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dc.contributor.authorGEFFEN, IRIS
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 48-09, Section: B, page: 2633.
dc.description.abstractThe primary translation product of bovine preproglucagon mRNA is a precursor of 21 kDa. The molecular basis for the post-translational processing and the regulation of secretion of the precursor and the processed products are unclear. To investigate these aspects of polypeptide hormone expression, we introduced preproglucagon cDNA, using gene transfer, into several cell lines, some of which do not normally synthesize and secrete hormones. These include fibroblasts (COS-7 and NIH-3T3), and Growth Hormone (GH) secreting pituitary cells (GH{dollar}\sb 3{dollar}). Cells either transiently or permanently expressing preproglucagon cDNA were pulse labeled with {dollar}\sp{lcub}35{rcub}{dollar}S-methionine and the intracellular and secreted material was analyzed by immunoprecipitation with appropriate antibodies, SDS-PAGE, and HPLC. The intracellular transit of the immunoreactive proteins was examined by analysis of secretion kinetics and sensitivity to treatment with secretagogues.;COS-7 and NIH-3T3 cells faithfully cleaved the signal peptide and secreted proglucagon, the latter process being particularly efficient in 3T3 cells; no further proteolytic processing was detected. In contrast, GH{dollar}\sb 3{dollar} cells further processed the precursor to a smaller immunoreactive peptide most of which was stored intracellularly. The mobility of this peptide on SDS-PAGE and its HPLC elution pattern indicate that the processing product was native glucagon. Proteolytic processing of proglucagon in GH{dollar}\sb 3{dollar} cells was a rather surprising finding, since these cells are not known to cleave any endogenous precursors at paired basic processing sites. A low percentage of proglucagon was secreted and this occurred with identical kinetics to those of growth hormone release. However, the kinetics of proglucagon proteolytic processing were slower than GH secretion, indicating segregation of these proglucagon molecules which are destined for post-translational proteolysis. Whereas 80-90% of growth hormone and unprocessed proglucagon were secreted constitutively, mature glucagon secretion was mostly regulated and its release, and that of the stored growth hormone and proglucagon, could be triggered by treatment with secretagogues. These observations indicate that GH3 cells possess the cellular machinery for sorting two polypeptide hormones into distinct cellular compartments that are differentially regulated. This "compartmentalization" seems to be related to proteolytic processing of only one of the hormones. (Abstract shortened with permission of author.).
dc.publisherProQuest Dissertations & Theses

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