Nerve growth (NGF) is a well-studied neurotrophic factor that functions in both the peripheral and central nervous systems. It, and related substances, appear to be important in neurodegenerative disease and aging. Experiments are proposed dealing with structural aspects important in receptor binding and in the molecular mechanisms of signal transductions. The latter work extensively employs receptor chimeras and their stable expression in PC12 cells. Structural and mutagenesis studies of the alpha and lambdaNGF subunits (mouse) will ascertain contact sites and act as probes of receptor binding sites for the biologically active ~-subunit. Mutagenesis as well as chemical modification experiments on betaNGF will specifically address these same interactions as well as the role of quaternary structure in receptor binding. The EGF/EGF-BP complex will be similarly examined. Using the extracellular (ligand binding domain) of the human platelet-derived growth factor (3 receptor (hPDGFbetaR), chimeric receptors with the transmembrane and intracellular domains of TrkA (NGF receptor), TrkE (a putative NGF receptor), EGFR, p75 (the low molecular weight NGFR) and c-Src will be constructed and transfected into PCI2 cells (as well as other cell types) with retroviruses to determine activity and the induction of various signalling pathways (Ras-MAPK and STAT) and immediate early/late gene responses. These constructs will be further altered by site-directed mutagenesis to ascertain the role of individual receptor tyrosine residues. Soluble versions of the receptor tyrosine kinases for TrkA, EGFR and FGFR1 will also be expressed and characterized. The role of c-Src in NGF responses in PC l 2 cells will be further probed with an anti-sense sequence to the SH2-SH3 region and by expression of TrkA in c-Src-fibroblasts. Other experiments will probe the possible induction and role of Crk in NGF responses. Proteins specifically effected in PCI2 cells during the first 6 hrs of induction by NGF and FGF (neurotrophic), as well as those by NGF, FGF and EGF (mitogenic). will be identified from 2D gels, using mass spectrometry and microsequencing. Finally, the role of NGF1-A (TIS8) in inducing tumor suppressors, in particular retinoblastoma proteins, as the molecular switch controlling NGF-induced neurodifferentiation in PCI2 cells will be examined.
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