Trafficking of non-regulated secretory proteins in insulin secreting (INS-1) cells |
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Authors: | M Molinete V Lilla R Jain P B M Joyce S-U Gorr M Ravazzola P A Halban |
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Institution: | (1) Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland, CH;(2) Department of Biological and Biophysical Sciences, University of Louisville School of Dentistry, Louisville, Kentucky, USA, US;(3) Department of Chemistry and Biochemistry and Centre for Structural and Functional Genomics, Concordia University, Montreal, Quebec, Canada, CA;(4) Department of Morphology, University Medical Centre, Geneva, Switzerland, CH |
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Abstract: | Aims/hypothesis. Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core
granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous
secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1
cells. Methods. SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced
fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with
recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by
its activity. Results. Secreted alkaline phosphatase showed high basal secretion (6.6 % total) but only modest (3.6-fold) stimulation of secretion
by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern
similar to insulin, with low basal secretion (0.8 % total) and 16-fold stimulation by secretagogues. Granular localization
of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention
of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked
oligomers. This could be implicated in its regulated secretion. Conclusion/interpretation. These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and
stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins
to granules in INS-1 cells or their retention therein or both. Diabetologia (2000) 43: 1157–1164]
Received: 13 December 1999 and in final revised form: 8 May 2000 |
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Keywords: | GFP insulin regulated secretory pathway secretory granules secretory alkaline phosphatase |
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