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Biochemical Journal logoLink to Biochemical Journal
. 1993 Apr 1;291(Pt 1):225–233. doi: 10.1042/bj2910225

Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

J N Bidard 1, F de Nadai 1, C Rovere 1, D Moinier 1, J Laur 1, J Martinez 1, J C Cuber 1, P Kitabgi 1
PMCID: PMC1132506  PMID: 8471039

Abstract

Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with 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 by 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. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon 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 radioimmunoassay 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 micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at their C-termini were present in lower amounts. The NN-like sequence was internal to all the large fragments. 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 system.(ABSTRACT TRUNCATED AT 400 WORDS)

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Selected References

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  1. Blache P., Kervran A., Martinez J., Bataille D. Development of an oxyntomodulin/glicentin C-terminal radioimmunoassay using a "thiol-maleoyl" coupling method for preparing the immunogen. Anal Biochem. 1988 Aug 15;173(1):151–159. doi: 10.1016/0003-2697(88)90172-8. [DOI] [PubMed] [Google Scholar]
  2. Brakch N., Boussetta H., Rholam M., Cohen P. Processing endoprotease recognizes a structural feature at the cleavage site of peptide prohormones. The pro-ocytocin/neurophysin model. J Biol Chem. 1989 Sep 25;264(27):15912–15916. [PubMed] [Google Scholar]
  3. Carraway R. E., Mitra S. P. Differential processing of neurotensin/neuromedin N precursor(s) in canine brain and intestine. J Biol Chem. 1990 May 25;265(15):8627–8631. [PubMed] [Google Scholar]
  4. Carraway R. E., Mitra S. P. Purification of large neuromedin N (NMN) from canine intestine and its identification as NMN-125. Biochem Biophys Res Commun. 1991 Aug 30;179(1):301–308. doi: 10.1016/0006-291x(91)91369-n. [DOI] [PubMed] [Google Scholar]
  5. Carraway R. E., Mitra S. P. The use of radioimmunoassay to compare the tissue and subcellular distributions of neurotensin and neuromedin N in the cat. Endocrinology. 1987 May;120(5):2092–2100. doi: 10.1210/endo-120-5-2092. [DOI] [PubMed] [Google Scholar]
  6. Carraway R., Leeman S. E. Radioimmunoassay for neurotensin, a hypothalamic peptide. J Biol Chem. 1976 Nov 25;251(22):7035–7044. [PubMed] [Google Scholar]
  7. Cuber J. C., Herrmann C., Kitabgi P., Bosshard A., Bernard C., De Nadai F., Chayvialle J. A. Neuromedin-N is not released with neurotensin from rat ileum. Endocrinology. 1990 Mar;126(3):1584–1592. doi: 10.1210/endo-126-3-1584. [DOI] [PubMed] [Google Scholar]
  8. Cupo A., Pontarotti P. A., Jarry T., Delaage M. A new immunological approach to the detection and the quantitation of the Met5-enkephalin precursors in rat brain. Neuropeptides. 1984 Sep;4(5):375–387. doi: 10.1016/0143-4179(84)90113-6. [DOI] [PubMed] [Google Scholar]
  9. Cupo A., Rougon-Rapuzzi G., Delaage M. A. Immunochemical detection of vasopressin precursors: artificial processing and quantification along the hypothalamo-hypophysial axis. Eur J Biochem. 1981 Mar 16;115(1):169–174. doi: 10.1111/j.1432-1033.1981.tb06213.x. [DOI] [PubMed] [Google Scholar]
  10. Dobner P. R., Barber D. L., Villa-Komaroff L., McKiernan C. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor. Proc Natl Acad Sci U S A. 1987 May;84(10):3516–3520. doi: 10.1073/pnas.84.10.3516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Douglass J., Civelli O., Herbert E. Polyprotein gene expression: generation of diversity of neuroendocrine peptides. Annu Rev Biochem. 1984;53:665–715. doi: 10.1146/annurev.bi.53.070184.003313. [DOI] [PubMed] [Google Scholar]
  12. Granier C., van Rietschoten J., Kitabgi P., Poustis C., Freychet P. Synthesis and characterization of neurotensin analogues for structure/activity relationship studies. Acetyl-neurotensin-(8--13) is the shortest analogue with full binding and pharmacological activities. Eur J Biochem. 1982 May;124(1):117–124. doi: 10.1111/j.1432-1033.1982.tb05913.x. [DOI] [PubMed] [Google Scholar]
  13. Kislauskis E., Bullock B., McNeil S., Dobner P. R. The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences. J Biol Chem. 1988 Apr 5;263(10):4963–4968. [PubMed] [Google Scholar]
  14. Kitabgi P., De Nadai F., Cuber J. C., Dubuc I., Nouel D., Costentin J. Calcium-dependent release of neuromedin N and neurotensin from mouse hypothalamus. Neuropeptides. 1990 Feb;15(2):111–114. doi: 10.1016/0143-4179(90)90047-3. [DOI] [PubMed] [Google Scholar]
  15. Kitabgi P., Masuo Y., Nicot A., Berod A., Cuber J. C., Rostène W. Marked variations of the relative distributions of neurotensin and neuromedin N in micropunched rat brain areas suggest differential processing of their common precursor. Neurosci Lett. 1991 Mar 11;124(1):9–12. doi: 10.1016/0304-3940(91)90810-g. [DOI] [PubMed] [Google Scholar]
  16. Odum L., Rehfeld J. F. Expression and processing of procholecystokinin in a rat medullary thyroid carcinoma cell line. Biochem J. 1990 Oct 1;271(1):31–36. doi: 10.1042/bj2710031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shaw C., McKay D., Johnston C. F., Halton D. W., Fairweather I., Kitabgi P., Buchanan K. D. Differential processing of the neurotensin/neuromedin N precursor in the mouse. Peptides. 1990 Mar-Apr;11(2):227–235. doi: 10.1016/0196-9781(90)90075-g. [DOI] [PubMed] [Google Scholar]
  18. Swank R. T., Munkres K. D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem. 1971 Feb;39(2):462–477. doi: 10.1016/0003-2697(71)90436-2. [DOI] [PubMed] [Google Scholar]
  19. Zeytinoğlu F. N., Brazeau P., Mougin C. Regulation of neurotensin secretion in a mammalian C cell line: effect of dexamethasone. Regul Pept. 1983 May;6(2):147–154. doi: 10.1016/0167-0115(83)90007-1. [DOI] [PubMed] [Google Scholar]
  20. Zeytinoğlu F. N., Gagel R. F., Tashjian A. H., Jr, Hammer R. A., Leeman S. E. Characterization of neurotensin production by a line of rat medullary thyroid carcinoma cells. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3741–3745. doi: 10.1073/pnas.77.6.3741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. de Nadai F., Cuber J. C., Kitabgi P. The characterization and regional distribution of neuromedin N-like immunoreactivity in rat brain using a highly sensitive and specific radioimmunoassay. Comparison with the distribution of neurotensin. Brain Res. 1989 Oct 23;500(1-2):193–198. doi: 10.1016/0006-8993(89)90313-2. [DOI] [PubMed] [Google Scholar]

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