Identification of the thyrotropin-releasing hormone-prohormone and its posttranslational processing in a transfected AtT20 tumoral cell line.

By using an AtT20 cell line transfected with complementary DNA for preproTRH, we have identified the proTRH polyeptide precursor [26 kilodaltons (kDa)] and shown that this molecule gives rise to the proTRH derived sequences as determined by pulse-chase and trypsinization studies. The predicted proTRH precursor composed of 231 amino acids with 5 copies of a TRH progenitor sequence (Gln-His-Pro-Gly) and 7 other cryptic peptides was demonstrated by: 1) Western Blot analysis of an AtT20 cell extract with anti-pCC10 antibodies (an antibody that recognizes the intact prohormone as well as some intermediate products of processing); 2) Immunoprecipitation of the radiolabelled 26 kDa protein with anti-pCC10 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis; 3) Gel filtration chromatography of the radiolabeled 26 kDa extracted from SDS-PAGE. 4) RIA with anti-pCC10 antiserum against peptides extracted from adult rat hypothalamus and olfactory lobe after SDS-PAGE. 5) Trypsinization of the proTRH precursor which generated the proTRH cryptic peptides preproTRH25-50 (pYE27) and preproTRH53-74 (pFT22). These moieties were also produced during trypsinization of intermediate products of processing. By means of pulse-chase studies, the 26 kDa polypeptide was shown to be the biosynthetic precursor to all the proTRH derived cryptic peptides. Cleavage at two positions in the center of the molecule (Lys107-Arg108 and Lys152-Arg153) generated two moieties of 16.5 and 15 kDa. The 15 kDa N-terminal fragment is later cleaved to a 6 kDa peptide that includes the proTRH derived peptides, pYE27, pFT22, and pEH24. The carboxy-terminal 16.5 kDa fragment of the prohormone is processed to a 9.6 kDa fragment which contains the proTRH derived peptide pST10 (preproTRH160-169) and a fragment of 5.4 kDa that may be the C-terminal peptide preproTRH208-255 recognized by antisera pAC12 and pYE17. In further processing, the 9.6 kDa molecule is cleaved to produce a 5.4 kDa peptide from either sequences 115-169 or 160-199.