IDENTIFICATION OF SITES ON THE HUMAN FC-EPSILON-RI-ALPHA SUBUNIT WHICH ARE INVOLVED IN BINDING HUMAN AND RAT IGE

The high affinity IgE Fe receptor (FcepsilonRI), found on mast cells a nd basophils, is a tetrameric receptor complex. The extracellular port ion of the FcepsilonRIalpha subunit consists of two immunoglobulin-lik e domains and binds IgE in the absence of the other subunits. To local ize the high affinity IgE binding site within the FcepsilonRIalpha sub unit, we generated a series of chimeric receptor constructs where one of the two immunoglobulin-like domains was either deleted or substitut ed with those from the human FcgammaRIIIAalpha or the rat FcepsilonRIa lpha subunit. The chimeric receptors were monitored for their capacity to bind human and rat IgE, and their reactivity with different antire ceptor antibodies. Domain I substitutions maintained high affinity hum an IgE binding. Domain II substitutions resulted in a total loss of bo th human and rat IgE binding. Single-domain alpha subunits could not b ind IgE, suggesting that both extracellular domains are required for p roper protein folding or IgE binding. To further localize the IgE bind ing sites, homolog-scanning mutagenesis was performed. At least three independent regions of domain II encompassing residues 118-129, 136-15 0, and 148-162 were required for IgE binding. Our results suggest that domain II of the human FcepsilonRIalpha confers most of the important contributions to the binding of the human IgE Fc molecule, whereas do main I of the rat FcepsilonRIalpha makes important contributions to th e binding of rat IgE.