Pro‐Thyrotropin‐Releasing Hormone Processing by Recombinant PC1

Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH ; pGlu-His-Pro-NH 2 ), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [ 3 H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH 25-152 or preproTRH 25-158 ) and a 10-kDa C-terminal peptide (preproTRH 154-255 or preproTRH 160-255 ). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (preproTRH 25-76 or preproTRH 25-82 ) and a 3.8-kDa peptide (preproTRH 83-108 ), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (preproTRH 206-255 ). The optimal pH for these cleavages was 5.5. ZnCl 2 , EDTA, EGTA, and the omission of Ca 2+ inhibited the formation of pYE 27 (preproTRH 25-50 ), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.