The role of the 7B2 CT peptide in the inhibition of prohormone convertase 2 in endocrine cell lines

Prohormone convertase (PC)2 plays an important role in the processing of ne uropeptide precursors via the regulated secretory pathway in neuronal and e ndocrine tissues. PC2 interacts with 7B2, a neuroendocrine protein that is cleaved to a 21-kDa domain involved in proPC2 maturation and a carboxyl-ter minal peptide (CT peptide) that represents a potent inhibitor of PC2 in vit ro. A role for the CT peptide as an inhibitor in vivo has not yet been esta blished. To study the involvement of the CT peptide in PC2-mediated cleavag es in neuroendocrine cells, we constructed a mutant proenkephalin (PE) expr ession vector containing PE with its carboxyl-terminal peptide (peptide B) replaced with the 7B2 inhibitory CT peptide. This PECT chimera was stably t ransfected into two PC2-expressing cell lines, AtT-20/PC2 and Rin cells. Al though recombinant PECT proved to be a potent (nM) inhibitor of PC2 in vitr o, cellular PC2-mediated cleavages of PE were not inhibited by the PECT chi mera, nor was proopiomelanocortin cleavage las assessed by adrenocorticotro pin cleavage to alpha-melanocyte-stimulating hormone) inhibited further tha n in control cells expressing only the competitive substrate PE. Tests of s timulated secretion showed that both the CT peptide and the PE portion of t he chimera were stored in regulated secretory granules of transfected clone s. In both AtT-20/PC2 and Rin cells expressing the chimera, the CT peptide was substantially internally hydrolyzed, potentially accounting for the obs erved lack of inhibition. Taken together, our data suggest that overexpress ed CT peptide derived from PECT is unable to inhibit PC2 in mature secretor y granules, most likely due to its inactivation by PC2 or by other enzyme(s ).