INTERNAL CLEAVAGE OF THE INHIBITORY 7B2 CARBOXYL-TERMINAL PEPTIDE BY PC2 - A POTENTIAL MECHANISM FOR ITS INACTIVATION

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-t erminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the met abolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and al pha TC1-6 cells. Extracts obtained from cells labeled for 6 h with [H- 3]valine were subjected to immunoprecipitation using an antibody raise d against the extreme carboxyl terminus of r7B2, and immunoprecipitate d peptides were separated by gel filtration. All three cell Lines yiel ded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an i nterior Lys-Lys site. These results were corroborated using a newly de veloped RIA against the carboxyl terminus of the CT peptide which show ed that the intact CT peptide represented only about half of the store d CT peptide immunoreactivity, with the remainder present as the 1.5-k Da peptide, Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itsel f could be responsible for this cleavage by performing in vitro experi ments. When I-125-labeled CT peptide was incubated with purified recom binant PC2, a smaller peptide was generated. Analysis of CT peptide de rivatives for their inhibitory potency revealed that CT peptide 1-18 ( containing Lys-Lys at the carboxyl terminus) represented a potent inhi bitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptida se E (CPE) in the reaction greatly diminished the inhibitory potency o f the CT peptide against PC2, in Line with the notion that the CT pept ide cleavage product is not inhibitory after the removal of terminal l ysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granu les; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.