Mutational analysis of PC1 (SPC3) in PC12 cells. 66-kDa PC1 is fully functional.
The proteinase mPC1, a neuroendocrine member of the mammalian family of subtilisin-like enzymes, has previously been shown to be converted to a carboxyl-terminally truncated 66-kDa form during transport through the secretory pathway. The cleavage site and the function of this carboxyl-terminal truncation event are unknown. We have performed site-directed mutagenesis of two paried basic sites in the mPC1 carboxyl-terminal tail and expressed these constructs in PC12 cells, a rat pheochromocytoma known to lack endogenous PC1. We found that the most likely site for the truncation event was at Arg590-Arg591 since mutation of this site to Lys-His prevented processing of 87-kDa PC1. A PC1 mutant carboxyl-terminally truncated at this site and expressed in PC12 cells was efficiently routed to the secretory pathway and stored in secretory granules, indicating that the carboxyl-terminal extension is not required for sorting of this enzyme. The function of the various PC1 constructs was assessed by analyzing proneurotensin cleavage to various forms. The carboxyl-terminally truncated PC1 mutant was found to perform most of the cleavages of this precursor as well as wild-type PC1; however, the blockade mutant processed proneurotensin much less efficiently. Differences between the site preferences of the various enzymes were noted. Our results support the notion that carboxyl-terminal processing of PC1 serves to regulate PC1 activity.
Zhou
Y
Y
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112, USA.
Rovere
C
C
Kitabgi
P
P
Lindberg
I
I
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000502:physiology*; D001483:Base Sequence; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D051381:Rats; D013329:Structure-Activity Relationship
D043484:Proprotein Convertases; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases