Detection of TRH extended peptides in rat hypothalamus using an antibody raised to pGluHisProGlyLys

An antibody was raised to the synthetic pentapeptide pGluHisProGlyLys which, in radioimmunoassay (RIA), could detect the pentapeptide at a level of 10 fmole per tube and exhibited <0.5 per cent cross reactivity with a series of related peptides. The RIA was used to demonstrate the presence of C-terminally extended forms of thyrotropin releasing hormone (TRH) in rat hypothalamus. After extraction, the endogenous peptides were resolved by gel exclusion chromatography and TRH-extended peptides were revealed by trypsin digestion to release the pentapeptide. The TRH extended peptides occurred in substantial quantity, approximately 11 pmoles/g, indicating that only partial processing of the gene duplicated prohormone takes place.     

Processing of the thyrotropin releasing hormone (TRH) precursor in Xenopus skin and bovine hypothalamus: evidence for the existence of extended forms of TRH

Acid extracts of Xenopus laevis skin were fractionated by gel filtration on Sephadex G50 ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC). Peptides related to thyrotropin releasing hormone (TRH) were identified in the eluted fractions by trypsin digestion and radioimmunoassay (RIA) using antibodies to the TRH tripeptide pGlu-His-Pro amide or to a TRH-related pentapeptide pGlu-His-Pro-Gly-Lys. In addition to the tripeptide hormone, evidence was obtained for the presence of peptides containing 10-20 amino acid residues which were extended on the NH2-terminal or COOH-terminal side of TRH. The peptides extending on the NH2-terminal side predominated and were shown to comprise 5 components present in differing concentrations, indicating that the processing sites in the TRH prohormone vary in their susceptibility to proteolysis. Evidence was also obtained for the presence of small amounts of the TRH-related pentapeptide pGlu-His-Pro-Gly-Lys. Using similar procedures it was demonstrated that TRH extended peptides were present in bovine hypothalamus. In this species the peptides extended at the NH2-terminus of TRH occurred in similar concentrations to the peptides extended at the COOH-terminus. The results show that processing of the TRH prohormone in Xenopus and ox leads to the formation of peptides intermediate in size between the prohormone and the tripeptide amide; the TRH extended peptides occur in significant quantity and in Xenopus are formed with a high degree of specificity.     

Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates pro-TRH-connecting peptides. Identification and characterization of prepro-TRH-(160-169) and prepro-TRH-(178-199) in the rat nervous system

Rat thyrotropin-releasing hormone prohormone (pro-TRH) contains five separate copies of the TRH progenitor sequence: Gln-His-Pro-Gly. Each of the five sequences is flanked by pairs of basic residues and linked together by one of several predicted connecting sequences. Two of the pro-TRH-connecting peptides, prepro-TRH-(160-169) and prepro-TRH-(178-199), were detected in extracts of rat neural tissues by radioimmunoassay using antibodies directed against the corresponding synthetic probes. Endogenous prepro-TRH-(160-169) and prepro-TRH-(178-199) were purified by gel exclusion chromatography, reverse-phase high pressure liquid chromatography, and ion-exchange chromatography. Structural identification of each peptide was achieved by chromatographic comparison with synthetic standards, immunological analysis, and tryptic mapping. Equimolar amounts of these connecting fragments were observed in hypothalamus and spinal cord. Quantification of TRH in spinal cord and hypothalamus extracts revealed the presence of 4.9-6.3 mol of TRH/mol of prepro-TRH-(178-199) and 4.4-6 mol of TRH/mol of prepro-TRH-(160-169), respectively. By using the indirect immunofluorescence technique, prepro-TRH-(178-199) immunoreactive cell bodies were found in the paraventricular nucleus of the hypothalamus, and a dense plexus of immunopositive nerve terminals was observed in the external zone of the median eminence, in a distribution similar to that described for TRH. These studies demonstrate that prepro-TRH-(160-169) and prepro-TRH-(178-199) are, together with TRH, predominant storage forms of the TRH precursor in hypothalamus and spinal cord, being present in molar ratios corresponding to those expected for a nearly complete processing of the prohormone molecule. The presence of pro-TRH-connecting peptides in various brain regions, including the median eminence, suggests that these peptides might act as neuromodulators in the central nervous system and/or neuroendocrine signals at the pituitary level. In the olfactory lobes, prepro-TRH is processed differently since a C-terminally extended form of TRH, prepro-TRH-(172-199), is found as a major end product along with lower but significant amounts of prepro-TRH-(178-199) and prepro-TRH-(160-169). The striking difference in pro-TRH processing patterns among the various tissues examined suggests differential regulating mechanisms for TRH and/or TRH-related activities.     

