Neuronal cell lines expressing PC5, but not PC1 or PC2, process pro-CCK into glycine-extended CCK 12 and 22

Endocrine turner cells in culture and in vitro cleavage assays have shown t hat PC1 and PC2 are capable of processing pro-CCK into smaller, intermediat e and final, bioactive forms. Similar studies have shown that PC5 has the a bility to process a number of propeptides. Here, we use GT1-7 (mouse hypoth alamic) and SK-N-MC and SK-N-SH (human neuroblastoma) tumor cell lines to s tudy the ability of PC5 to process pro-CCK. RT-PCR and Western blot analysi s showed that the cells express PC5 mRNA and protein, but not PCI or PC2. T hey were engineered to stably overexpress CCK and cell media was analyzed f or pro-CCK expression and cleavage of the prohormone. Radioimmunoassays sho wed that pro-CCK was expressed, but no amidated CCK was detected. Lack of p roduction of amidated CCK may be due to the lack of the appropriate carboxy peptidase and amidating enzymes. Production of glycine-extended CCK process ing products was evaluated by treatment of media with carboxypeptidase B fo llowed by analysis with a CCK Gly RIA. Glycine-extended forms of the peptid e were found in the media. The predominant forms co-eluted with CCK 12 Gly and CCK 22 Gly on gel filtration chromatography. The results demonstrate th at these cell lines which express PC5 and not PCI or PC2 have the ability t o process pro-CCK into intermediate, glycine-extended forms more closely re sembling pro-CCK products in intestine than in brain. (C) 2001 Elsevier Sci ence Inc. All rights reserved.