Dr. Kaushansky and colleagues propose to use a novel approach to identify ligand binding sites on the interleukin 3 (IL-3) receptor (IL-3R). The approach is to restore activity to a mutated IL 3 selecting for receptors bearing accommodating mutations. It is predicted that the nature and location of the mutation in the receptor will provide evidence for a direct interaction of that site with the altered site on the receptor mutant. To identify the receptor residues which bind to the site modified in a series of IL-3 loss-of-function muteins the PI will generate libraries of altered IL-3R a and bc subunits and utilize a selection method to identify reactivating modifications. The IL-3/IL-3Ra/bc interactions will then be modeled to generate further testable hypotheses. Dr. Kaushansky also proposes to use the recently developed technique of fluorescence resonance energy transfer (FRET) to dissect the sites at which the IL-3Ra and IL-3Rb subunits interact in the activated form of the receptor. Specifically, it is proposed to: 1.Determine whether the wild type residue altered in each of a series of loss-of-function human IL-3 muteins normally binds to the a or b chain of the IL-3 receptor. 2.Construct CDNA expression libraries containing multiple alterations of the IL-3 receptor a and b subunit sequences at a large number of sites, including those identified in other receptors to bind their corresponding ligands. 3.Screen the libraries for receptor clones, selecting for those that allow cellular proliferation in the presence of mutant IL-3 molecules that have otherwise lost activity on wild type IL-3 receptor-bearing cells.4.Model the interaction of IL-3 and its receptor, and test the model with additional IL-3 muteins and altered IL-3 receptor subunits.5.Determine the sites at which the IL-3Ra and bc subunits interact by site-specific mutagenesis, screening for receptor mutants that lose the capacity to heterodimerize based on the loss of energy transfer between fluorochrome-labeled monoclonal antibodies to each of the receptor subunits.
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