Fine Structure Analysis of Interaction of Fcepsilon RI with IgE*

Mark D. HulettDagger §, Ross I. Brinkworth, Ian F. C. McKenzieDagger , and P. Mark HogarthDagger parallel

From Dagger  The Austin Research Institute, Austin Hospital, Studley Road, Heidelberg, Victoria 3084, Australia and  The Centre for Drug Design and Development, University of Queensland, Brisbane, Queensland 4072, Australia

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The high affinity receptor for IgE (Fcepsilon RI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human Fcepsilon RI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the alpha -subunit (Fcepsilon RIalpha ). In this study, the IgE binding site of human Fcepsilon RIalpha 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 Fcepsilon RIalpha and the functionally distinct but structurally homologous low affinity receptor for IgG (Fcgamma RIIa) have been used to localize two IgE binding regions of Fcepsilon RIalpha to amino acid segments Tyr129-His134 and Lys154-Glu161. Both regions were capable of independently binding IgE upon placement into Fcgamma RIIa. Molecular modeling of the three-dimensional structure of Fcepsilon RIalpha -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 Fcepsilon RIalpha was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159 decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116 in 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 Fcepsilon RIalpha -IgE interaction for the possible treatment of IgE-mediated allergic disease.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fcepsilon RI binds monomeric IgE with high affinity (Ka = 1010 M-1) and is expressed on mast cells, basophils (1, 2), Langerhans cells (3, 4), peripheral blood dendritic cells (5), eosinophils (6), and monocytes (7). The receptor can exist in two distinct multimeric forms, a tetrameric complex comprising an alpha -subunit, a beta -subunit, and a disulfide-bonded homodimer of two gamma -subunits (1, 2) or a trimeric complex (alpha gamma 2), which lacks the beta -subunit (5, 7). The alpha -subunit of Fcepsilon RI (Fcepsilon RIalpha ) is the IgE binding chain and is structurally related to the Ig binding chains of the leukocyte receptors for IgG (Fcgamma R) and IgA (Fcalpha R), containing an extracellular region of two Ig-like domains (1). The associated beta - and gamma -subunits plays crucial roles in both cell surface expression and signal transduction of the receptor (8).

The binding of IgE by Fcepsilon 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 Fcepsilon 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 Fcepsilon RI may prove useful in the treatment of allergic disease. Clearly, understanding the molecular basis of the interaction of Fcepsilon 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-Fcepsilon RIalpha monoclonal antibodies have identified the second extracellular domain of Fcepsilon RIalpha 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 Fcepsilon RIalpha -D2 have been implicated in the binding of IgE. In a series of "gain of function" experiments using chimeric Fcepsilon RIalpha /Fcgamma RIIa receptors, we identified three relatively large regions of Fcepsilon RIalpha -D2, each capable of independently binding IgE (9). The Fcepsilon RIalpha regions encompassed by residues Trp87-Lys128, Tyr129-Asp145, or Ser146-Val169 when inserted into Fcgamma RIIa were each able to impart IgE binding to the receptor. Mallamaci et al. (10) have used a similar approach with chimeric Fcepsilon RIalpha /Fcgamma RIII receptors, however, in "loss of function" experiments and identified four regions of Fcepsilon RIalpha -D2 that putatively contribute to IgE binding. The replacement of each of the Fcepsilon RIalpha regions encompassed by residues Ser93-Phe104, Arg111-Glu125, Asp123-Ser137, and Lys154-Ile167 with the corresponding regions of Fcgamma RIII, was found to result in reduced IgE binding. In addition, a recent study by McDonnell et al. (13) has demonstrated that residues Ile119-Tyr129 of Fcepsilon RIalpha -D2, when synthesized as a conformationally constrained peptide, can inhibit the binding of IgE to Fcepsilon RI.

