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Abstract
Bioactivation of prohormones occurs in the granules of the regulated secretory pathway of endocrine cells, which release hormones in response to external stimulation. How secretory granules are formed and how the cargo is selected is still unclear, but it has been shown for several prohormones and processing enzymes that domains within the prohormone structure can act as "sorting signals" for this pathway. The domains mediate interactions with other proteins or with the membrane or facilitate aggregation of the (pro)peptides. We have now searched for domains in progastrin that are active in sorting the prohormone into secretory granules. Truncation studies showed that the N-terminal 30 residues of progastrin are dispensable, whereas the last 49 residues are sufficient for correct biosynthesis of bioactive gastrin. Thus, further N-terminal truncation abolished gastrin expression. C-terminal truncation of 8 residues resulted in an increase in basal secretion as did point mutations in the dibasic processing sites of progastrin. These mutants, however, still responded to secretagogues, suggesting a residual sorting capacity to the regulated pathway. Amino acid substitutions in an acidic, polyglutamate motif within gastrin-17, the main bioactive, cellular gastrin form, did not alter secretion per se, but when these residues were substituted in C-terminally truncated mutants, double mutants increased in basal secretion and did not respond to secretagogue stimulation. This implies that the mutants are constitutively secreted. Our data suggest that the dibasic processing sites constitute the most important sorting domain of progastrin, and these sites act in synergy with the acidic domain.
Highlights
Tory granules has been debated extensively in recent years
Using N- and C-terminal truncations and amino acid substitutions, we have found that two domains are implicated in progastrin sorting
As part of the N-terminal end of progastrin is well conserved during evolution, we did N-terminal truncations to analyze whether this region is necessary for sorting to the regulated secretory pathway
Summary
Materials—Culture media and supplements for cell culture were obtained from Invitrogen. Media were changed the following day, and the cells were allowed 2 days for expression before harvesting. Stimulation experiments were initiated 1 day after transfection by the addition of 0.5 mM IBMX (Sigma) and incubated for peptide expression for 24 h, when cells and culture media were harvested. Culture media were recovered and, cells were removed by the addition of phosphate-buffered saline added to 2 g/liters EDTA, pelleted, and kept at Ϫ20 °C until peptide extraction. Nomenclature—The nomenclature used in this study is as follows (see Fig. 1 for residue numbering and progastrin fragment references). HIT-T15 was transfected in parallel and cells and culture media were analyzed for amidated gastrins (mean Ϯ S.E., n ϭ 4). The secretion ratio was calculated as the ratio of amidated gastrins in the media divided with cell contents
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