Evidence That PC2 Is the Endogenous Pro-neurotensin Convertase in rMTC 6-23 Cells and That PC1- and PC2-transfected PC12 Cells Differentially Process Pro-neurotensin

Abstract

The neuropeptide precursor proneurotensin/neuromedin N (pro-NT/NN) is mainly expressed and differentially processed in the brain and in the small intestine. We showed previously that rMTC 6-23 cells process pro-NT/NN with a pattern similar to brain tissue and increase pro-NT/NN expression in response to dexamethasone, and that PC12 cells also produce pro-NT/NN but are virtually unable to process it. In addition, PC12 cells were reported to be devoid of the prohormone convertases PC1 and PC2. The present study was designed to identify the proprotein convertase(s) (PC) involved in pro-NT/NN processing in rMTC 6-23 cells and to compare PC1- and PC2-transfected PC12 cells for their ability to process pro-NT/NN. rMTC 6-23 cells were devoid of PC1, PC4, and PC5 but expressed furin and PC2. Stable expression of antisense PC2 RNA in rMTC 6-23 cells led to a 90% decrease in PC2 protein levels that correlated with a > 80% reduction of pro-NT/NN processing. PC2 expression was stimulated by dexamethasone in a time- and concentration-dependent manner. Stable PC12/PC2 transfectants processed pro-NT/NN with a pattern similar to that observed in the brain and in rMTC 6-23 cells. In contrast, stable PC12/PC1 transfectants reproduced the pro-NT/NN processing pattern seen in the gut. We conclude that (i) PC2 is the major pro-NT/NN convertase in rMTC 6-23 cells; (ii) its expression is coregulated with that of pro-NT/NN in this cell line; and (iii) PC2 and PC1 differentially process pro-NT/NN with brain and intestinal phenotype, respectively.

The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer's disease

“…For both convertases, Western blotting revealed similar molecular forms in human cerebral cortex to those previously reported in various mammalian tissues, as well as in cells transfected with the cDNA encoding each enzyme (Paquet et al, 1996;Rovere et al, 1996;Dong et al, 1997;Muller et al, 1998). The slower migration on SDS-PAGE of rat proPC2 as compared to human is most likely due to the fact that the rat prosegment sequence is more acidic (pI 10.0) than that of human (pI 10.7).…”

Molecular Characterization and Differential Gene Induction of the Neuroendocrine‐Specific Genes Neurotensin, Neurotensin Receptor, PC1, PC2, and 7B2 in the Human Ocular Ciliary Epithelium

“…Recently, cathepsin E has also been suggested to be specific in NT/NN processing (Kageyama et al, 1995), as well as the prohormone convertases PC1 and PC2 (Rovere et al, 1996). Now there exists evidence that the ocular cibiary epithelium expresses numerous proteases, including (1) cathepsin D (Ortego et al, 1997); (2) the neuropeptide-processing enzymes CPE and PAM (Ortego et al, 1996); and (3) the endopeptidases furin, PCi, and PC2 (this study).…”

“…That is, when transfected with PCi DNA, pro-NT!NN is processed in a pattern similar to the intestine, and when transfected with PC2 DNA, pro-NT! NN is processed as in the brain (Rovere et al, 1996). If this is the case, the human ocular cell line ODM-2 serves as a unique cell system to determine whether PCI and PC2 might be under different mechanisms of activation.…”

Mutations in the Catalytic Domain of Prohormone Convertase 2 Result in Decreased Binding to 7B2 and Loss of Inhibition with 7B2 C-terminal Peptide

“…The presence of a basic residue at position P4 is favored by both enzymes for cleavage of an artificial substrate (19 -21). However, the two enzymes show important differences in specificity as regards natural substrates; processing of endogenous substrates such as proenkephalin (PE), proopiomelanocortin (POMC), proglucagon, procholecystokinin, proneurotensin, and promelaninconcentrating hormone by PC1 or by PC2 results in differing product patterns (12,(22)(23)(24)(25)(26)(27)(28)(29)(30). In the majority of the studies performed to date, PC1 exhibits a more restricted substrate specificity than PC2.…”