Processing of rat preprocortistatin in mouse AtT-20 cells.
Preprocortistatin (PPCST) has been recently identified as a novel somatostatin (SST)-related gene expressed only in brain. PPCST shares 11 of 14 residues with SST-14 at its C-terminal segment, where it features Lys-Lys and Lys-Arg basic sites for cleavage to putative cortistatin (CST)-14 and CST-29 peptides, respectively. Although synthetic replicates of the two putative CST peptides interact with SST receptors, they also display novel effects suggesting independent biological functions. Nothing is currently known about the naturally occurring mature cleavage products of PPCST posttranslational processing. Here we have cloned rat PPCST cDNA, stably expressed it in AtT-20 pituitary cells, and characterized the cellular and releasable products 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 +/- 33 pg per plate with forskolin stimulation (p < 0.05). HPLC chromatograms of cell extracts revealed three peaks corresponding to CST-14, CST-29, and unprocessed 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-stimulated 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 equally, CST-14 is released preferentially via the regulated secretory pathway.
Puebla
L
L
Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada.
Mouchantaf
R
R
Sasi
R
R
Khare
S
S
Bennett
H P
HP
James
S
S
Patel
Y C
YC
D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005576:Colforsin / Q000494:pharmacology; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D011863:Radioimmunoassay; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013004:Somatostatin / Q000096:biosynthesis; D014162:Transfection
D018076:DNA, Complementary; D011498:Protein Precursors; C107518:preprocortistatin; D005576:Colforsin; D013004:Somatostatin