Peptide biosynthetic processing: distinguishing prohormone convertases PC1 and PC2

Abstract

To determine whether manipulation of time, temperature and intragranular pH could be used to distinguish the actions of two subtilisin-related endoproteases, PC1 and PC2, in peptide biosynthesis, the biosynthetic processing of proneuropeptide Y (proNPY) and proopiomelanocortin (POMC) was examined in pituitary cell lines. AtT-20 cells express PC1 and POMC endogenously; stably transfected AtT-20 lines expressing NPY or PC2 were studied. GH3 cells express PC2 endogenously; NPY-expressing GH3 transfectants were investigated. PC1 mediated rapid processing of NPY and POMC; PC1-dependent cleavages were relatively insensitive to 20 degrees C blockade (which arrests secretory pathway transport at the trans-Golgi network) and do not require an acidic intracellular compartment (as in secretory granules). PC2 mediated much slower processing of proNPY and POMC which was totally blocked at 20 degrees C and required an acidic intracellular compartment. Thus, kinetics, abolition of intracellular pH gradients, and incubation at reduced temperatures can be used to distinguish PC1 and PC2 actions in neuroendocrine cells.