Despite the localization of multiple binding regions in Fcepsilon RIalpha -D2, the interaction of Fcepsilon RI with IgE at the level of individual residues has not been defined. In this study, we have identified small IgE binding subregions of Fcepsilon RIalpha -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 Fcepsilon RIalpha binds IgE and contribute to our understanding of the interaction of the leukocyte FcR family with their Ig ligands.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Chimeric Fcepsilon RIalpha /Fcgamma RIIa and Mutant Fcepsilon RIalpha Receptor cDNA Expression Constructs

Chimeric Fcepsilon RIalpha /Fcgamma RIIa or mutant Fcepsilon RIalpha cDNAs were constructed by splice overlap extension PCR1 (14) using an expressible form of the Fcepsilon RIalpha chain (15) or Fcgamma RIIaNR cDNA (16) as templates. The expressible form of the Fcepsilon RIalpha chain consists of the extracellular region of Fcepsilon RIalpha linked to the transmembrane and cytoplasmic tails of Fcgamma RIIa and is expressed on the cell surface and binds monomeric hIgE with an affinity comparable with that of the wild-type Fcepsilon RIalpha chain, as described previously (15). Splice overlap extension PCR was performed as follows. Two PCR reactions were used to amplify the Fcepsilon RIalpha -Fcgamma RIIa or Fcepsilon RIalpha fragments to be spliced together. The reactions were performed on 100 ng of the Fcepsilon RIalpha 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 MgCl2 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 5'-end (the 5'-flanking oligonucleotide primer containing an EcoRI recognition site) and a SalI site at their 3' end (the 3'-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 SequenaseTM (U.S. Biochemical Corp.) as described (19).

Monoclonal Antibodies and Ig Reagents

The anti-Fcepsilon RIalpha mAb 3B4 and the anti-Fcgamma RIIa mAb 8.2 were produced in this laboratory (20). The anti-Fcepsilon RIalpha 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 IgG1 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 NH4SO4, 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 enzyme-linked immunosorbent assay, and contaminating IgG was estimated at <1%.

Transfection

COS-7 cells (30-50% confluent per 5-cm2 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/5-cm2 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 Na2HPO4, 3.25 mM NaH2PO4, 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 µg/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 IgG1 anti-2,4,6-trinitrophenyl mAb (23). Two ml of 2% EAs (v/v) were added per 5-cm2 dish of transfected cells and incubated for 5 min at 37 °C. Plates were then centrifuged at 500 × 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 phosphate-buffered saline, 0.5% bovine serum albumin; and resuspended at 107 cells/ml in L-15 medium, 0.5% bovine serum albumin. 50 µl of cells were incubated with 50-µl serial dilutions of 125I-hIgE for 120 min at 4 °C. 125I-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 125I-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 125I-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 Fcepsilon RIalpha receptors were determined by assessing the binding of the anti-Fcepsilon RIalpha mAb 22E7 (shown to bind distantly to the binding site; see Ref. 12) at 2 µg/ml in a direct binding assay as described for the binding of hIgE. Any variation in cell surface receptor expression between the mutant Fcepsilon RIalpha and wild-type Fcepsilon RIalpha COS-7 cell transfectants (levels ranged from 80 to 120% of wild-type Fcepsilon RIalpha ) was then normalized, and the binding of hIgE by the mutant Fcepsilon RIalpha receptors was corrected using the following formula: (mutant - mock IgE binding) × ((wild type - mock 22E7 binding)/(mutant - mock 22E7 binding)).

Generation of Fcepsilon RIalpha Domain 2 Model Structure

Molecular modeling of domain 2 (D2) of human Fcepsilon RIalpha 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 Fcepsilon RIalpha -D2 was constructed by mutation of our previously described model of human Fcgamma 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 Fcepsilon RIalpha -D2 with Fcgamma RIIa-D2 and CD4-2 (28), regions of Fcgamma RIIa-D2 aligned with the beta -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 Fcgamma 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 Calpha -Cbeta bonds. The structure was then minimized using the Discover module to a maximum root-mean-square derivative of 0.0001. The Fcgamma RIIa-D2 model was then converted to a Fcepsilon RIalpha -D2 model by mutation of the residues followed by further minimization of the structure using the above protocol.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Modeling of the Extracellular Domains of Fcepsilon RIalpha -- The three-dimensional structure of Fcepsilon RIalpha has not yet been solved. To aid in the localization of putative IgE binding regions of Fcepsilon RIalpha , we have generated a three-dimensional model of the second extracellular domain (D2) of Fcepsilon RIalpha 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 Fcepsilon RIalpha -D2 with CD4-2 suggests that this domain will adopt a similar folding pattern (25, 28). The structure of the Fcepsilon RIalpha -D2 model is characteristic of the C2 Ig-fold, comprising seven beta -strands (A, B, C, C', E, F, G) that form two antiparallel beta -sheets of ABE and CC'FG (Fig. 1A).


