Chromogranin A processing and secretion - Specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway

Chromogranins A and B and secretogranin II are a family of acidic proteins found in neuroendocrine secretory vesicles; these proteins contain multiple potential cleavage sites for proteolytic processing by the mammalian subtilisin-like serine endoproteases PC1 and PC2 (prohormone convertases 1 and 2), and furin. We explored the role of these endoproteases in chromogranin processing in AtT-20 mouse pituitary corticotropes. Expression of inducible antisense PC1 mRNA virtually abolished PC1 immunoreactivity on immunoblots. Chromogranin A immunoblots revealed chromogranin A processing, from both the NH, and COOH termini, in both wild-type AtT-20 and AtT-20 antisense PC1 cells, After antisense PC1 induction, an similar to 66-kD chromogranin A NH2-terminal fragment as well as the parent chromogranin A molecule accumulated, while an similar to 50 kD NH2-terminal and an similar to 30 kD COOH-terminal fragment declined in abundance, Chromogranin B and secretogranin II immunoblots showed no change after PC1 reduction. [S-35]Methionine/cysteine pulse-chase metabolic labeling in AtT-20 antisense PC1 and antisense furin cells revealed reciprocal changes in secreted chromogranin A COOH-terminal fragments (increased similar to 82 kD and decreased similar to 74 kD forms, as compared with wild-type AtT-20 cells) indicating decreased cleavage, while AtT-20 cells overexpressing PC2 showed increased processing to and secretion of similar to 71 and similar to 27 kD NH2-terminal chromogranin A fragments, Antisense PC1 specifically abolished regulated secretion of both chromogranin A and beta-endorphin in response to the usual secretagogue, corticotropin-releasing hormone. Moreover, immunocytochemistry demonstrated a relative decrease of chromogranin A in processes (where regulated secretory vesicles accumulate) of AtT-20 cells overexpressing either PC1 or PC2. These results demonstrate that chromogranin A is a substrate for the endogenous endoproteases PC1 and furin in vivo, and that such processing influences its trafficking into the regulated secretory pathway; furthermore, lack of change in chromogranin B and secretogranin II cleavage after diminution of PC1 suggests that the action PC1 on chromogranin A may be specific within the chromogranin/secretogranin protein family.