectants (levels ranged from 80 to 120% of wild-type Fc⑀RI␣) was then normalized, and the binding of hIgE by the mutant Fc⑀RI␣ receptors was corrected using the following formula: (mutant Ϫ mock IgE binding) ϫ ((wild type Ϫ mock 22E7 binding)/(mutant Ϫ mock 22E7 binding)).

Generation of Fc⑀RI␣ Domain 2 Model Structure
Molecular modeling of domain 2 (D2) of human Fc⑀RI␣ was performed using the Homology and Discover modules of the InsightII environment of Molecular Simulations Inc. on a Silicon Graphics Indigo workstation. The model of Fc⑀RI␣-D2 was constructed by mutation of our previously described model of human Fc␥RIIa-D2 (25), which was based on the crystal structure of domain 2 of CD4 (protein data base file pbd2cd4.ent; Brookhaven National Laboratory, Upton, NY) (26,27). Briefly, using a sequence alignment of Fc⑀RI␣-D2 with Fc␥RIIa-D2 and CD4-2 (28), regions of Fc␥RIIa-D2 aligned with the ␤-sheet residues of CD4-2 were designated as structurally conserved residues, with other residues designated as loops. The coordinates for the atoms of the structurally conserved residues of Fc␥RIIa were assigned from those of the equivalent residues in the pbdcd4 file, with the coordinates of the side chain atoms assigned through mutation of the pbdcd4 side chains. Using the Homology Loop Search function, segments of Protein Data Bank files with the correct number of residues and appropriate gap distance were obtained. Incorporation of the loops was then followed by elimination of severe atomic overlaps ("bumps") by altering the torsion angles of side chain C␣-C␤ bonds. The structure was then minimized using the Discover module to a maximum root-mean-square derivative of 0.0001. The Fc␥RIIa-D2 model was then converted to a Fc⑀RI␣-D2 model by mutation of the residues followed by further minimization of the structure using the above protocol.

Molecular Modeling of the Extracellular Domains of
Fc⑀RI␣-The three-dimensional structure of Fc⑀RI␣ has not yet been solved. To aid in the localization of putative IgE binding regions of Fc⑀RI␣, we have generated a three-dimensional model of the second extracellular domain (D2) of Fc⑀RI␣ based on the known structure of a related domain, CD4-2. CD4-2 belongs to the C2 set of Ig superfamily members, and sequence alignment of Fc⑀RI␣-D2 with CD4-2 suggests that this domain will adopt a similar folding pattern (25,28). The structure of the Fc⑀RI␣-D2 model is characteristic of the C2 Ig-fold, comprising seven ␤-strands (A, B, C, CЈ, E, F, G) that form two antiparallel ␤-sheets of ABE and CCЈFG (Fig. 1A).

Chimeric Receptors Identify Multiple Regions of Fc⑀RI␣ Involved in IgE
Binding-Based on the location of the previously described IgE binding regions (9 -13) on our three-dimensional molecular model of Fc⑀RI␣-D2 and by analogy with mapping studies of the homologous interaction of the Fc␥R with IgG (25, 28 -31), we targeted the B-C, CЈ-E, and F-G loop regions of Fc⑀RI␣-D2 as likely to be involved in the binding of IgE. In order to assess the contribution these three loops made to the binding of IgE by Fc⑀RI␣, chimeric receptors were generated, whereby Fc␥RIIa was used as a scaffold to accept each of these Fc⑀RI␣ loop regions. The three resultant chimeric receptors consisted of Fc␥RIIa containing the following regions of Fc⑀RI␣-D2: (i) the B-C loop, residues Gly 109 -Tyr 116 ; (ii) CЈ-E loop, residues Tyr 129 -His 134 ; and (iii) F-G loop, residues Lys 154 -Glu 161 , designated the ␥109 -116⑀, ␥129 -134⑀, and ␥154 -161⑀ chimeric receptors, respectively. COS-7 cells were transfected with expression constructs of these chimeric receptors and tested for their capacity to bind mouse IgE (or IgG 1 ) immune complexes by EA rosetting. Cells transfected with the ␥154 -161⑀ chimeric receptor bound IgE-EA ( Fig. 2A, Table I), and the binding was specific, since mock-transfected cells or cells transfected with Fc␥RIIa did not bind IgE-EA (Table I).
