Function and mechanism of NGF-mediated c-Ret activation

Washington University, Saint Louis MO

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Abstract

DESCRIPTION (provided by applicant): The functions of GDNF family of ligands (GFLs) in postnatal development of the nervous system is largely unknown because deletion of the RTK through which GFLs function, c-Ret, causes perinatal lethality. In sympathetic neurons both in culture and in vivo the amount of Ret phosphorylation increases with postnatal age, reaching a maximum by P21. Surprisingly, this increase in Ret phosphorylation is not mediated by GFLs. Rather, nerve growth factor (NGF), a neurotrophic factor critical for sympathetic neuron development, regulates Ret phosphorylation via a novel mechanism. The goal of Aim 1 is to determine the mechanism by which NGF regulates Ret phosphorylation. The tyrosine residues in the NGF receptor TrkA, and thus the signaling pathways, responsible for NGF-dependent Ret phosphorylation will be identified by expressing TrkA point mutants in the highly transfectable Neuro 2A neuroblastoma cell line. The goal of Aim 2 is to determine the function of NGF-dependent Ret phosphorylation using two complementary approaches. First, the function of NGF-dependent Ret phosphorylation in postnatal neurons will be determined by examining cultured 21 DIV sympathetic neurons from ret-/- animals. Preliminary data suggests that Ret-1 neurons have metabolic deficiencies. Second, transgenic mice will be generated that express dominant-negative forms of Ret in the postnatal nervous system, thereby avoiding abnormalities that lead to early lethality in order to determine the function of Ret in the postnatal nervous system.