Neurotrophins play a major role in development and maintenance of many types of neurons. The family of NGF related neurotrophins (BDNF, NT-3, NT-4/5) interact with a low affinity neurotrophin receptor (LANR) and with the family of trk proto-oncogene receptor tyrosine kinases (Trk) to establish signaling within responsive peripheral neurons (sensory, sympathetic) and central neurons (cholinergic, dopaminergic). The question addressed is the nature of the recognition, specificity, and signal transduction through the receptor to the inside of the cell, i.e., how conformational changes are communicated across the membrane. These projects will characterize the dynamics of protein-protein interactions involved in neurotrophin- receptor(s) binding by using chemical, immunochemical, recombinant, mutagenic, and spectroscopic techniques with kinetic and thermodynamic analyses on purified neurotrophin and purified receptor components.
The Specific Aims of this project are: (1) Recombinant, partially processed NGF (1-120) has diminished activity in certain bioassays suggesting that NGF activity may be regulated by extracellular proteolytic processing. The physiological importance of the NGF (1-120), containing the C- terminal dipeptide, will be determined by further characterizing its specificity, by making mutants at the C-terminus, and by measuring the amounts and distribution of C-terminal unprocessed NGF in nervous tissue. (2) The functional and structural role of the reverse turn loops of NGF in binding both LANR and Trk will be determined by mutagenesis. Interactions between neurotrophin receptors will be analyzed using multiple mutations of NGF with thermodynamic cycle linkage. (3) The contribution of the aromatic amino acids at positions 52-54 and the N-terminus of NGF to function, stability and structure will be determined by point and deletion mutagenesis. The conformation and stability of the mutants (in these first three aims) will be characterized by biophysical chemical techniques; their specificity will be determined by binding to recombinant Trk family receptors and by bioassays with PC12 cells, dorsal root and nodose sensory neurons, sympathetic neurons and dopaminergic neurons and (4) Potential conformational changes upon interaction of the neurotrophins with the LANR receptor extracellular domain (LAN-RED) and with the Trk receptor extracellular domain (TRK-RED) family will be determined using time- resolved fluorescence and circular dichroism studies. The projects outlined here are important in understanding the molecular basis of neurotrophin effects on specific neurons in the CNS and PNS that may be affected in aging or in neurological disorders such as Alzheimer's disease, Parkinsons disease, and neuropathies.
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