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Fig. 1.   Molecular modeling of the extracellular region of human Fcepsilon RIalpha domain 2 and location of residues putatively involved in the interaction with IgE. A, Fcepsilon RIalpha domain 2 model structure displayed as a ribbon diagram. The model is orientated such that it adjoins domain 1 at the top and the transmembrane region at the bottom. The three regions of domain 2 putatively involved in IgE binding (B-C, C'-E, and F-G loops) are shown in white. An additional region (C'-C loop) postulated to also be involved in IgE binding (13) is shown in magenta, with the remainder of the domain shown in green. B, location of residues putatively involved in the interaction of Fcepsilon RIalpha domain 2 with IgE. The side chains of amino acids implicated in IgE binding as described under "Results" are indicated. Those side chains that when substituted result in decreased or increased binding are shown in red or yellow, respectively. The computer model of Fcepsilon RIalpha domain 2 was generated by molecular modeling based on the structure of the related CD4 domain 2 as described under "Experimental Procedures."

Chimeric Receptors Identify Multiple Regions of Fcepsilon RIalpha Involved in IgE Binding-- Based on the location of the previously described IgE binding regions (9-13) on our three-dimensional molecular model of Fcepsilon RIalpha -D2 and by analogy with mapping studies of the homologous interaction of the Fcgamma R with IgG (25, 28-31), we targeted the B-C, C'-E, and F-G loop regions of Fcepsilon RIalpha -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 Fcepsilon RIalpha , chimeric receptors were generated, whereby Fcgamma RIIa was used as a scaffold to accept each of these Fcepsilon RIalpha loop regions. The three resultant chimeric receptors consisted of Fcgamma RIIa containing the following regions of Fcepsilon RIalpha -D2: (i) the B-C loop, residues Gly109-Tyr116; (ii) C'-E loop, residues Tyr129-His134; and (iii) F-G loop, residues Lys154-Glu161, designated the gamma 109-116epsilon , gamma 129-134epsilon , and gamma 154-161epsilon 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 IgG1) immune complexes by EA rosetting. Cells transfected with the gamma 154-161epsilon chimeric receptor bound IgE-EA (Fig. 2A, Table I), and the binding was specific, since mock-transfected cells or cells transfected with Fcgamma RIIa did not bind IgE-EA (Table I). These data indicate that the Lys154 to Glu161 region of Fcepsilon RIalpha can direct the binding of IgE. As expected, this chimeric receptor was unable to bind IgG1, since the previously described IgG binding region, residues Asn154-Ser161 (25), has been replaced with the homologous Fcepsilon RIalpha sequence (Table I). Similar experiments demonstrated that the gamma 129-134epsilon chimeric receptor could also specifically bind IgE-EA (Fig. 2B, Table I), indicating that the Tyr129-His134 region also contains an IgE binding site. As expected, this chimeric receptor was able to bind IgG1-EA (Table I) due to the presence of the Fcgamma RIIa Asn154-Ser161 IgG binding sequence.


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Fig. 2.   IgE complex binding of chimeric Fc receptors. COS-7 cell monolayers were transfected with the following chimeric cDNA constructs: gamma 154-161epsilon (A), gamma 129-134epsilon (B), and gamma 109-116epsilon (C). The binding of IgE immune complexes was assessed directly on monolayers by EA rosetting using mouse IgE-sensitized erythrocytes. The transfections were performed in a transient expression system, resulting typically in 30-50% of cells expressing the chimeric FcR. Cells were considered to be expressing functional receptors if >10 red blood cells were bound per cell.

                              
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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 Fcepsilon RIalpha chain, and unshaded regions are derived from Fcgamma RIIa. The relative positions of the putative beta -strands are shown above as labeled solid lines. The binding of mouse IgE and IgG1 (mIgE and mIgG) was assessed by EA rosetting as described under "Experimental Procedures." +, >10% of cells rosetting; -, no cells rosetting.

