Nerve Growth Factor (NGF) is an important protein involved in the development and maintenance of sensory and sympathetic neurons. Although much is known about the protein structure of the active beta-subunit and the 7S oligomeric form of the protein, the neurobiologic role during development, and the cell biology of its stimulus for differentiation of responsive cells, less is known about the structure-function relationships in the beta-NGF itself. This project is directed toward determining some of the features of the 13,000 Da polypeptide chain that are responsible for interaction with the cellular receptor and resultant effects on cell morphology.
The specific aims are: (1) to utilize site-specific mutagenesis to obtain beta-NGF variants; (2) to correlate these changes with biological and protein properties; (3) to obtain agonists and partial agonists of beta-NGF; (4) to produce site-specific antibodies directed against synthetic oligopeptides; and (5) to correlate the effects of these antibodies on biological properties of beta-NGF. Two approaches will be utilized to achieve these aims. (a) Immunochemical. Oligopeptides corresponding to particular sequences in the beta-NGF molecule will be synthesized and monospecific antibodies toward them will be produced. The determinants recognized by existing monoclonal antibodies will be determined. These antibodies will be used to study the functional role of various portions of the beta-NGF molecule. (b) Molecular engineering. We have inserted the cDNA for beta-NGF into an E. coli expression system. Site specific replacements in critical amino acid residues will be produced to determine the role of these residues in biological function. The effects of the antibodies and altered beta-NGF will be assessed in neurite outgrowth in Pheochromocytoma (PC12) cells and sensory neurons, in binding to cellular receptor of PC12 cells, in early cellular responses to NGF, in the interaction of the beta subunit with the other two subunits of the 7S oligomer, and on the dimerization of beta-NGF. Effects on the conformation of the recombinant beta-NGF will be determined by spectroscopic techniques. The results of these experiments should provide useful new agonists and antagonists of beta-NGF for developmental studies, as well as allow a critical understanding at the molecular level of the requisite features for interaction and stimulation of neuronal cells by NGF.
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