The events leading to neuronal differentiation have been shown clearly to depend upon interactions of growth factors with receptors on the neuronal cell surface. In both the peripheral and central nervous systems Nerve Growth Factor (NGF) is required for the development of distinct neuronal populations in vivo. The rat pheochromocytoma cell line, PC 12, is the premier cell culture model for the differentiating actions of the neurotrophin, NGF. A major action of NGF on PC12 cells is the induction of membrane excitability. Through collaborations described in this program project we have determined that the membrane excitability is the result of induced expression of CNS- and PNS-type sodium channel genes through distinct signal transduction pathways. We have also revealed a new signal transduction pathway utilized by NGF, as well as by FGF, EGF and the cytokine interferon-gamma, to selectively induce expression of the peripheral nerve type sodium channel gene, PN1. We have termed the new pathway the """"""""triggered"""""""" pathway because it is stimulated by only a one-minute exposure to the factors. A major goal of project 2 is to define the molecular intermediates responsible for triggered PN1 gene induction. We will determine which domains in the NGF and FGF receptors are responsible for PN1 regulation (aim 1), using techniques of site- directed mutagenesis and generation of transfected PC12 clones expressing the mutant receptor proteins. We will use techniques of microinjection, immunochemistry and genetics to examine a possible role for the STAT family of proteins in PN1 gene induction (aim 2). By defining the domains in the NGF and FGF receptors that lead to type II sodium channel induction (aim 3), we will begin to define the independent signaling pathways regulating the CNS (type II) and PNS (PN1) channel types. Our studies will provide insights into the unique signaling pathways regulating neuronal excitability in both the PNS and CNS, and into how these signaling pathways converge onto the transcriptional factors that ultimately regulate the sodium channel genes. Results from these studies will be integrated into projects 3 and 4, which are aimed at understanding growth factor regulation of excitability in primary neuronal cell cultures and in vivo.
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