RNAi-mediated silencing of prohormone convertase (PC) 5/6 expression leads to impairment in processing of cocaine- and amphetamine-regulated transcript (CART) precursor

Understanding the functions of the widely expressed PCs (prohormone/proprotein convertases), including PC5/6, furin and PACE4 (paired basic amino acid cleaving enzyme 4), in animal models is difficult since individual knockouts of these PCs in mice exhibit early embryonic lethality. To investigate the roles of PC5/6 in processing pro-CART (pro-cocaine- and amphetamine-regulated transcript), an important anorexigenic peptide precursor, we have generated GH3 cells silenced for PC5/6 expression by RNAi (RNA interference). We show, following transient knockdown of PC5/6 in these neuroendocrine cells, that generation of the two bioactive forms, CART I (amino acids 42-89/55-102) and CART II (amino acids 49-89/62-102), from pro-CART is impaired due to a lack particularly of the A isoform of PC5/6. The results indicate that PC5/6A shares specificities primarily with PC2 (PC5/6A < PC2) in cleaving the pairs of basic residues, KR (40, 41↓/53, 54↓) and KK (47, 48↓/60, 61↓), within the pro-CART isoforms [see Dey, Zhu, Carroll, Turck, Stein and Steiner (2003) J. Biol. Chem. 278, 15007-15014]. We do not find any significant role of PC5/6A in processing the RXXR (29-32↓) site for production of intermediate CART (amino acids 33-102) from long pro-CART. The findings taken altogether indicate that PC5/6 participates in normal processing of pro-CART.