Neurotrophins are a family of highly conserved polypeptide growth factors that play critical roles in the differentiation of neuroblasts and the survival of mature neurons. More recent studies have revealed additional actions in mediating axonal guidance, synaptic plasticity and injury protection. Neurotrophins are synthesized as preproproteins which are subsequently cleaved to smaller, mature forms which dimerize. At the molecular level, neurotrophins exert their effects by interacting with two structurally unrelated receptors: p75, a member of the TNF receptor superfamily, and the Trk receptor tyrosine kinases. Neuronal signaling and gene regulation mainly reflect Trk activation, while p75 can modulate ligand binding when both receptors are expressed. In addition, ligand activation of p75 can initiate apoptosis when p75 is expressed independently of Trk. Tyrosine phosphorylation of activated Trk recruits downstream signaling enzymes and adaptor proteins that contain protein interacting domains. Although the formation of receptor-adaptor-enzyme complexes is believed to mediate the numerous biological responses ascribed to the neurotrophins, only a limited number of signaling modules, such as the Ras-MAP kinase cascade and the PI3-kinase-Akt pathway, have been identified to date. Another confounding issue is that many of the downstream effectors of Trk are not unique to the neurotrophins, but are activated of other receptor tyrosine kinases to yield different biological endpoints. The diversity and specificity of neurotrophin actions on neuronal populations therefore suggest that additional mechanisms exist which determine the cellular consequences of p75 and/or Trk receptor activation. The long-term goal of our work is to understand the biochemical and molecular basis of neurotrophin function. Using the nerve growth factor (NGF) responsive cell line PC12 and the brain derived neurotrophic factor (BDNF) responsive primary cortical neurons, we have identified novel signaling paradigms at the levels of ligand:receptor interaction, post-receptor signaling and transcriptional activation. The combination of in vitro and in vivo approaches outlined below are designed to test three inter-related hypotheses. Specifically we propose to: 1. Define the biological activities of the pro-forms of NGF and BDNF in the selective activation of Trk or p75 and to determine their biological relevance in neuronal signal transduction. 2. Characterize the unique mechanism by which activated TrkA and TrkB receptor tyrosine kinases is linked to the cellular adaptor molecule CrkL to generate neuronal specific and transcriptional responses. 3. Define the mechanism and functional consequences of TrkA- and TrkB-mediated activation of the transcription factor STAT5.
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