The biosynthesis of mouse nerve growth factor, an important regulator of neuronal development will be examined by translation of hybrid selected NGF-mRNA and by transcription and translation of NGF-cDNA in a number of systems. The NGF precursor and the intermediates in the biosynthesis will be characterized by size and peptide composition and the mechanism of the processing by kallikrein enzymes explored. Radioimmune assays and single stranded probes will be used to determine if proteins analogous to the Alpha and Gamma subunits appear in sympathetic target tissues and to explore their synthesis. The corresponding proteins in the African rat submaxillary gland will be characterized. The mechanisms whereby nerve growth factor (NGF) induces and maintains the differentiated state of a clonal cell line of rat pheochromocytoma, PC12 will be studied. In order to approach the nature of the intracellular signals which direct this action the NGF receptors will be cloned by transfection of PC12 cell DNA into a mouse L cell recipient and selection of transfected cells expressing the receptor by cell sorting after binding of a suitable fluorescinated antibody to the receptor. The NGF receptors themselves will be purified from PC12 cells or from a transfected cell line expressing large numbers of receptors and compared by peptide mapping. The fate of the internalized NGF-NGF receptor will be followed by taking advantage of the differential trypsin sensitivity and molecular weights of the two forms. In PC12 cells NGF induces and maintains neurite formation. Removal of NGF or addition of microtubule depolymerizing drugs causes neurite disintegration. The mechanism by which NGF enhances microtubule stability will be explored by measurements of the levels of Alpha, Beta tubulin, tau and MAP1 proteins and of the specific mRNAs under various conditions and kinetics of neurite outgrowth.
Showing the most recent 10 out of 103 publications