As described above, the segment of Fcepsilon RIalpha -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 gamma 109-116epsilon chimeric receptor containing the Fcepsilon RIalpha 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 Gly109-Tyr116 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 Fcepsilon RIalpha IgE Binding Site-- To identify the key residues of the Fcepsilon RIalpha binding regions (C'-E loop, residues Tyr129-His134; F-G loop, residues Lys154-Glu161) involved in the interaction with IgE, site-directed mutagenesis was used to replace each residue in these regions with alanine. In addition, residues Trp113, Val115, and Tyr116 in the B-C loop were also substituted with alanine, since the Fcepsilon RIalpha -D2 model predicts this region is likely to be adjacent to the F-G and C'-E loops and may therefore contribute to IgE binding. The alanine substitution mutants of Fcepsilon RIalpha were expressed in COS-7 cells, and the binding of monomeric human IgE was examined in direct binding assays by titration of 125I-labeled hIgE (Fig. 3). The levels of cell surface expression of the Fcepsilon RIalpha mutants on the COS-7 cell transfectants were determined using the Fcepsilon RIalpha 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 Fcepsilon RI levels (data not shown).


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Fig. 3.   Monomeric human IgE binding by Fcepsilon RIalpha alanine point mutants. Radiolabeled monomeric human IgE was titrated on COS-7 cells transfected with wild-type Fcepsilon RIalpha or Fcepsilon RIalpha containing alanine point mutations. A, F-G loop mutants, wild-type Fcepsilon RIalpha (), Lys154 right-arrow Ala (×), Val155 right-arrow Ala (black-square), Trp156 right-arrow Ala (open circle ), Gln157 right-arrow Ala (diamond ), Leu158 right-arrow Ala (black-diamond ), Asp159 right-arrow Ala (triangle ), Tyr160 right-arrow Ala (black-triangle), Glu161 right-arrow Ala (). B, C'-E loop mutants, wild-type Fcepsilon RIalpha (), Tyr129 right-arrow Ala (), Trp130 right-arrow Ala (open circle ), Tyr131 right-arrow Ala (black-square), Glu132 right-arrow Ala (diamond ), Asn133 right-arrow Ala (triangle ), His134 right-arrow Ala (black-triangle); C, B-C loop mutants, wild-type Fcepsilon RIalpha (), Trp113 right-arrow Ala (black-square), Val115 right-arrow Ala (open circle ), Tyr116 right-arrow Ala (). A comparison of the levels of IgE binding with the Fcepsilon RIalpha mutants relative to wild-type Fcepsilon RIalpha is shown. D, F-G loop mutants. E, C'-E loop mutants. F, B-C loop mutants. The percentage of binding was calculated from hIgE bound at a concentration of 2 µg/ml. The binding of wild-type Fcepsilon RIalpha was taken as 100% and mock-transfected cells as 0% binding. Results are expressed as means ± S.E. To control for variable receptor expression between the mutant Fcepsilon RI COS-7 cell transfectants, levels of expression were determined using a radiolabeled monoclonal anti-Fcepsilon RI antibody 22E7, and IgE binding was normalized to that seen for wild-type Fcepsilon RI (see "Experimental Procedures").

First, the individual alanine substitution of residues Lys154-Glu161 in the F-G loop indicated that each mutant retained hIgE binding, with the striking exception of the Val155-Ala mutant, where binding of monomeric hIgE was almost totally abolished, this receptor exhibiting only 3.2 ± 2.1% (mean ± S.D.) binding relative to the wild-type receptor (Fig. 3, A and 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 Fcepsilon RI (data not shown). The substitution of Asp159 with alanine also resulted in diminished IgE binding, this receptor exhibiting 52.7 ± 7.2% binding of the wild-type receptor. The substitution of Lys154, Gln157, and Leu158 with alanine had no significant effect on the binding of IgE, these mutants exhibiting binding comparable with wild-type Fcepsilon RIalpha . In contrast, the replacement Trp156, Tyr160, or Glu161 with alanine produced the interesting effect of increasing the binding of IgE (132.7 ± 14.0, 123.7 ± 11.1, and 139 ± 15.0% of wild-type Fcepsilon RIalpha , respectively). Therefore, these findings clearly identify five individual residues of the F-G loop of Fcepsilon RIalpha (Val155, Trp156, Asp159, Tyr160, and Glu161 as playing critical roles in the binding of hIgE. The observation that substitution of Val155 and Asp159 decreased binding suggests that these residues may directly interact with hIgE. The increased binding observed upon substitution of Trp156, Tyr160, and Glu161 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 Lys154-Glu161 of the F-G loop, alanine substitution of residues Tyr129-His134 of the C'-E loop was found to result in loss or enhancement of hIgE binding. Substitution of Tyr131 and Glu132 substantially decreased hIgE binding to 30.3 ± 4.4 and 61.4 ± 3.9% that of wild-type Fcepsilon RIalpha (Fig. 3, B and E). In contrast, replacement of Trp130 dramatically increased binding by over 70% to 172.5 ± 8.8% binding of the wild-type receptor. The substitution of Tyr129, Asn133, and His134 had no significant effect on the binding of hIgE, since these mutants exhibited binding comparable with that seen for wild-type Fcepsilon RIalpha (data not shown). These findings suggest that Trp130, Tyr131, and Glu132 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 C'-E loop as also playing a role in the binding of IgE by Fcepsilon RIalpha .

