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.
Bibliographical noteFunding Information:
Acknowledgements. We are grateful to Ms I. Dillier for technical support and Dr K. Sadoul for helpful discussions. This work was supported by a Training and Mobility for Researchers programme grant from the European Union (ERB-4061-PL-95–0350) and the Swiss Federal Office for Education and Science (95.0654), mentor-based fellowship award No. 398 211 from the Juvenile Diabetes Foundation International, and grant numbers 31–50811–97 and 31–43 366.95 from the Swiss National Science Foundation. Partial support was received from the Jewish Hospital Foundation, Louisville, Kentucky and Public Health Service grant R01 DK 53367–01 (S.-U. Gorr).
- Regulated secretory pathway
- Secretory alkaline phosphatase
- Secretory granules