PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues.

Abstract

A recombinant vaccinia virus vector was used to coexpress the two candidate mouse prohormone convertases, PC1 and PC2, together with mouse proopiomelanocortin (POMC) in the constitutively secreting cell line BSC-40 and in the endocrine tissue-derived cell lines PC12 and AtT-20, which exhibit regulated secretion. Monitoring of POMC processing demonstrated the distinct cleavage specificities of PCi and PC2, since in the cell lines analyzed (i) PCi cleaves POMC into corticotropin and fB-lipotropin, (ii) PC2 cleaves POMC into (3-endorphin, an N-terminally extended corticotropin containing the joining peptide, and either aMSH or desacetyl-aMSH, and (iii) PC2 cleaves POMC at the five pairs of basic residues analyzed, whereas PC1 cleaves two of them preferentially, suggesting that PC2 has a broader spectrum of activity than PC1. These data are consistent with our hypothesis on the physiological role of PCi and PC2 as distinct proprotein convertases acting alone or together to produce a set of tissuespecific maturation products in the brain and in peripheral tissues.Limited proteolysis of inactive precursors at pairs of basic residues or, less frequently, at single basic amino acids (1, 2) is a general mechanism by which the cell produces a variety of active proteins and peptides. Until recently, the molecular nature of the processing enzyme(s) was not identified except for the yeast pro-a mating factor precursor, where a subtilisin-like serine proteinase called KEX2 was proven to be the physiological convertase (3). In the last year, different groups obtained the complete cDNA structures of three distinct candidate mammalian convertases, furin (4), PC1 (5, 6), and PC2 (5, 7). The putative convertase function of these proteins was largely based on sequence homology with KEX2 (8, 9) and on their tissue and cellular distribution, especially for PC1 and PC2 (5, 6). Here we show that the expression of PC1 or PC2 by vaccinia virus (VV) recombinants results in the production of distinct proteolytic enzymes in three cell lines: the mouse pituitary corticotroph line AtT-20 and two that do not express these enzymes endogenously, the African green monkey kidney epithelial cell line BSC-40 and the rat pheochromocytoma cell line PC12. These proteinases cleave proopiomelanocortin (POMC) into the same set of distinct peptides known to be produced in vivo in the hypothalamus, adenohypophysis (AP), and pars intermedia of the pituitary, including adrenocorticotropin (ACTH), p-lipotropin (,8LPH), ,f-endorphin (f3End), and a-melanotropin (aMSH) or desacetyl-aMSH.MATERIALS AND METHODS Ws. Purified recombinant VVs using the full-length mouse (i) PC1 and mPC2 cDNA inserts (5, 6) (VV:mPC1 and VV:mPC2) were prepared as described (10). VV:POMC (mouse) was a gift of G. Thomas (Vollum Institute, Portland, OR).Northern Blot Analysis. Total RNA was prepared as described (11). The [a-32P]UTP-labeled cRNA probes were generated from cDNA sequences of mPC1 (6) and mPC2 (5) inserted in plasmid Rc/CMV vector (Invitrogen, San Diego) and linearized...

Sorting and processing of secretory proteins

“…PCI and POMC have been co-localized in the anterior corticotrophs, while PC2 and POMC mRNAs were rarely observed together. These results are in agreement with the actions of PCI and PC2 on POMC [224], and with the co-ordinate regulation of POMC, PCI and PC2 mRNAs that has been demonstrated in the pituitary [253].…”

“…These convertases have thus been well conserved throughout evolution. In keeping with their proposed role in proprotein conversion, these enzymes correctly cleave precursor proteins at paired basic residues [196,197,[221][222][223][224]. Kex2, furin and PC6B have hydrophobic transmembrane domains, while PCI, PC2, PC4, PC5, PC6A and PACE 4 do not.…”

“…The cleavage specificity of PCI and PC2 was first demonstrated by their co-transfection with mouse POMC and POMC mutants into the constitutively secreting BSC-40 cells and cell lines derived from endocrine tissue, namely PC12 and AtT20 cells, using the vaccinia expression system [223,224]. PC2 efficiently produced a-MSH and ,-endorphin-(1-3 1), while PCI generated ACTH and ,i-LPH.…”

Malignant Gastric Carcinoid Causing Ectopic ACTH Syndrome: Discrepancy of Plasma ACTH Levels Measured by Different Immunoradiometric Assays

“…In contrast to high ACTH levels determined by Allegro ® assay kit, those determined by Yuka ® assay kit in the same patient's plasma samples were almost normal. Two-site IRMA kit by Allegro ® employs 125 I-labeled antibody against Nterminal fragment (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and antibody against Cterminal fragment(34-39) of ACTH, while that by Yuka ® employs 125 I-labeled antibody against Nterminal fragment (20)(21)(22)(23) and antibody against Cterminal fragment(35-39) of ACTH, respectively. These data strongly suggest that various circulating forms of ACTH molecule in patient's plasma could be differentially recognized by these two different IRMA kits.…”

“…In the human pituitary, POMC is enzymatically processed to pro-ACTH, N-terminal POMC (N-POC), b-lipotropic hormone (b-LPH), ACTH(1-39), and b-endorphin. Processing of POMC in the pituitary takes place by the protease prohormone convertase (PC) 1/3 located within mature dense core granules via the regulated secretory pathway [7,8].…”