The proposed program will analyze the biological role of multiple Nerve Growth Factor (NGF) precursors in neural development. Mature NGF protein supports the survival of sympathetic and sensory neurons. NGF mRNA has however been found in several tissues not innervated by sympathetic or sensory neurons (e.g., brain, placenta). These findings suggest additional roles for NGF, or for other proteins encoded by the NGF gene. The original mouse NGF cDNA clones predict that this biologically active molecule results from the processing of a larger precursor protein; the mature NGF protein is located at the carboxy-terminus of the predicted precursor. Little is known about the function of the non-NGF moiety of the precursor. Our discovery of different RNA species, which encode different NGF precurosrs, raises the possibility of complex and novel regulatory strategies associated with this hormone. The long NGF mRNA contains two potential initiation codons and the second precedes the only substantial hydrophobic sequence, presumably the signal sequence for secretion, in the precursor protein. A short NGF transcript contains only the 2nd AUG, the first having been spliced out, and thus encodes a protein truncated at the amino-terminus, with the putative signal peptide directly following the initiator methionine. We postulate that the differences between the two NGF transcripts affect the cellular localization of NGF (e.g. membrane-bound in the larger mRNA vs secreted in the shorter), or change the biological activity of the precursor or of its cleavage products. This would account for the expression of NGF in supporting cells (glia, fibroblasts) in the case of a membrane-associated form and in target cells in the case of the secreted protein. Using an antibody to the NGF precursor expressed as a fusion protein in bacteria (no such antibody is yet available), we will identify the cellular localization and processing of the NGF-associated polypeptides in mammalian cells transfected with the different NGF cDNAs. We will purify the precursors and characterize their biological activity. Last, transgenic animals made to overexpress NGF in specific tissues will facilitate an analysis of the full role played by the NGF and non-NGF moieties of the precursor in development. The results of this study will elucidate aspects of normal neural development in molecular detail. It will also potentially indicate the locus of the primary defect in a developmental or degenerative neurological disorder and may well suggest avenues for intervention in many human conditions through the enhancement of regeneration.
| Edwards, R H; Selby, M J; Mobley, W C et al. (1988) Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector. Mol Cell Biol 8:2456-64 |
| Edwards, R H; Selby, M J; Garcia, P D et al. (1988) Processing of the native nerve growth factor precursor to form biologically active nerve growth factor. J Biol Chem 263:6810-5 |
| Edwards, R H; Rutter, W J (1988) Use of vaccinia virus vectors to study protein processing in human disease. Normal nerve growth factor processing and secretion in cultured fibroblasts from patients with familial dysautonomia. J Clin Invest 82:44-7 |
| Selby, M J; Edwards, R H; Rutter, W J (1987) Cobra nerve growth factor: structure and evolutionary comparison. J Neurosci Res 18:293-8 |
