IDENTIFICATION OF THE REGION WITHIN THE NEUROENDOCRINE POLYPEPTIDE 7B2 RESPONSIBLE FOR THE INHIBITION OF PROHORMONE CONVERTASE PC2

The highly conserved polypeptide 7B2 and the subtilisin-related prohor mone convertases PC1/PC3 and PC2 are broadly distributed in neurons an d endocrine cells and are localized to secretory granules. We recently showed that recombinant 7B2 is in vitro a potent inhibitor of PC2 act ivity, but not of PC1/PC3, and that newly synthesized 7B2 is transient ly associated with proPC2 in vivo. In the present study, in vitro muta genesis was used to identify the region within the 7B2 sequence respon sible for the inhibition of PC2. Mutant proteins were produced in a pr okaryotic expression system and their effects on PC1/PC3 and PC2 activ ities were studied by two different in vitro enzyme assays. None of th e 7B2 mutant proteins inhibited PC1/PC3 activity. Truncation studies r evealed that a short segment within the COOH-terminal portion of 7B2 i s critical for its inhibitory effect on PC2. This segment contains a p air of basic amino acid residues which may represent a recognition mot if for PC2. Single amino acid substitutions within this Lys(171)-Lys(1 72) site strongly diminished and a double mutation abolished the inhib itory potency of 7B2. Our results indicate that, although amino acid r esidues directly surrounding this dibasic pair also contribute to PC2 inhibition, the Lys(171)-Lys(172) site is particularly important for t he ability of 7B2 to inhibit PC2.