Biosynthesis and posttranslational processing of the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line. Effect of dexamethasone.
Neurotensin (NT) and Neuromedin N (NN) are two biologically active peptides present in one copy each in the C-terminal region of a 169-residue precursor. Four basic Lys-Arg doublets occur within the precursor and represent putative processing sites. We investigated the effects of dexamethasone on the biosynthesis and the posttranslational processing of the NT/NN precursor in the rat medullary thyroid carcinoma 6-23 cell line (rMTC 6-23). Western blot analysis and RIA coupled to HPLC and arginine-directed tryptic cleavage of precursor forms were performed with antisera specific for precursor sequences adjacent to the four basic doublets. These studies revealed that rMTC 6-23 cells synthesized the NT/NN precursor in response to dexamethasone and had the capability to process this precursor at the three Lys-Arg doublets that flank and separate NT and NN, thus yielding authentic NT, NN, and several larger products. The most N-terminal Lys-Arg doublet was not processed in this system. Dexamethasone increased in a concentration- and time-dependent manner the levels of all the NT/NN precursor-derived products. This increase did not affect the relative proportion of the different products. We also showed by Northern blot analysis that both the 1.1-kilobase and 1.5-kilobase NT/NN precursor messenger RNAs were present in the rMTC 6-23 cell line and that the time course and dose response of dexamethasone-induced messenger RNA synthesis were in good agreement with those observed for dexamethasone-induced increase in processing products. The rMTC 6-23 cell line represents a good model to elucidate the steps involved in the posttranslational processing of the NT/NN precursor.
de Nadai
F
F
Institut de Pharmacologie Moléculaire et Cellulaire, Université de Antipolis, Valbonne, France.
Rovere
C
C
Bidard
J N
JN
Laur
J
J
Martinez
J
J
Cuber
J C
JC
Kitabgi
P
P
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002277:Carcinoma / Q000378:metabolism* / Q000473:pathology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003907:Dexamethasone / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009496:Neurotensin / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010449:Peptide Mapping; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013964:Thyroid Neoplasms / Q000378:metabolism* / Q000473:pathology; D014357:Trypsin; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D011498:Protein Precursors; D012333:RNA, Messenger; C042090:neuromedin N; D009496:Neurotensin; D003907:Dexamethasone; D014357:Trypsin