The mechanisms of signal transduction from cytokine and growth factor receptors are important for understanding the physiological roles of these important mediators of cell growth, differentiation, and response to pathogens. We have been defining the biochemistry of signal transduction for interferons (IFNs) and have extended the paradigm discovered in this system to the epidermal and platelet-derived growth factors (EGF & PDGF). The proposed research will define, clone and characterize novel transcription factors regulated by these cytokines; determine the biochemical mechanisms involved in coupling cell surface receptors to activation of transcription; identify and isolate target genes activated by cytokines functioning through this pathway; and create mutant cell lines and mouse strains specifically deficient in this signaling pathway by gene targeting through homologous recombination. Understanding the biochemical mechanisms of growth factor and cytokine signaling and their target genes will allow definition of steps deregulated in pathological states such as cancer where over activity of growth factors or under activity of growth inhibitory cytokines leads to abnormal cell growth. Creation of mutant cell lines and animals with specific lesions in cytokine pathways will define the physiological consequences of cytokine functioning under normal and abnormal conditions and will provide new models for disease. New members of the ISGF3 transcription factor family will be cloned and analyzed for structure-function relationships, for their ability to form multimers with novel DNA-binding specificity, for their ability to be activated by different cytokines, and for their differential, tissue- specific patterns of expression. The biochemistry of signal coupling allowing cytokine and growth factor receptors to activate transcription factors will be defined by determining the linkage between receptors, the Jak family kinases, and the transcription factor substrates. Structure- function studies of the EGF and PDGF receptor and Jak1 kinase will define physical interactions linking these proteins and the mechanisms of ligand- induced activation. Similarly, studies of the p9l and p93 proteins of ISGF3 will define how they are specifically recognized by growth factor and/or IFN receptors. Target genes activated by growth factors will be identified by arbitrarily-primed PCR fingerprinting of RNA from cells treated under different conditions. Expression patterns from wildtype and mutant EGF and PDGF receptors will define genes regulated via the ISGF3 pathway. Altered embryonic stem cells with disruption of the ISGF3 p9l gene by homologous recombination will be used to create homozygous mutant animals. Homozygous null cells will be analyzed for their capacity to differentiate in vitro and for ability to colonize specific tissues in chimeric mice, with particular attention to hematopoiesis.
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