IMMUNOLOGICAL AND BIOCHEMICAL-CHARACTERIZATION OF PROCESSING PRODUCTSFROM THE NEUROTENSIN NEUROMEDIN-N PRECURSOR IN THE RAT MEDULLARY-THYROID CARCINOMA-6-23 CELL-LINE

Neurotensin (NT) and neuromedin N (NN) are two related biologically ac tive peptides that are encoded in the same, precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting wit h an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it b y a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. F inally, an Arg-Arg pair is present within the NT moiety. The four Lys- Arg doublets represent putative processing sites in the precursor mole cule. The present study was designed to investigate the post-translati onal processing of the NT/NN precursor in the rat medullary thyroid ca rcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT u pon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmu noassay studies in combination with gel filtration, reverse-phase h.p. l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micro-method by which trypsin specifically cleaves at arg inine residues was developed. The results show that dexamethasone-trea ted rMTC 6-23 cells produced comparable amounts of NT, NN and a peptid e corresponding to a large N-terminal precursor fragment lacking the N N and NT moieties. This large fragment was purified. N-Terminal sequen cing revealed that it started at residue Ser23 of the prepro-NT/NN seq uence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing re spectively the NN and NT sequences at their C-termini were present in lower amounts. The NN-like sequence was internal to all the large frag ments. There was no evidence for the presence of peptides with the NN- like sequence at their N-termini. This shows that, in rMTC 6-23 cells, the precursor is readily processed at the three Lys-Arg doublets that flank and separate the NT and NN sequences. In contrast, the Lys-Arg doublet that precedes the NN-like sequence is not processed in this sy stem. The tools and methods developed here which allow detection of pr ecursor forms at the fentomolar level will be useful for the study of the post-translational processing of the NT/NN precursor in tissues th at express the NT/NN gene.