Although the chimeric receptor strategy failed to reveal a direct binding role for the B-C loop (residues Gly109-Tyr116), mutagenesis of residues Trp113, Val115, and Tyr116 within this loop suggests that it may also contribute to IgE binding by Fcepsilon RIalpha . This was demonstrated, since the substitution of Trp113 for alanine resulted in a dramatic loss of IgE binding, this mutant receptor exhibiting only 18.6 ± 3.2% of IgE binding relative to the wild-type receptor. Substitution of Val115 or Tyr116 with alanine did not significantly alter IgE binding (Fig. 3, C and F).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two approaches have been used to identify and analyze the IgE binding site of Fcepsilon RIalpha . First, to localize IgE binding regions of Fcepsilon RIalpha , a series of chimeric FcRs were engineered by exchange of segments between the second domain of Fcepsilon RIalpha and Fcgamma 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 Fcepsilon RIalpha using alanine scanning mutagenesis. These approaches have enabled the localization of IgE binding regions in Fcepsilon RIalpha to subregions of the second extracellular domain and identified key residues putatively involved in the interaction with IgE. Based on a molecular model of Fcepsilon RIalpha -D2, these data suggest that the IgE binding regions comprise the F-G, C'-E, and B-C loops and adjacent strand regions of this domain. Both the F-G and C'-E loops were directly implicated in the interaction with IgE, since insertion of these regions into Fcgamma 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 Trp113 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 Fcepsilon RIalpha -D2 suggests that the F-G, C'-E, and B-C loops of Fcepsilon RIalpha -D2 are likely to be juxtaposed at the membrane distal end of the domain at the interface with domain 1. The localization of the Fcepsilon RIalpha -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 Fcepsilon RIalpha with IgE. In support of this model, the anti-Fcepsilon RIalpha mAb 15A5, which recognizes an epitope in the B-C loop region of Fcepsilon RIalpha -D2, is able to block the binding of IgE to Fcepsilon 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 Fcepsilon RIalpha -D2 has also implicated the C'-C loop (residues Ile119-Tyr129 as playing a role in the binding of IgE (13). A peptide encompassing this region and designed to mimic the conformation of the C'-C loop was demonstrated to competitively inhibit IgE binding to Fcepsilon RIalpha and prevent IgE-mediated mast cell degranulation in vitro. Thus, the inclusion of the C'-C loop with the B-C, C'-E, and F-G loops described herein extends the putative region of contact of Fcepsilon RIalpha with IgE. These data therefore suggest that the entire four-stranded beta -sheet face of Fcepsilon RIalpha -D2, namely the C-C'-F-G strands and adjacent loops, may be important in the interaction of Fcepsilon RIalpha with IgE.

The alanine scanning mutagenesis of the F-G, C'-E, and B-C loops of Fcepsilon RIalpha -D2 has identified a number of residues that may contribute to the binding of IgE. The substitution of amino acids Trp113, Tyr131, Glu132, Val155, and Asp159 with alanine decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. Based on the three-dimensional model of Fcepsilon RIalpha -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-C'-F-G region (Fig. 1B). The majority of these residues are aromatic (Trp113, Trp130, Tyr131, Trp156, Tyr160) or charged (Glu132, Asp159) and are likely candidates for direct contact with IgE.

