The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcepsilonRI) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of IgE-Fc (Cepsilon3) as the site of the interaction with FcepsilonRI. We have prepared a series of site-directed mutants of human IgE-Fc, particularly focusing on the N-terminal "linker" region and AB loop of Cepsilon3. The kinetics of binding IgE and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of IgE-Fc for FcepsilonRI by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in Fcepsilon3-4, a truncated form of IgE-Fc which lacks the Cepsilon2 domain pair, and thus it does not exert its effect through altering the quaternary structure of IgE-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of FcepsilonRII (CD23), the low-affinity receptor for IgE, demonstrating the structural integrity of the mutated IgE-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of IgE-Fc or Fcepsilon3-4. These results demonstrate the importance of the N-terminal linker region of Cepsilon3 in the interaction of IgE with FcepsilonRI.

The high-affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is an alphabetagamma2 tetramer found on mast cells, basophils, and several other types of immune effector cells. The interaction of IgE with the alpha-subunit of FcepsilonRI is central to the pathogenesis of allergy. Detailed knowledge of the mode of interaction of FcepsilonRI with IgE may facilitate the development of inhibitors for general use in the treatment of allergic disease. To this end we have performed site-directed mutagenesis on a soluble form of the FcepsilonRI alpha-chain (sFcepsilonRIalpha). The effects of four mutations in the second immunoglobulin-like domain of sFcepsilonRIalpha upon the kinetics of binding to IgE and fragments of IgE have been analyzed using surface plasmon resonance. As described in the preceding paper of this issue [Henry, A. J., et al. (1997) Biochemistry 36, 15568-15578], biphasic binding kinetics was observed. Two of the mutations had significant effects on binding: K117D reduced the affinity of sFcepsilonRIalpha for IgE by a factor of 30, while D159K increased the affinity for IgE by a factor of 7, both principally through changes in the rates of dissociation of the slower phase of the interaction. Circular dichroism spectra of sFcepsilonRIalpha incorporating either of these mutations were indistinguishable from those of wild-type sFcepsilonRIalpha, demonstrating that the native conformation had not been disrupted. Our results, together with those from site-directed mutagenesis on fragments of IgE presented in the accompanying paper, define the contact surfaces in the IgE:sFcepsilonRIalpha complex.

Soluble fragments of the α-chain of FcεRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcεRIα is used in this study, in conjunction with the crystal structure of the FcεRIα/IgE complex, to define the relative importance of specific residues within human FcεRIα for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcεRIα and attenuate allergic disease. Three residues within the C-C′ region of the FcεRI α2 domain and two residues within the α2 proximal loops of the α1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three α2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in α1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the α1/α2 interface. Our results demonstrate that the effects of mutations in human FcεRIα on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.