The mechanisms involved in synthesis and secretion of specific transmitters play a central role in determining the properties of neuronal cells and their functional role in the nervous system. My long-term objective is to understand, at a molecular level, the processes involved in the packaging and secretion of neurotransmitters.
The specific aims of this proposal are (1) to define the steps involved in packaging of mature secretory peptides into vesicles, (2) to determine the capacity of neuronal cells to segregate these secretory peptides into one population of vesicles, and (3) to generate a stable cell line that will permit the introduction of antibodies into the cell cytoplasm to investigate the mechanisms involved in cellular secretion. In addition to their general interest in the cell biology of neurons, these aims relate specifically to an understanding of pathological processes involving inappropriate processing of peptides (one example is acromegaly) and inadequate secretion of transmitters (possibly including degenerative diseases and affective disorders). To achieve these goals, methods of recombinant DNA and cell biology will be combined with a cell culture system that permits genetic manipulation of cells with a neuronal phenotype. The genes for foreign secretory peptides, including human growth hormone and insulin, will be introduced into the rat pheochromocytoma cell line, PC-12, which responds to NGF by changing from an endocrine to a neuronal phenotype. The products of these introduced genes will be followed using metabolic labeling and immunoprecipitation to determine how they are processed. Finally, this technology will be used to create a PC-12 cell line expressing a viral fusion protein on its surface, which will permit the introduction of antibodies, by red cell-mediated fusion, into the cytoplasm. This cell line will be used to identify molecular components of the secretory apparatus.
