TRH Precursor Peptide

Distribution of thyrotropin-releasing hormone (TRH) and precursor peptide (TRH-Gly) in adult rat tissues

TRH (pGlu-His-Pro-NH2) arises from the post-translational processing of a larger precursor peptide containing multiple copies of the TRH progenitor sequence, Gln-His-Pro-Gly. Concentrations of TRH and its precursor peptide (TRH-Gly) were determined in serum and a variety of tissues of the rat using specific RIA systems. TRH and TRH-Gly immunoreactivities were detectable in almost all tissues studied. TRH was distributed mainly in neural tissues, with the highest mean concentration (126 pg/mg tissue) in hypothalamus. In extra-neural tissues, mean TRH levels ranged from 0.6-4.8 pg/mg tissue; the mean serum concentration was 12.4 pg/ml. In contrast to the distribution of TRH, relatively higher mean TRH-Gly concentrations were observed in serum (76.5 pg/ml) and in extraneural tissues, including prostate (83.3 pg/mg tissue), spleen (19.0 pg/mg), adrenal (16.2 pg/mg), kidney (13.3 pg/mg), and gastrointestinal tract (6.3-19.8 pg/mg). Among brain tissues, the TRH-Gly concentration was highest in pituitary gland (13.1 pg/mg). The mean ratio of TRH-Gly/TRH concentrations was less than 1 in neural tissues and pancreas. The lowest ratio (0.04) was observed in hypothalamus, and the highest ratio (66) in prostate gland. Assuming that tissue TRH-Gly levels reflect TRH synthesis, these results suggest that 1) the processing of TRH-Gly to TRH varies among tissues, 2) TRH-Gly to TRH conversion occurs most efficiently in neural tissues, and 3) TRH-Gly to TRH conversion may be a rate-limiting step in TRH biosynthesis.

Controversies in TRH biosynthesis and strategies towards the identification of a TRH precursor

It is now clear that TRH is derived from posttranslational processing of a precursor polyprotein like other hypothalamic releasing factors and not by a soluble nonribosomal enzymatic mechanism. With an oligonucleotide probe directed against a presumptive TRH progenitor sequence, a frog skin cDNA library was screened and a clone identified coding for a peptide of 123 amino acids containing four copies of the TRH progenitor sequence (Gln-His-Pro-Gly) flanked by paired basic amino acid residues. The amphibian probe did not, however, hybridize with mammalian hypothalamus. To identify the TRH precursor in the rat hypothalamus, an antiserum was raised against the synthetic decapeptide sequence, Cys-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-Cys. It was hypothesized that the N- and C-terminal cysteines would cyclize, permitting an antibody to be generated against the midregion of the molecule and its extended counterpart sequences in nature pro-TRH. Such an antiserum was generated that recognized the intact or partially processed precursor immunohistochemically and was used to identify the prepro-TRH cDNA on screening of a rat hypothalamic lambda gt11 expression library. The rat precursor is similar to the amphibian only insofar as multiple copies of the TRH sequence are encoded in each. Thus, the resolution of the contentious question of the mode of TRH biosynthesis in the rat hypothalamus required the development of a novel antiserum, screening by immunocytochemistry and the application of modern molecular biological techniques.

Thyrotropin-releasing hormone (TRH) precursor processing. Characterization of mature TRH and non-TRH peptides synthesized by transfected mammalian cells

Prepro-thyrotropin-releasing hormone (TRH) contains five TRH progenitor sequences and at least six other potential peptides (Lechan, R. M., Wu, P., Jackson, I. M. D., Wolf, H., Cooperman, S., Mandel, G., and Goodman, R. H. (1986a) Science 231, 159-161). Previous studies using radioimmunoassays developed against discrete regions of prepro-TRH have demonstrated that several of the potential peptides are present in rat brain and pancreas (Wu, P., Lechan, R. M., and Jackson, I. M. D. (1987) Endocrinology 121, 108-115; Wu, P. and Jackson, I. M. D. (1988a) Brain Res. 456, 22-28; Wu, P., and Jackson, I. M. D. (1988b) Regul. Pept. 22, 347-360). However, the low level of peptides present in intact tissues has made isolation of the peptides difficult. CA77 cells, a medullary thyroid carcinoma cell line, also express prepro-TRH and display processing similar to that found in tissues. However, peptide content in this tumor cell line is enhanced only 3-fold compared with normal tissues (Sevarino, K. A., Wu, P., Jackson, I. M. D., Roos, B. A., Mandel, G., and Goodman, R. H. (1988) J. Biol. Chem. 263, 620-623). To achieve higher levels of expression for facilitating peptide sequencing studies and to see if alternate processing of prepro-TRH could be detected in different cell types, we transfected into 3T3, GH4, AtT20, and RIN 5F cells a cDNA vector under control of the cytomegalovirus immediate-early promoter. 3T3 and GH4 cells failed to process prepro-TRH beyond cleavage of the signal sequence. Both AtT20 and RIN 5F cells efficiently cleaved the precursor at dibasic sites to generate mature TRH and the non-TRH peptides previously identified in vivo. Peptide content was up to 30 times greater than in hypothalamic extracts and 10 times greater than in CA77 cells. Secretion experiments with transfected AtT20 cells demonstrated that both mature TRH and the non-TRH peptides were secreted via a regulated secretory pathway similar to that utilized by endogenously synthesized peptides. We isolated several of the non-TRH peptides synthesized by transfected AtT20 cells and characterized these peptides by sequential Edman degradation. These studies identified the signal sequence cleavage site and determined that the non-TRH peptides are generated by cleavage at the dibasic sites flanking the five TRH progenitor sequences. Further, we determined that processing occurs at the Arg51-Arg52 site located in the amino-terminal portion of the precursor, the only dibasic site not flanking a TRH progenitor sequence.

Molecular cloning in the marmoset shows that semenogelin is not the precursor of the TRH-like peptide pGlu-Glu-Pro amide

Two peptides with similar structures to thyrotropin-releasing hormone (TRH), pGlu-Glu-Pro amide and pGlu-Phe-Pro amide, have been identified in human seminal fluid and it has been shown that one of these peptides, pGlu-Glu-Pro amide, has the ability to increase the capacitation of sperm cells, consistent with a role in fertility. In order to select a species in which there is a high degree of expression of the genes that code for 'TRH-like' peptides, we have determined the levels of these peptides in the prostate, pancreas and thyroid of a range of species including rat, rabbit, ox, marmoset, macaque and man. The peptides were extracted from the tissues and purified before determination by RIA with TRH antibody. In addition, trypsin digestion and TRH RIA was used to investigate the presence of N-extended forms. The highest concentrations of TRH-immunoreactive peptides were found in the tissues of the marmoset, Callithrix jacchus. Ion-exchange chromatography demonstrated that marmoset thyroid contained principally authentic TRH, the pancreas contained both TRH and TRH-like peptides while the prostate contained TRH-like peptides alone. Further purification by HPLC showed that the main TRH-immunoreactive peptide in marmoset prostate was pGlu-Glu-Pro amide and a second component was identified as pGlu-Phe-Pro amide. The results indicate that the biosynthesis of these peptides could be studied to advantage in the marmoset. The biosynthetic precursors of the TRH-like peptides have not been identified. To examine whether pGlu-Glu-Pro amide might originate from semenogelin, we determined the sequence of semenogelin in the marmoset. It exhibited a high degree of homology with human semenogelin-I, but in place of the Lys-Gln-Glu-Pro sequence that might give rise to pGlu-Glu-Pro amide, marmoset semenogelin possessed the sequence Ser-Gln-Asp-Gln which cannot serve as a precursor for a TRH-like peptide. Further evidence was obtained by Northern blot analysis of a range of marmoset tissues. The results showed that semenogelin is not present in marmoset prostate. It is concluded that pGlu-Glu-Pro amide originates from a precursor distinct from semenogelin, both in marmoset and in man.

Effects of dexamethasone on TRH and TRH precursor peptide (Lys-Arg-Gln-His-Pro-Gly-Arg-Arg) levels in various rat organs

The effect of an acute dexamethasone administration on thyrotropin-releasing hormone (TRH) and TRH precursor peptide (Lys-Arg-Gln-His-Pro-Gly-Arg-Arg) (p-8) levels in various rat organs has been studied. Rats were injected i.p. with 25 micrograms of dexamethasone/100 g body weight (group A), 500 micrograms of dexamethasone/100 g body weight (group B) or saline (group C). The rats were serially decapitated after the injection. TRH and p-8 levels in the hypothalamus, cerebrum, cerebellum and brain stem, stomach and eye and plasma TRH and thyrotropin (TSH) levels were measured by individual radioimmunoassays. P-8 levels in the hypothalamus decreased significantly in both group A and B at 1-4 hours after the injection, and then returned to pretreated levels at 24 hours after the injection. TRH levels in the hypothalamus increased significantly in both group A and group B at 1-4 hours after dexamethasone injection. No changes in p-8 and TRH levels were observed in other organs. In group A, plasma TRH levels tended to decrease at 1-2 hours, then to increase at 3 hours. In group B, plasma TRH levels decreased 1-4 hours after the dexamethasone injection, then increased at 24 hours. The plasma TSH levels decreased significantly at 1-4 hours in group A and group B, returned to pretreatment levels at 24 hours in group A, and increased significantly in group B at 24 hours after dexamethasone injection.(ABSTRACT TRUNCATED AT 250 WORDS)