Interferon-gamma (IFN-gamma) plays a pivotal role in promoting host defense to a variety of intracellular and extracellular microbial pathogens and neoplastic cells. For the past 12 years my laboratory has focused its efforts on elucidating IFNgamma's molecular mechanism of action and physiologic role in vivo. During the current funding period we cloned and characterized the ligand binding chain of the human and murine IFNgamma receptors (now termed the alpha chain), demonstrated the requirement for a second species matched polypeptide (now termed the beta chain), demonstrated the ligand dependent tyrosine phosphorylation of the IFNgamma receptor alpha chain and defined the mechanism by which IFNgamma receptor alpha chain tyrosine phosphorylation couples the receptor to its signal transduction system. On the basis of this work and work from other laboratories we have formulated a model of IFNgamma signal transduction that involves the following steps: (l) ligand induced assembly of an activated receptor complex (2) activation of receptor associated tyrosine kinases (3) phosphorylation of a specific tyrosine residues in the intracellular domain of the receptor a chain, (4) binding of a latent cytosolic/membrane associated transcription factor known as p9l to the phosphorylated receptor a chain, (5) phosphorylation/activation of p9l by the receptor associated active tyrosine kinases and (6) assembly of an activated p9l containing transcription factor complex that then translocates to the nucleus where it transcriptionally activates IFNgamma inducible genes. The focus of the research proposed in this renewal application is to critically test the model described above and to elucidate at the molecular level the interactions that occur between the IFNgamma receptor subunits and specific pathway components. To accomplish this long range goal we intend to pursue the following three specific aims. I. We will elucidate the structure and function of the human IFNgamma receptor beta chain. Here we will use mutagenesis approaches to identify the structure-function relationships that are operative within the intracellular domain of the IFNgamma receptor beta chain and explore how this region of the molecule participates in the signal transduction process. We will also assess the mechanism by which the beta chain is recruited into the signaling process by ligand. II. We will define in detail the interaction of p9l with the phosphorylated IFNgamma receptor a chain. We intend to finish the description of the interaction by exploring whether p9l can be coprecipitated with the IFNgamma receptor a chain, define whether the interaction of p9l with the phosphorylated receptor a chain occurs directly or requires an adapter protein, and study the structures on p9l (and/or the adapter protein) needed to mediate the interaction. III. We will define the molecular basis of the IFNgamma inducible tyrosine kinase activity. This study will establish the identity of the tyrosine kinase(s) that associate with the IFNgamma receptor alpha chain, define the basis of the specificity of the receptor-kinase associations, and elucidate the mechanism of kinase activation. These studies are certain to provide new insights that will further elucidate the molecular mechanisms underlying IFNgamma's important immunomodulatory and proinflammatory activities.
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