Processing of rat preprocortistatin in mouse AtT-20 cells

Preprocortistatin (PPCST) has been recently identified as a novel somatosta tin (SST)-related gene expressed only in brain. PPCST shares 11 of 14 resid ues with SST-14 at its C-terminal segment, where it features Lys-Lys and Ly s-Arg basic sites for cleavage to putative cortistatin (CST)-14 and CST-29 peptides, respectively. Although synthetic replicates of the two putative C ST peptides interact with SST receptors, they also display novel effects su ggesting independent biological functions. Nothing is currently known about the naturally occurring mature cleavage products of PPCST posttranslationa l processing. Here we have cloned rat PPCST cDNA, stably expressed it in At T-20 pituitary cells, and characterized the cellular and releasable product s of PPCST processing by HPLC and radioimmunoassay using a SST-14 antibody that recognizes synthetic CST-14 and CST-29. Transfected cells released 120 +/- 21 pg of total CST-LI per plate basally, with an increase to 204 +/- 3 3 pg per plate with forskolin stimulation (p < 0.05). HPLC chromatograms of cell extracts revealed three peaks corresponding to CST-14, CST-29, and un processed PPCST (ratio, 41.55:4.5). CST was released preferentially as CST- 14 (63-70%) compared with CST-29 (30-37%) under basal and forskolin-stimula ted conditions. These studies demonstrate efficient processing of PPCST to both CST-14 and CST-29 through putative cleavage at both C-terminal dibasic sites of PPCST. Although the two peptides are synthesized approximately eq ually, CST-14 is released preferentially via the regulated secretory pathwa y.