Abstract
The high affinity IgE Fc receptor (Fc epsilon RI), found on mast cells and basophils, is a tetrameric receptor complex. The extracellular portion of the Fc epsilon RI alpha subunit consists of two immunoglobulin-like domains and binds IgE in the absence of the other subunits. To localize the high affinity IgE binding site within the Fc epsilon RI alpha subunit, we generated a series of chimeric receptor constructs where one of the two immunoglobulin-like domains was either deleted or substituted with those from the human Fc gamma RIIIA alpha or the rat Fc epsilon RI alpha subunit. The chimeric receptors were monitored for their capacity to bind human and rat IgE, and their reactivity with different antireceptor antibodies. Domain I substitutions maintained high affinity human IgE binding. Domain II substitutions resulted in a total loss of both human and rat IgE binding. Single-domain alpha subunits could not bind IgE, suggesting that both extracellular domains are required for proper protein folding or IgE binding. To further localize the IgE binding sites, homolog-scanning mutagenesis was performed. At least three independent regions of domain II encompassing residues 118-129, 136-150, and 148-162 were required for IgE binding. Our results suggest that domain II of the human Fc epsilon RI alpha confers most of the important contributions to the binding of the human IgE Fc molecule, whereas domain I of the rat Fc epsilon RI alpha makes important contributions to the binding of rat IgE.
Highlights
One of these is characterized by anoninhibitory class of monoclonal antibodies which does not block the binding of IgE to the a subunit
Residues 118-129, 136-160, and 148-162 were re- In the currentstudy we have exchanged the Ig-like domains quired for IgE bindinOg.ur resultssuggest that domain of the human FctRIa subunit with the corresponding domains
Characterization of IgE Binding to Chimeric Receptors-To determine the siteson the human FccRIa that areimportant for binding IgE, chimeric receptors were generated by the substitution of regions of the human FccRIa with complementary regions derived from the human FcyRIIIAa [19] or rat FccRIa [20] (Fig. 1).The human FcyRIIIAa!subunit does not bind human orrat IgE [1].The ratFccRIa binds rat IgE but not human IgE [21]
Summary
Characterization of IgE Binding to Chimeric Receptors-To determine the siteson the human FccRIa that areimportant for binding IgE, chimeric receptors were generated by the substitution of regions of the human FccRIa with complementary regions derived from the human FcyRIIIAa [19] or rat FccRIa [20] (Fig. 1).The human FcyRIIIAa!subunit does not bind human orrat IgE [1].The ratFccRIa binds rat IgE but not human IgE [21]. The human FccRIa binds bothhumanand rat IgE with apparent affinities that are approximately 10-fold different [22]. Chimeras were generated and expressed in COS cells and the cell surface binding of both human and rat IgE, as well as a panel of inhibitory and noninhibitory anti-FctRIa mAbs [11]were examined. The amount of radiolabeled IgE or mAbs bound to each chimera was determined and compared with the binding of the wild type receptor. Chimeras which bound IgE or mAbs at levels 40% or greater, compared with the binding of the wild type receptor were considered unaffected by the substitutions. If the relative binding for a chimera fell between 5 and 40%, the specific binding was interpreted as being reduced. If the relative binding fell below5%, the ability of the chimera to
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