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 peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences, which are thought to represent putative processing sites, 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 response to a combination of nerve growth factor, dexamethasone, forskolin, and Li+. Here, we investigated the effects of this combination of inducers on the posttranslational processing of the neurotensin/neuromedin N precursor in PC12 cells. Radioimmunoassays coupled to HPLC and arginine-directed tryptic cleavage of cell extracts were performed with five antisera specific for precursor sequences adjacent to basic doublets. These studies revealed that PC12 cells synthesized huge amounts (> 100 pmol/mg of protein) of unprocessed neurotensin/neuromedin N precursor in response to inducers, but largely lacked the capability to process this precursor at any of the four Lys-Arg doublets. Thus, mature neurotensin and neuromedin N represented less than 1% of the total precursor content in PC12 cells. The PC12 cell line may represent an interesting model with which one could transfect the recently cloned prohormone convertases PC1 and PC2, thereby allowing the study of the role of these enzymes in the processing of the neurotensin/neuromedin N precursor.