Show simple item record

dc.contributor.authorStoller, Timothy J.
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 49-10, Section: B, page: 4163.;Advisors: Dennis Shields.
dc.description.abstractSomatostatin (SRIF) is a 14 amino acid peptide hormone that is synthesized as a larger precursor: preproSRIF. The goals of this project were: (i) To determine if cells which synthesize hormones that are not posttranslationally cleaved have processing enzymes capable of efficiently cleaving foreign prohormones; (ii) Investigate the role of the SRIF proregion in mediating ER to Golgi transport and targeting to the regulated secretory pathway; (iii) Determine the specificity of endoproteolytic cleavage using site-directed mutagenesis to alter the paired-basic residues in the processing site.;cDNA encoding preproSRIF was introduced into two mammalian cell lines, GH{dollar}\sb3{dollar} (rat anterior pituitary cell which synthesized growth hormone) and 3T3 (mouse fibroblasts) using a recombinant retrovirus. Clonal lines expressing preproSRIF were established and the proteolytic processing efficiency, specificity and kinetics of secretion determined. In GH{dollar}\sb3{dollar} cells, preproSRIF was efficiently processed to the mature hormone as determined by HPLC analysis and microsequencing. Surprisingly, mature SRIF was stored more efficiently than the endogenous growth hormone, 55% versus 5%.;Using in vitro site-directed mutagenesis, the paired-basic amino acids were changed to ArgArg and LysArg. Microsequencing showed that the mutant cleavage sites were accurately processed in GH{dollar}\sb3{dollar} cells, indicating either a relative lack of cleavage specificity or the presence of multiple amino acid-specific processing enzymes.;The role of the proregion in mediating intracellular transport and sorting was tested directly by expressing two fusion proteins: a chimera of the SRIF preproregion and ape {dollar}\alpha{dollar}-globin and ape {dollar}\alpha{dollar}-globin fused to an N-terminal signal sequence. The signal peptide of the latter chimera was accurately cleaved but globin was not secreted and rapidly turned over intracellularly (t{dollar}\sb{lcub}1/2{rcub}{dollar} = 5 min). In contrast, 30% of the newly synthesized proregion-globin was transported through the secretory pathway. Most significantly, 40% of the proSRIF-globin was proteolytically processed at the native cleavage site generating mature globin which was secreted. These results demonstrate that the propeptide can act as a universal transport sequence. (Abstract shortened with permission of author.).
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleThe role of prohormones in mediating intracellular sorting, processing and secretion

Files in this item


There are no files associated with this item.

This item appears in the following Collection(s)

Show simple item record