PC12 CELLS CAN BE INDUCED TO PRODUCE, BUT DO NOT PROCESS, THE NEUROTENSIN NEUROMEDIN N PRECURSOR

Neurotensin and neuromedin N are two biologically active, related pept ides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-te rminal region one copy each of neurotensin and neuromedin N. Four Lys- Arg sequences, which are thought to represent putative processing site s, occur in the precursor molecule. Studies by others have shown that rat pheochromocytoma PC12 cells produced neurotensin and dramatically increased their neurotensin/neuromedin N precursor mRNA content in res ponse to a combination of nerve growth factor, dexamethasone, forskoli n, and Li+. Here, we investigated the effects of this combination of i nducers on the posttranslational processing of the neurotensin/neurome din N precursor in PC12 cells. Radioimmunoassays coupled to HPLC and a rginine-directed tryptic cleavage of cell extracts were performed with five antisera specific for precursor sequences adjacent to basic doub lets. These studies revealed that PC12 cells synthesized huge amounts (> 100 pmol/mg of protein) of unprocessed neurotensin/neuromedin N pre cursor in response to inducers, but largely lacked the capability to p rocess this precursor at any of the four Lys-Arg doublets. Thus, matur e neurotensin and neuromedin N represented less than 1% of the total p recursor content in PC12 cells. The PC12 cell line may represent an in teresting model with which one could transfect the recently cloned pro hormone convertases PC1 and PC2, thereby allowing the study of the rol e of these enzymes in the processing of the neurotensin/neuromedin N p recursor.