Monoclonal antibodies and synthetic peptides define the active site of Fc(epsilon)RI and a potential receptor antagonist

Defining the structure of the human high-affinity receptor for IgE, Fc,RI, is crucial to understand the receptor:ligand interaction, and to develop dr ugs to prevent IgE-dependent allergic diseases. To this end, a series of fo ur anti-Fc(epsilon)RI monoclonal antibodies (mAbs), including three new mAb s, 47, 54, and 3B4, were used in conjunction with synthetic Fc(epsilon)RI p eptides to define functional regions of the Fc IgE-binding site and identif y an antagonist of IgE binding. The spatial orientation of the epitopes det ected by these antibodies and their relationship to the IgE-binding region of Fc(epsilon)RI was defined by a homology model based on the closely relat ed Fc(gamma)RIIa. Using recombinant soluble F6(epsilon)RI-alpha as well as Fc(epsilon)RI-alpha expressed on the cell surface, a series of direct and c ompetitive binding experiments indicated that the mAbs detected nonoverlapp ing epitopes. One antibody (15-1), previously thought to be located close t o the IgE-binding site, was precisely mapped to a single loop within the Ig E-binding site by both mutagenesis and overlapping synthetic peptides encom passing the entire extracellular domain. A synthetic peptide epsilonRI-11, containing the amino acids 101-120 and the mAb 15-1 epitope, inhibited IgE binding and may form the basis for the development of a useful receptor-bas ed therapy.