Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes.
Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrine-precursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursor-processing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processing-deficient BSC-40 cells cleaved native mouse POMC at the -Lys-Arg- sites flanking corticotropin. The -Lys-Arg- within beta-lipotropin was less efficiently cleaved to release beta-endorphin. Expression of PC2 together with PC3 resulted in efficient conversion of beta-lipotropin, as occurs in pituitary melanotrophs. Furthermore, coexpression of PC2 together with mouse POMC in bovine adrenomedullary chromaffin cells resulted in conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin in the regulated secretory pathway. Finally, the processing selectivities of PC3 and PC2 expressed together in BSC-40 cells were determined by using a series of mutant mouse POMCs containing all possible pairs of basic residues at certain sites. The observed pattern of cleavage site selectivities mimicked that of the endogenous endoproteases of the insulinoma and bovine adrenomedullary chromaffin cells, suggesting that PC2 and PC3 may represent important core endoproteases in the catalysis of prohormone processing in many neuroendocrine cell types.
Thomas
L
L
Vollum Institute, Oregon Health Sciences University, Portland 97201.
Leduc
R
R
Thorne
B A
BA
Smeekens
S P
SP
Steiner
D F
DF
Thomas
G
G
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics; D008322:Mammals; D010957:Plasmids; D011333:Pro-Opiomelanocortin / Q000378:metabolism*; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051578:Proto-Oncogene Proteins c-fes; D011863:Radioimmunoassay; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D001615:beta-Endorphin / Q000378:metabolism; D008083:beta-Lipotropin / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D004247:DNA; D008083:beta-Lipotropin; D011505:Protein-Tyrosine Kinases; D051578:Proto-Oncogene Proteins c-fes; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D045706:Proprotein Convertase 2