These data indicate that the Lys 154 to Glu 161 region of Fc⑀RI␣ can direct the binding of IgE. As expected, this chimeric receptor was unable to bind IgG 1 , since the previously described IgG binding region, residues Asn 154 -Ser 161 (25), has been replaced with the homologous Fc⑀RI␣ sequence (Table I). Similar experiments demonstrated that the ␥129 -134⑀ chimeric receptor could also specifically bind IgE-EA (Fig. 2B, Table I), indicating that the Tyr 129 -His 134 region also contains an IgE binding site. As expected, this chimeric receptor was able to bind IgG 1 -EA (Table I) due to the presence of the Fc␥RIIa Asn 154 -Ser 161 IgG binding sequence.
As described above, the segment of Fc⑀RI␣-D2 encompassed by residues 87-128 had previously been shown to contain an IgE binding site, which we predicted to be the B-C loop (28). However, when transfected into cells, the ␥109 -116⑀ chimeric receptor containing the Fc⑀RI␣ B-C loop did not bind IgE-EA (Fig. 2C). Since the receptor was clearly expressed on the cell surface, demonstrated by its ability to bind IgG-EA (Table I), these results suggest that the Gly 109 -Tyr 116 region is insufficient to bind IgE in its own right and therefore that the IgE binding region in the 87-128 segment is either not the B-C loop or requires the B-C loop in combination with additional surrounding region(s). This was further investigated by site-directed mutagenesis (see below).
Fine Structure Analysis of the Fc⑀RI␣ IgE Binding Site-To identify the key residues of the Fc⑀RI␣ binding regions (CЈ-E loop, residues Tyr 129 -His 134 ; F-G loop, residues Lys 154 -Glu 161 ) involved in the interaction with IgE, site-directed mutagenesis was used to replace each residue in these regions with alanine. In addition, residues Trp 113 , Val 115 , and Tyr 116 in the B-C loop were also substituted with alanine, since the Fc⑀RI␣-D2 model predicts this region is likely to be adjacent to the F-G and CЈ-E loops and may therefore contribute to IgE binding. The alanine substitution mutants of Fc⑀RI␣ were expressed in COS-7 cells, and the binding of monomeric human IgE was examined in direct binding assays by titration of 125 I-labeled hIgE (Fig. 3). The levels of cell surface expression of the Fc⑀RI␣ mutants on the COS-7 cell transfectants were determined using the Fc⑀RI␣ mAb 22E7, shown to detect an epitope distant to the binding site (12). Using these results, the binding of hIgE was corrected for variation in expression between the mutant receptors, which ranged from 80 to 120% of wild-type Fc⑀RI levels (data not shown).
First, the individual alanine substitution of residues Lys 154 -Glu 161 in the F-G loop indicated that each mutant retained hIgE binding, with the striking exception of the Val 155 -Ala mutant, where binding of monomeric hIgE was almost totally abolished, this receptor exhibiting only 3.2 Ϯ 2.1% (mean Ϯ S.D.) binding relative to the wild-type receptor (Fig. 3, A and  The binding of wild-type Fc⑀RI␣ was taken as 100% and mock-transfected cells as 0% binding. Results are expressed as means Ϯ S.E. To control for variable receptor expression between the mutant Fc⑀RI COS-7 cell transfectants, levels of expression were determined using a radiolabeled monoclonal anti-Fc⑀RI antibody 22E7, and IgE binding was normalized to that seen for wild-type Fc⑀RI (see "Experimental Procedures").

