Trafficking of non-regulated secretory proteins in insulin secreting (INS-1) cells

Aims/hypothesis. Sorting of proinsulin to the regulated secretory pathway o f pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these e vents, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investig ated 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 s ecreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transf ection and by infection with recombinant adenovirus, respectively. Secretio n 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% to tal) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By cont rast SEGFP had a secretory pattern similar to insulin, with low basal secre tion (0.8% total) and 16-fold stimulation by secretagogues. Granular locali zation of SEGFP was confirmed by high resolution electron microscopy immuno cytochemistry. Pulse-chase experiments indicated retention of SEGFP in gran ules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its r egulated secretion. Conclusion/interpretation. These data indicate that in INS-1 cells SEGFP, b ut not SEAP, is unexpectedly handled as a regulated secretory protein and s tored along with insulin in granules. This raises questions about the speci ficity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both.