Studies examining the binding regions on the Fc portion of IgE for Fcepsilon RIalpha have identified a number of putative interactive sites (32-36). The third constant domain (Cepsilon 3) appears to be the principle Fcepsilon RIalpha binding domain, containing major binding sites in the Cepsilon 2/Cepsilon 3 junction and the Cepsilon 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 Fcepsilon RIalpha . Interestingly, the Cepsilon 2/Cepsilon 3 junction region is located distally to the Cepsilon 3 A-B loop, suggesting a discontinuous binding site in IgE-Fc. This implies that the Cepsilon 2/Cepsilon 3 and Cepsilon 3 A-B loop may interact with different regions of Fcepsilon RIalpha . Since the Fcepsilon RIalpha binding site appears to comprise a single continuous region in the C-C'-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 Fcepsilon RIalpha and IgE awaits the elucidation of the structure of Fcepsilon RIalpha -IgE complexes.

The findings described herein for Fcepsilon RIalpha when compared with similar studies of the structurally related Fcgamma Rs, i.e. Fcgamma RI (37), Fcgamma RIIa (25, 29), and Fcgamma 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 Fcepsilon RIalpha , Fcgamma RIIa, and Fcgamma RIII and the first two domains of the three domains of Fcgamma 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 Fcepsilon RIalpha , Fcgamma RIIa, and Fcgamma 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 Fcepsilon RIalpha , namely the F-G, C'-E, and B-C, have also been implicated as crucial in the binding of IgG by Fcgamma RIIa (25, 29) or Fcgamma RIII (30, 31) (Fig. 4). The C'-C loop of Fcepsilon RIalpha and Fcgamma RIII also contributes to Ig binding in both of these receptors (13, 30, 31); however, it does not appear to be involved in Fcgamma RIIa (29) (Fig. 4). Thus, the focus of the interaction of Fcepsilon RIalpha and Fcgamma RIII with Ig exhibits some differences to that of Fcgamma 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 beta -sheet (C-C'-F-G) face, constitute the major Ig interactive regions of these receptors. Thus, despite Fcepsilon RIalpha exhibiting a distinctly different specificity and affinity for Ig to the Fcgamma R, structural similarities are likely to be maintained between these receptors in their interaction with Ig.


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Fig. 4.   Ig binding regions of leukocyte FcR. A schematic diagram is shown of model structures proposed for domain 2 of human Fcepsilon RI, Fcgamma RII, and Fcgamma RIII, showing the location of amino acid residues implicated in Ig binding. The models are based on the known structure of CD4-domain 2 (see Refs. 26 and 27) and are shown in ribbon form with beta -strands labeled and depicted as arrows. The models are oriented with the four beta -strand C'CFG face at the front and adjoin domain 1 at the top and the transmembrane region at the bottom. The predicted positions of amino acids implicated in Ig binding through mutagenesis studies (see "Fine Structure Analysis of the Fcepsilon RIalpha IgE Binding Site" for details) are indicated with red circles and labeled in single letter code with their residue number. The C'-C loop region of Fcepsilon RI implicated in IgE binding using peptide inhibition studies is highlighted in red. The data are compiled from this paper and Refs. 13, 25, 29, 30, and 31.

Understanding the molecular basis of the interaction of Fcepsilon RIalpha with IgE will assist in the design of therapeutic strategies to treat IgE-mediated allergic disease by blocking the binding of IgE by Fcepsilon RIalpha . The contribution to the definition of the IgE binding site of Fcepsilon RIalpha 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 C'-C loop of Fcepsilon RIalpha -D2 can inhibit IgE binding to Fcepsilon RI highlights the feasibility of a rational approach (13). The IgE binding loops of Fcepsilon RIalpha -D2 identified herein, i.e. F-G and C'-E, may represent other candidate regions for similar studies. The ability of recombinant soluble Fcepsilon RIalpha to inhibit the binding of IgE to cell surface Fcepsilon RI has also been demonstrated (38-40). The engineering of higher affinity forms of soluble Fcepsilon RIalpha , such as the Trp130 right-arrow Ala, Trp156 right-arrow Ala, Tyr160 right-arrow Ala, and Glu161 right-arrow Ala as described in this study, may provide more effective therapeutic agents.

    ACKNOWLEDGEMENTS

We thank Jim Karkaloutsos and Ewa Witort for technical assistance.

    FOOTNOTES

* This work was supported by 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. The article must therefore be 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.

parallel To whom correspondence should be addressed. Tel.: 61-3-9287-0666; Fax: 61-3-9287-0600.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; mAb, monoclonal antibody; hIgE, human IgE; EA, erythrocyte-antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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