TABLE I Chimeric FcR composition and Ig complex binding
A schematic representation of the domain 2 composition of the chimeric receptors is shown. Shaded regions are derived from the Fc⑀RI␣ chain, and unshaded regions are derived from Fc␥RIIa. The relative positions of the putative ␤-strands are shown above as labeled solid lines. The binding of mouse IgE and IgG 1 (mIgE and mIgG) was assessed by EA rosetting as described under "Experimental Procedures." ϩ, Ͼ10% of cells rosetting; Ϫ, no cells rosetting. D). The loss of hIgE binding by this mutant receptor was not due to decreased cell surface expression as demonstrated by its expression on the cell surface in levels comparable with that of wild-type Fc⑀RI (data not shown). The substitution of Asp 159 with alanine also resulted in diminished IgE binding, this receptor exhibiting 52.7 Ϯ 7.2% binding of the wild-type receptor. The substitution of Lys 154 , Gln 157 , and Leu 158 with alanine had no significant effect on the binding of IgE, these mutants exhibiting binding comparable with wild-type Fc⑀RI␣. In contrast, the replacement Trp 156 , Tyr 160 , or Glu 161 with alanine produced the interesting effect of increasing the binding of IgE (132.7 Ϯ 14.0, 123.7 Ϯ 11.1, and 139 Ϯ 15.0% of wild-type Fc⑀RI␣, respectively). Therefore, these findings clearly identify five individual residues of the F-G loop of Fc⑀RI␣ (Val 155 , Trp 156 , Asp 159 , Tyr 160 , and Glu 161 as playing critical roles in the binding of hIgE. The observation that substitution of Val 155 and Asp 159 decreased binding suggests that these residues may directly interact with hIgE. The increased binding observed upon substitution of Trp 156 , Tyr 160 , and Glu 161 also suggests an important role for these residues, which is possibly a contribution to the structural integrity of the binding site, although a direct role in hIgE binding cannot be excluded. As observed for residues Lys 154 -Glu 161 of the F-G loop, alanine substitution of residues Tyr 129 -His 134 of the CЈ-E loop was found to result in loss or enhancement of hIgE binding. Substitution of Tyr 131 and Glu 132 substantially decreased hIgE binding to 30.3 Ϯ 4.4 and 61.4 Ϯ 3.9% that of wild-type Fc⑀RI␣ (Fig. 3, B and E). In contrast, replacement of Trp 130 dramatically increased binding by over 70% to 172.5 Ϯ 8.8% binding of the wild-type receptor. The substitution of Tyr 129 , Asn 133 , and His 134 had no significant effect on the binding of hIgE, since these mutants exhibited binding comparable with that seen for wild-type Fc⑀RI␣ (data not shown). These findings suggest that Trp 130 , Tyr 131 , and Glu 132 may play an important role in the binding of hIgE. Again, a distinction between a possible direct binding role or contribution to structural integrity of the receptor cannot be made. However, as for the mutagenesis studies of the F-G loop, these results clearly identify the CЈ-E loop as also playing a role in the binding of IgE by Fc⑀RI␣.
