The overall aim of our studies has been to elucidate the biological significance of the presence in higher organisms (including human) of both the hormone-like polypeptide, epidermal growth factor (EGF), and receptors for this peptide. At the molecular level we hope to understand the biochemical steps leading from the binding of EGF by receptors on the cell membrane to the activation of RNA and protein synthesis and to the final induction of DNA synthesis. Key elements in the molecular study of EGF action have been our isolation of the receptor for EGF, the finding that the receptor is a tyrosine-specific EGF-activated protein kinase, and the demonstration that EGF together with its receptor/kinase are internalized by way of endocytic vesicles. In this proposal we plan to exploit our new finding that in the living mouse, novel tyrosine phosphorylated substrates can be detected in all tissues examined following the intraperitoneal administration of EGF. In view of the large amount of tissue available, the ease of manipulation and the physiological relevance of the in situ detected substrates, the detailed examination of signal transduction in this system should prove very fruitful. We have already identified SHC as an EGF dependent tyrosine phosphorylated substrate in the mouse liver and have detected a 92 kDa tyrosine phosphorylated protein that appears in liver nuclei within minutes following the administration of EGF.
My specific aims are: (1) Isolate and characterize the tyrosine- phosphorylated 92 kDa protein that we have detected in nuclei of liver cells following the administration of EGF to adult mice and prepare polyclonal antibodies to this protein. (2) Determine whether the same nuclear response can be detected in other tissues of the adult mouse, as well as the placenta and fetal tissue during development. (3) Identify other EGF induced tyrosine phosphorylated proteins that we have detected in various tissues of the mouse. (4) Attempt to replicate our results in vitro, using liver slices, liver minces, primary liver cells, or liver cell lines where labeling experiments are easily performed. (5) Attempt cell-free reconstitution experiments to begin to understand the biochemical mechanisms involved in signal transduction.
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