Cellular and subcellular aspects of the synthesis, storage, and secretion of several biologically active peptides will be probed. The ACTH/endorphin system of peptides, as expressed in AtT-20 mouse pituitary tumor cells and in rat anterior and intermediate pituitary cells, will provide the foundation for these studies. The production and secretion of insulin by AtT-20ins4b/1 cells and of neuropeptide Y in AtT-20 cells transfected with the appropriate vectors (to be prepared in collaboration with Dr. Jack Dixon, Purdue University) will be examined, as well as expression of the ACTH/endorphin gene in normal and mutant yeast cells. In all of these studies, the precision of the co- and post-translational processing steps and the fidelity of the storage and secretion mechanisms will be examined in detail. Neuropeptide Y provides a particularly advantageous system for comparison to ACTH/endorphin because the NPY precursor is subject to proteolysis, amidation, and possibly phosphorylation, but probably gives rise to only two major peptide products. AtT-20 cells will be transfected with vectors containing normal NPY DNA and NPY DNA that has been altered by single base changes at sites involved in proteolysis, phosphorylation, glycosylation and amidation; the altered DNA molcules and transfected cell lines will also be prepared in collaboration with Dr. Jack Dixon (Purdue). The expression of the normal and altered NPY DNA in fibroblasts will also be analyzed. In order to compare the processing and secretory events seen in cells that do and cells that do not normally produce bioactive peptides. The peptides produced by the new cell lines will be examined to look for predictable and specific alterations. Many existing antibodies will be employed, and additional rabbit antibodies to peptides and monoclonal antibodies to secretory granule proteins will be produced as needed. Where appropriate, pharmacological manipulations of the relevant cells and subcellular fractionations will be utilized.
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