The membrane-proximal part of Fc epsilon RI alpha contributes to human IgEand antibody binding - implications for a general structural motif in Fc receptors

The high affinity receptor for human IgE (Fc epsilon RI) on tissue mast cel ls and blood basophils is responsible for immediate hypersensitivity reacti ons. Binding of human IgE (hIgE) to Fc epsilon RI has been shown to be medi ated via three independent regions in the extracellular part of the alpha-s ubunit of Fc epsilon RI (ecFc epsilon RI alpha). By site-directed mutagenes is we investigated the contribution of amino acids within the ecFc epsilon RI alpha FG loop (residues Lys(154)-Leu(165)) to binding to hIgE and two mo noclonal anti-Fc epsilon RI alpha antibodies (15/1, 5H5/F8). The mutated re ceptors were expressed and secreted from eukaryotic cells as aminoterminal 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/Fc epsil on RI alpha interaction, maps to this region whereby a single W156A mutatio n results in complete loss of mAb 15/1 binding. In contrast, hIgE binding i s not affected by the W156A mutation indicating that different amino acid r esidues within the loop are recognized by the mAbs 15/1 and hIgE, MAb 5H5/F 8 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(173)REKY(177) whereas one of the 15/1 bindin g 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 polyclo nal antisera raised against two ecFc epsilon RI alpha peptides, we propose a structural element in the membrane proximal part of ecFc epsilon RI alpha which forms a 3D structure which might facilitate specific and efficient a ttachment of hIgE. (C) 1998 Federation of European Biochemical Societies.