The goal of the research described in this application is to elucidate the mechanism of action of members of the recently described cytokine receptor superfamily, using the human interleukin 4 receptor (HIL4R) as a model. This will be accomplished first through the establishment of a cloned cell line transfected with a functional HIL4R that will serve as an invaluable model for in vitro studies. We will map the functional domains of the HIL4R through an analysis of deletion mutants and then assess the dominance of those non-functional mutants in cotransfection experiments with wild type HIL4R. The HIL4-dependent cell line will be used to study the cascade of events that occurs subsequent to the binding of HIL4. An anti-HIL4R monoclonal antibody will be raised and used to immunoprecipitate HIL4R-associated molecules in crosslinking studies. Commercial antiphosphotyrosine antibodies will be used to identify phosphoproteins generated by HIL4 binding. Molecules of interest identified in these two approaches will be purified by affinity and conventional chromatography, N-terminal amino acid sequence will be obtained, and the genes will be cloned. Alteration in mRNA levels of transcription factors and nuclear protooncogenes occurring after HIL4 binding will be analyzed. The findings from these studies will be validated in studies of normal and malignant human cells. If the mutant HIL4R or a homologous mutant mouse IL4R is capable of competitively inhibiting the response of the wild type receptor in a """"""""dominant negative"""""""" fashion in vitro, in the second phase of studies we will apply this approach in transgenic mice in an attempt to ablate IL4 function in an intact animal system. Lineage specificity of the """"""""dominant negative"""""""" phenotype will be obtained through the choice of promoter for the transgene. We will also attempt to study the tumorigenicity of constitutively activated HIL4R in an animal model. The findings from all of these studies will be applied to other members of the cytokine receptor superfamily.
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