title: Chromogranin A processing and secretion: specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway.
abstract: 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 NH2 and COOH termini, in both wild-type AtT-20 and AtT-20 antisense PC1 cells. After antisense PC1 induction, an approximately 66-kD chromogranin A NH2-terminal fragment as well as the parent chromogranin A molecule accumulated, while an approximately 50 kD NH2-terminal and an approximately 30 kD COOH-terminal fragment declined in abundance. Chromogranin B and secretogranin II immunoblots showed no change after PC1 reduction. [35S]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 approximately 82 kD and decreased approximately 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 approximately 71 and approximately 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 PCl suggests that the action of PC1 on chromogranin A may be specific within the chromogranin/secretogranin protein family.
authors.affiliation: Department of Medicine and Center for Molecular Genetics, University of California, San Diego, USA.
authors.forename: N L
authors.initials: NL
authors.lastname: Eskeland
authors.forename: A
authors.initials: A
authors.lastname: Zhou
authors.forename: T Q
authors.initials: TQ
authors.lastname: Dinh
authors.forename: H
authors.initials: H
authors.lastname: Wu
authors.forename: R J
authors.initials: RJ
authors.lastname: Parmer
authors.forename: R E
authors.initials: RE
authors.lastname: Mains
authors.forename: D T
authors.initials: DT
authors.lastname: O'Connor
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D053379:Chromogranin A; D002864:Chromogranins / Q000302:isolation & purification / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D045683:Furin; D051379:Mice; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism; D016372:RNA, Antisense; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D053379:Chromogranin A; D002864:Chromogranins; D011506:Proteins; D016372:RNA, Antisense; C506155:chromogranin A, mouse; D043484:Proprotein Convertases; D013381:Subtilisins; D045683:Furin; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases