Nerve growth factor (NGF) is a polypeptide growth factor that plays critical roles in the differentiation of immature neuroblasts and the subsequent survival of a subpopulation of mature neurons in both the peripheral as well as central nervous systems. Although the precise mechanism of NGF actions remains incompletely undefined, NGF initiates neuronal diffentiation by binding to the receptor tyrosine kinase (TRK A). This induces the phosphorylation of TRK A on intracellular tyrosine residues which recruits downstream signal transducing enzymes as well as adaptor proteins that contain only the Src homology (SH) domains 2 and 3. Formation of this receptor-signal transducing complex mediates the numerous cellular responses that ultimately lead to a diffentiated neuronal phenotype. Paradoxically, activation of other receptor tyrosine kinases by their cognate ligands, such as the binding of epidermal growth factor (EGF) to its receptor, also appears to initiate a similar spectrum of downstream biochemical events and yet, these growth factors induce mitogenesis rather than differentiation. This dichotomy of action suggests that downstream events are critical in determining the cellular consequences of receptor tyrosine kinase activation. The long term goal of our work is to understand the biochemical and molecular events accompanying NGF actions, and to delineate how NGF signaling promotes neuritogenesis. We have established a model system which can augment, or inhibit NGF induced neurite formation by the ectopic expression of the v-Crk adaptor molecule, or SH2 mutant v-Crk proteins, respectively, in the neural chest cell line PC12. This model system will be used to dissect the mechanisms encoding differentiative signaling . Specifically we propose to: (1.) Identify the intracellular substrates that interact with v-Crk to determine which pathways are critical for neuritogeneis. V-crk, or mutant v-Crk proteins will be expressed in PC12 cells by gene transfer to delineate the domains of v-Crk which regulate neuronal differentiation. We will also explore the potential role of v-Crk associated phosphatase/s in modulating trk receptor signaling . (2.) Determine the role of native adaptor proteins and v-Crk in linking activated receptor tyrosine kinased to the cytoskeleton. (3.) Characterize the trk A/crk/cytoskeletal interactions which result in enhanced receptor internalization.
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