Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells.

Abstract

A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Sorting and processing of secretory proteins

“…In transfected GH4C1 cells such 75, 69 and 60 kDa forms were secreted, but were possibly inactive [231]. In AtT20 cells, by contrast, a 66 kDa product was the major secreted form when secretion was stimulated, and while the 87 kDa form was membrane-associated in the cells, the 66 kDa form was found in soluble and membrane-bound fractions [265]. A similar 66 kDa form is found in rat islets [250,252] [267], which suggests that cleavage might be autocatalytic or that another unidentified proteinase is responsible for this processing [266,267].…”

“…An initial cleavage event C-terminal to Arg-SerLys-Arg'10 in mouse (or Arg'09 in bovine) PCI leads to the removal of the N-terminal pro-region residues [185, 232,264] and the generation of 80-85 kDa forms [231,264] or an 87 kDa form [232,265]. The discrepancy in size of mouse PCI reported by the different authors might be due to tissue-specific post-translational modification.…”

“…The discrepancy in size of mouse PCI reported by the different authors might be due to tissue-specific post-translational modification. These forms are released from GH4C1 and AtT20 cells [231,264,265] but are found within cells only in a membranebound form [265]. Further truncation can occur at the Cterminus, leading to the production of several smaller products [266].…”

“…The kinetics of processing of endogenous PCI [252] and PC2 [252,270] have been followed in rat islets (the latter in greater detail), and those of PCI have been studied in AtT20 cells [265]. The processing of both enzymes has also been studied in considerable detail following their expression by vaccinia infection ofGH4CI cells [267].…”

Prohormone Convertase 1 Is Necessary for the Formation of Cholecystokinin 8 in Rin5F and STC-1 Cells

“…Western Blot Analysis-Polyclonal antibodies recognizing PC1 and PC2 proteins were generously provided by Iris Lindberg at the Louisiana State Medical Center (38,39). Western analysis was performed with slight modifications following the protocol sent by her laboratory.…”