Processing of Prothyrotropin-releasing Hormone by the Family of Prohormone Convertases

Abstract

The post-translational processing of prothyrotropinreleasing hormone (pro-TRH 25-255 ) has been extensively studied in our laboratory, and the processing pathway to mature TRH has been elucidated. We have also demonstrated that recombinant PC1 and PC2 process partially purified pro-TRH to cryptic peptides in vitro and that pro-TRH and PC1 mRNAs are coexpressed in primary cultures of hypothalamic neurons. To further define the role of each convertase, and particularly PC1 and PC2, in pro-TRH processing, recombinant vaccinia viruses were used to coexpress the prohormone convertases PC1, PC2, PACE4, PC5-B, furin, or control dynorphin together with rat prepro-TRH in constitutively secreting LoVo cells or in the regulated endocrine GH4C1 cell line.Radioimmunoassays from LoVo-derived secreted products indicated that furin cleaves the precursor to generate both N-and C-terminal intermediates. PC1, PC2, and PACE4 only produced N-terminal intermediates, but less efficiently than furin. In GH4C1 cells, PC1, PC2, furin, PC5-B, and PACE4 produced both N-terminal and C-terminal forms. Significantly, TRH-Gly and TRH were mostly produced by PC1, PC2, and furin. Utilizing gel electrophoresis to further analyze the cleavage specificities of PC1 and PC2, we found that PC1 seems primarily responsible for cleavage to both intermediates and mature TRH, since it generated all products at significantly higher levels than PC2. The addition of 7B2 to the coinfection did not augment the ability of PC2 to cleave pro-TRH to either N-or C-terminal forms.Many neuropeptides are first synthesized as large prohormones, precursors that must be post-translationally proteolyzed to elaborate smaller bioactive peptides. This processing occurs through limited endoproteolytic cleavage at paired basic residues, either Lys-Arg or Arg-Arg, with cleavage at monobasic sites occurring less frequently (1, 2). Through such posttranslational processing, prothyrotropin-releasing hormone (pro-TRH) 1 is cleaved to mature TRH. Mature TRH is a modified 3-amino acid neuropeptide (pyroglutamate-histidine-proline-amide) that stimulates the synthesis and release of thyrotropin, prolactin, and growth hormone from the mammalian pituitary (3-6). In the rat, mature TRH is derived from a 231-amino acid prohormone, which contains five TRH progenitor sequences (Gln-His-Pro-Gly) (7,8). Each one of these sequences is flanked by dibasic residues, and another dibasic residue exists that does not flank a TRH progenitor sequence (7). Cleavage at all dibasic residues yields five copies of the TRH progenitor and seven cryptic peptides (7, 9). Mature TRH is achieved by exoproteolytic excision of the basic residues flanking the TRH progenitor by carboxypeptidase E, amidation of the carboxyl-terminal proline by peptidyl glycine ␣-amidating monooxygenase, and cyclization of the glutamine (10 -12).Much of the characterization of pro-TRH and its post-translational processing and sorting have been done using transfected AtT-20 cells, which yield high levels of prohormone expression (7). U...

Dynamic expression of Thyrotropin‐releasing hormone in the mouse definitive endoderm

“…Thyrotropin-releasing hormone (TRH) is a weakly basic tripeptide (pyroglutamyl-histidyl-proline amide) and plays a critical role in regulation of the hypothalamic-pituitary-thyroid (HPT) axis through the regulation of the synthesis and secretion of thyroid-stimulating hormone (TSH) in the anterior pituitary (reviewed in O' Leary and O'Connor, 1995;Nillni and Sevarino, 1999). The active form of TRH arises from the posttranslational cleavage of a large precursor protein by proprotein convertases PC1 and PC2 involving 3 cleavage steps at 14 cleavage sites within the precursor peptide (Sevarino et al, 1989;Schaner et al, 1997). TRH acts through two receptors, TRHR and TRHR2, resulting in the production of second messengers and the subsequent activation of several downstream kinases ultimately leading to TSH release (reviewed in O' Leary and O'Connor, 1995;Nillni and Sevarino, 1999).…”

Leptin Regulates Prothyrotropin-releasing Hormone Biosynthesis

“…Anti-pAV 37 antibody recognizes the TRH prohormone, and anti-pST 10 antibody recognizes the end product of processing prepro-TRH-(160 -169) (11). The conjugation of Texas Red (red color) to these antibodies, as well as their ability to obtain successful colocalization with other proteins, was described previously by us in experiments demonstrating the colocalization of pro-TRH with the prohormone convertase-1 (18).…”

“…Radioactive or unlabeled samples were fractionated by mobility by loading onto a discontinuous Tricine/SDS-PAGE system for separation of low molecular mass peptides (18). A stacking gel was made to 3% cross-linking (acrylamide/bisacrylamide solution), and the separating gel was made to 6% cross-linking (acrylamide/bisacrylamide solution).…”

“…Hypothalamic neurons (3 ϫ 10 5 ) from 12-day-old cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline and subjected to an immunocytochemistry protocol as described previously (11,18). CHO cells transfected with an empty vector or expressing the short form of the leptin receptor were subjected to immunostaining using an antibody against the common extracellular domain of the leptin receptor.…”

“…After 12 days in culture, primary cultures of hypothalamic neurons were stimulated with leptin for 6 h in the presence of radioactive leucine (leucine is present 21 times in the pro-TRH sequence). After treatment, radioactive peptides were acid-extracted and subjected to double immunoprecipitation with an antibody, previously characterized by us (18), directed against prepro-TRH-(116 -151) (anti-pAV 37 antibody) that recognizes the TRH precursor (26 kDa) and an intermediate form of its processing of ϳ16 kDa. The immunoprecipitates were then subjected to separation on a Tricine/SDS-PAGE system, followed by slicing and radioactive counting.…”