The IgE receptor on mast cells and basophils (Fcepsilone R-I) is directly involved in the IgE-mediated activation of these cells. Recently, cDNA coding for the alpha-subunit of the rat Fcepsilone R-I (Fcepsilone R-Ialpha) has been cloned. RNA heterogeneity was subsequently detected, suggesting the presence of variants of Fc epsilone R-Ialpha, including the possible presence of intracellular forms and secretory truncated forms of Fc epsilone R-Ialpha. A new IgE-binding protein, epsilone BP, was found to contain interesting structural features, but its function is yet to be defined. In this research program, we will continue our studies of both Rc epsilone R-I and epsilone BP. First, structure-function relationship of rodent Fcepsilone R-I alpha will be established. Expression of cloned cDNA in mammalian cells by gene transfection will be conducted. Expression of both the surface membrane-bound form and soluble form of the receptor will be pursued. Mutant Fc epsilone R-I alpha in normal rat mast cells. Various protein products predicted from the cDNA cloning results, including the intracellular and secetory truncated forms of Fcepsilone R-I alphs will be generated for structure-function analysis to identify structural elements in FcepsiloneR-Ialphs that are involved in binding to IgE. Second, various forms of FcepsiloneR-Ialpha will be identified. RNase protection analysis and polymerase chain reaction methodology will be employed to identify various RNA forms related to FcepsiloneR-Ialpha in normal rat mast cells. Various protein products predicted from the cDNA cloning results, including the intracellular and secretory truncated forms of FcepsiloneR-Ialpha will be detected in RBL cells. Genomic DNA coding for mouse FCepsiloneR-Ialpha will be cloned and used to extend the above analysis to the mouse system. Third, function of the newly defiend IgE-binding protein (epsiloneBP) will be delineated. Cell types expressing epsilone and the location of epsilone in cells will be identified. If it is established that epsilone is not a cell suface receptor, its role as a soluble protein, regulating IgE synthesis or histamine release form mast cells and basophils will be explored. The long-term goals of this research are: 1) establishment of structure, function and regulation of key components of the IgE system; and 2) development of therapeutic methods for the treatment of human allergic disorders.
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