The physiological state of a cell is modulated in response to specific extracellular signals through changes in metabolic processes resulting, in part, from differential regulation of gene expression. The broad objective of this proposal is to characterize the molecular mechanisms involved in signal transduction initiated by polypeptide ligands binding to their specific cell surface receptors. Interferons (IFNs) area a class of polypeptide cytokines which induce rapid and coordinate transcriptional activation of a set of dispersed interferon stimulated genes (ISGs), leading to profound effects on the growth, differentiation, and metabolism of sensitive cells. The activation of these genes is completely dependent on the presence of IFN bound to its cell surface receptor and is mediated through the action of pre-existing protein components which change in activity, rather than in abundance, following treatment with IFN. Although the cis-acting DNA sequences regulating these genes have been characterized and trans-acting nuclear and cytoplasmic factors have been identified, the protein components of the signal transduction pathway from the cell surface to the nucleus, the nature of the biochemical events which carry the signal, and the mechanisms of transcriptional control of ISGs remain at present largely undefined. The major goal of this project is to develop a comprehensive understanding of IFN-induced transcription which leads to production of gene products necessary for growth control and for viral resistance. We propose to determine the molecular mechanics of IFN-induced signal transduction by defining genes and gene products in the pathway, by purifying and characterizing constituent proteins, and by defining the biochemical steps involved in signal generation. In this proposal, the individual components of signal transduction will be isolated and purified in order to reconstitute the process in vitro and to study the biochemical steps involved. This will be approached by isolating, purifying, and cloning the positive transcription factor which activates ISG expression in the nucleus, the protein precursors to this transcription factor which exists in the cytoplasm, the proteins which interact with these components to achieve activation and nuclear translocation, the type I IFN cell surface receptor which initially generates the signal, and membrane and cytoplasmic receptor-proximal proteins which serve as links in the signal chain. These proteins will be analyzed in vitro and in vivo by mutagenesis to define functional domains involved in IFN binding, signal transduction, subunit association, nuclear translocation, DNA binding, and transcriptional activation. Knowledge of the normal mechanism of action of IFN will provide a basis for understanding its role in viral inhibition and control of proliferation and will lead to investigations of defects in these processes which may contribute to impaired resistance to infectious disease and to abnormal cell growth. These studies may also be broadly applicable signal transduction systems involving polypeptide hormones and growth factors.
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