Although the chimeric receptor strategy failed to reveal a direct binding role for the B-C loop (residues Gly 109 -Tyr 116 ), mutagenesis of residues Trp 113 , Val 115 , and Tyr 116 within this loop suggests that it may also contribute to IgE binding by Fc⑀RI␣. This was demonstrated, since the substitution of Trp 113 for alanine resulted in a dramatic loss of IgE binding, this mutant receptor exhibiting only 18.6 Ϯ 3.2% of IgE binding relative to the wild-type receptor. Substitution of Val 115 or Tyr 116 with alanine did not significantly alter IgE binding (Fig. 3, C and F). DISCUSSION Two approaches have been used to identify and analyze the IgE binding site of Fc⑀RI␣. First, to localize IgE binding regions of Fc⑀RI␣, a series of chimeric FcRs were engineered by exchange of segments between the second domain of Fc⑀RI␣ and Fc␥RIIa. Second, fine structure analysis of these binding regions, and an additional region likely to be in juxtaposition, was performed by generating 17 point mutants in Fc⑀RI␣ using alanine scanning mutagenesis. These approaches have enabled the localization of IgE binding regions in Fc⑀RI␣ to subregions of the second extracellular domain and identified key residues putatively involved in the interaction with IgE. Based on a molecular model of Fc⑀RI␣-D2, these data suggest that the IgE binding regions comprise the F-G, CЈ-E, and B-C loops and adjacent strand regions of this domain. Both the F-G and CЈ-E loops were directly implicated in the interaction with IgE, since insertion of these regions into Fc␥RIIa was able to impart IgE binding to this receptor. In contrast, insertion of the B-C loop was itself insufficient to direct the binding of IgE. However, site-directed mutagenesis of this region identified the residue Trp 113 as playing an important binding role, which provides evidence to suggest that the B-C loop also contributes to the interaction with IgE.
The molecular model of Fc⑀RI␣-D2 suggests that the F-G, CЈ-E, and B-C loops of Fc⑀RI␣-D2 are likely to be juxtaposed at the membrane distal end of the domain at the interface with domain 1. The localization of the Fc⑀RI␣-D2 IgE interactive sites to this region, together with the finding that domain 1 also plays a key role in maintaining high affinity binding of the receptor (9,11), suggests that the interdomain region between domains 1 and 2 comprises an important region of interaction of Fc⑀RI␣ with IgE. In support of this model, the anti-Fc⑀RI␣ mAb 15A5, which recognizes an epitope in the B-C loop region of Fc⑀RI␣-D2, is able to block the binding of IgE to Fc⑀RI completely (12), suggesting that the multiple IgE binding regions are likely to be situated in close proximity to one another.
Interestingly, a recent study examining the IgE inhibitory capacity of synthetic peptides designed to mimic regions of Fc⑀RI␣-D2 has also implicated the CЈ-C loop (residues Ile 119 -Tyr 129 as playing a role in the binding of IgE (13). A peptide encompassing this region and designed to mimic the conformation of the CЈ-C loop was demonstrated to competitively inhibit IgE binding to Fc⑀RI␣ and prevent IgE-mediated mast cell degranulation in vitro. Thus, the inclusion of the C'-C loop with the B-C, CЈ-E, and F-G loops described herein extends the putative region of contact of Fc⑀RI␣ with IgE. These data therefore suggest that the entire four-stranded ␤-sheet face of Fc⑀RI␣-D2, namely the C-CЈ-F-G strands and adjacent loops, may be important in the interaction of Fc⑀RI␣ with IgE.
The alanine scanning mutagenesis of the F-G, CЈ-E, and B-C loops of Fc⑀RI␣-D2 has identified a number of residues that may contribute to the binding of IgE. The substitution of amino acids Trp 113 , Tyr 131 , Glu 132 , Val 155 , and Asp 159 with alanine decreased the binding of IgE, whereas substitution of Trp 130 , Trp 156 , Tyr 160 , and Glu 161 increased binding. Based on the three-dimensional model of Fc⑀RI␣-D2, the side chains of these residues are exposed predominantly on the surface of the domain and contribute to a continuous face in the C-CЈ-F-G region (Fig. 1B). The majority of these residues are aromatic (Trp 113 , Trp 130 , Tyr 131 , Trp 156 , Tyr 160 ) or charged (Glu 132 , Asp 159 ) and are likely candidates for direct contact with IgE.
Studies examining the binding regions on the Fc portion of IgE for Fc⑀RI␣ have identified a number of putative interactive sites (32)(33)(34)(35)(36). The third constant domain (C⑀3) appears to be the principle Fc⑀RI␣ binding domain, containing major binding sites in the C⑀2/C⑀3 junction and the C⑀3 A-B loop region. Both of these regions contain a number of exposed aromatic and charged residues that may form a complementary surface for interaction with that described herein for Fc⑀RI␣. Interestingly, the C⑀2/C⑀3 junction region is located distally to the C⑀3 A-B loop, suggesting a discontinuous binding site in IgE-Fc. This implies that the C⑀2/C⑀3 and C⑀3 A-B loop may interact with different regions of Fc⑀RI␣. Since the Fc⑀RI␣ binding site appears to comprise a single continuous region in the C-CЈ-F-G face of domain 2, it is therefore possible that a second binding site distant from this region (e.g. in domain 1) may also exist.
The definition of the precise molecular basis of the interaction between Fc⑀RI␣ and IgE awaits the elucidation of the structure of Fc⑀RI␣-IgE complexes.
The findings described herein for Fc⑀RI␣ when compared with similar studies of the structurally related Fc␥Rs, i.e. Fc␥RI (37), Fc␥RIIa (25,29), and Fc␥RIII (30,31), reveal a number of similarities in the molecular basis of how these receptors interact with their respective ligands. The two Ig-like domains of the extracellular regions of Fc⑀RI␣, Fc␥RIIa, and Fc␥RIII and the first two domains of the three domains of Fc␥RI clearly represent a structurally conserved Ig binding motif of this receptor family. In each of these receptors, it is the second extracellular domain that is responsible for the direct binding of Ig, with the first domain making an as yet undefined contribution to maintain optimal binding affinity. The localization of Ig-binding regions in domain 2 of Fc⑀RI␣, Fc␥RIIa, and Fc␥RIII has identified common regions of these receptors that are involved in the interaction with their Ig ligands (Fig. 4). The three loop regions identified herein as involved in the binding of IgE by Fc⑀RI␣, namely the F-G, CЈ-E, and B-C, have also been implicated as crucial in the binding of IgG by Fc␥RIIa (25,29) or Fc␥RIII (30, 31) (Fig. 4). The CЈ-C loop of Fc⑀RI␣ and Fc␥RIII also contributes to Ig binding in both of these receptors (13,30,31); however, it does not appear to be involved in Fc␥RIIa (29) (Fig. 4). Thus, the focus of the interaction of Fc⑀RI␣ and Fc␥RIII with Ig exhibits some differences to that of Fc␥RIIa. However, it is clear from all of these studies that the above mentioned loop regions of the second extracellular domain, which contribute to the four-strand ␤-sheet (C-CЈ-F-G) face, constitute the major Ig interactive regions of these receptors. Thus, despite Fc⑀RI␣ exhibiting a distinctly different specificity and affinity for Ig to the Fc␥R, structural similarities are likely to be maintained between these receptors in their interaction with Ig.
Understanding the molecular basis of the interaction of Fc⑀RI␣ with IgE will assist in the design of therapeutic strategies to treat IgE-mediated allergic disease by blocking the binding of IgE by Fc⑀RI␣. The contribution to the definition of the IgE binding site of Fc⑀RI␣ as described herein represents a step toward the possibility of rational design of such therapeutic agents. The recent demonstration that the structure-based design of a constrained peptide of the CЈ-C loop of Fc⑀RI␣-D2 can inhibit IgE binding to Fc⑀RI highlights the feasibility of a rational approach (13). The IgE binding loops of Fc⑀RI␣-D2 identified herein, i.e. F-G and CЈ-E, may represent other candidate regions for similar studies. The ability of recombinant soluble Fc⑀RI␣ to inhibit the binding of IgE to cell surface Fc⑀RI has also been demonstrated (38 -40). The engineering of higher affinity forms of soluble Fc⑀RI␣, such as the Trp 130 3 Ala, Trp 156 3 Ala, Tyr 160 3 Ala, and Glu 161 3 Ala as described in this study, may provide more effective therapeutic agents.