The membrane-proximal part of FcϵRIα contributes to human IgE and antibody binding – implications for a general structural motif in Fc receptors

Abstract

The high affinity receptor for human IgE (FcϵRI) on tissue mast cells and blood basophils is responsible for immediate hypersensitivity reactions. Binding of human IgE (hIgE) to FcϵRI has been shown to be mediated via three independent regions in the extracellular part of the α-subunit of FcϵRI (ecFcϵRIα). By site-directed mutagenesis we investigated the contribution of amino acids within the ecFcϵRIα FG loop (residues Lys 154–Leu 165) to binding to hIgE and two monoclonal anti-FcϵRIα antibodies (15/1, 5H5/F8). The mutated receptors were expressed and secreted from eukaryotic cells as amino-terminal fusion to HSA. We show that the proposed loop region contributes partly to hIgE binding and that the epitope of mAb 15/1, which inhibits hIgE/FcϵRIα interaction, maps to this region whereby a single W156A mutation results in complete loss of mAb 15/1 binding. In contrast, hIgE binding is not affected by the W156A mutation indicating that different amino acid residues within the loop are recognized by the mAbs 15/1 and hIgE. MAb 5H5/F8 does not recognize a receptor mutant truncated to Ile 170. By screening a random dodecapeptide library displayed on bacterial flagella the epitope for mAb 5H5/F8 was mapped to P 173REKY 177 whereas one of the 15/1 binding clones displayed a peptide with an amino acid sequence homologous to Leu 158–Ile 167. Based on the epitopes identified for the inhibitory mAb 15/1 and the non-inhibitory mAb 5H5F8 and on binding data obtained with polyclonal antisera raised against two ecFcϵRIα peptides, we propose a structural element in the membrane proximal part of ecFcϵRIα which forms a 3D structure which might facilitate specific and efficient attachment of hIgE.