Two major lines of experimentation on nerve growth factor (NGF) will be pursued. The first deals with the biosynthesis, processing and release of the hormone, including structural features of the neuronally active Beta-subunit and associated polypeptides. The NGF from four different species will be examined with the most detailed studies focused on the guinea pig prostate (gp) molecule. The gp BetaNGF will be further characterized with respect to the C-terminal sequence and the high molecular weight form examined with respect to the number and kinds of subunits. Using mouse BetaNGF cDNA probe, the corresponding gp mRNA will be identified, cloned and sequenced. The gp preproBetaNGF cDNA will be incorporated into the overproducing vector pAS1 and cloned into E. coli to allow the expression of preparative amounts of the precursor. This molecule will be purified to homogeneity and used as substrate to detect potential processing agents (proteases) found in both the soluble and membraneous fractions of prostate cells. Synthetic prepro and proBetaNGF will be crystallized to determine the 3-dimensional structure by x-ray crystallography. Bovine seminal plasma NGF and C. adamanteus NGF will be isolated and characterized with respect to their C-terminal sequences and their associated polypeptides. Finally, the Alpha- and Gamma-subunits of mouse submandibular NGF will be deglycosylated and electrophoretically homogeneous forms crystallized for x-ray defraction analysis. Northern blot analyses using synthetic oligonucleotides as well as full length cDNA probes will be used to detect mRNA for the Alpha and Gamma-subunits in a variety of mouse tissues using the BetaNGF cDNA probe as a control. The second main area of study involves the functional aspects of NGF and will deal with both cell surface and nuclear receptors in peripheral neurons and in cultured cells (melanoma and neuroblastoma). The anti-receptor monoclonal antibody already established will be further characterized and new monoclonal antibodies raised to both the surface and nuclear receptors. The surface receptors of neuroblastoma and melanoma cells isolated, characterized and compared to that of sympathetic and sensory neurons, particularly with respect to tyrosine-specific kinase activity. The receptor for human NGF will be cloned using NGF receptor-less mutants of pheohromocytoma cells.
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