TRH-extended peptides in the olfactory lobe are formed by incomplete cleavage at pairs of arginine residues in the TRH prohormone

High concentrations of thyrotrophin-releasing hormone (TRH) are known to be present in the olfactory lobe, and the processing of the TRH prohormone in this region of the brain has been examined in this study. TRH-extended peptides have been detected in the rat olfactory lobe: these peptides accounted for approximately 11% of the total TRH immunoreactivity present in the tissue and contained the sequence pGlu-His-Pro-Gly-Arg exclusively at their N-termini. Extended peptides containing pGlu-His-Pro-Gly-Lys at their N-termini were not detected suggesting that incomplete cleavage occurs only at Arg-Arg residues in the TRH-prohormone. In view of the highly specific processing of the prohormone, it is likely that the TRH-extended peptides play important physiological roles.     

Effect of precipitated morphine withdrawal on post-translational processing of prothyrotropin releasing hormone (proTRH) in the ventrolateral column of the midbrain periaqueductal gray

We have demonstrated that during opiate withdrawal, preprothyrotropin releasing hormone (preproTRH) mRNA is increased in neurons of the midbrain periaqueductal gray matter (PAG) while the concentration of TRH remained unaltered, suggesting that the processing of proTRH may be different in this region of the brain. The aim of the present study was to determine which of the proTRH-derived peptides are affected by opiate withdrawal in the PAG. These changes were compared to other TRH-containing areas such as the hypothalamic paraventricular nucleus (PVN), median eminence (ME) and the lateral hypothalamus (LH). Control and morphine-treated rats 24 h following naltrexone-precipitated withdrawal were decapitated and the brain microdissected. Pooled samples from each animal group were acid extracted, and peptides were electrophoretically separated then analyzed by specific radioimmunoassay. Opiate withdrawal caused a significant change in the level of some post-translational processing products derived from the TRH precursor. In the PAG, opiate withdrawal resulted in an accumulation of the intervening preproTRH(83-106) peptide from the N-terminal side of the prohormone, while the levels of the C-terminal preproTRH(208-285) peptide were reduced, with no change in preproTRH(25-50) or TRH, itself, as compared to control animals. Immunohistochemical analysis also showed significant increases in cellular preproTRH(83-106) peptide immunolabeling in the PAG. Opiate withdrawal in the lateral hypothalamus, unlike from the PAG, was accompanied by an increase in the concentration of TRH. In addition, western blot analysis showed that during opiate withdrawal, the mature form of the prohormone convertase 2 (PC2) increased only in PAG as compared with their respective controls. Thus, these results demonstrate a region-specific regulation of TRH prohormone processing in the brain, which may engage PC2, further suggesting a role for specific proTRH-derived peptides in the manifestations of opiate withdrawal.     

Nicotine-induced alterations in brain regional concentrations of native and cryptic Met- and Leu-enkephalin

The distribution of cryptic forms (larger enkephalin-containing peptides) in neostriatum, hypothalamus, spinal cord T₃-L₁ and neurointermediate lobe of pituitary were determined by radioimmunoassay. Optimal conditions for enzymic hydrolysis of the cryptic enkephalins by trypsin and carboxypeptidase B were established. The proportion of total Met- and Leu-enkephalin represented by native pentapeptide varied markedly among these central nervous system regions. Also, the distributions of native and cryptic Met-enkephalin were distinct from that of Leu-enkephalin. Chromatographic separation by HPLC of immunoreactive Met-enkephalin peptides revealed only two peaks corresponding to Met-enkephalin and Met-enkephalin sulfoxide in rather equal amounts. Hydrolysis of cryptic Met-enkephalin also produced only two HPLC-separable peaks of immunoreactive Met-enkephalin, again corresponding to Met-enkephalin and Met-enkephalin sulfoxide. Bioactivity of cryptic striatal Met-enkephalin after hydrolysis was demonstrated by antinociception and catalepsy in rats following its intracerebroven-tricular injection. Repeated short-term administration of nicotine, 0.1 mg/kg IP six times at 30 min intervals, produced significant increases in native and cryptic Met-enkephalin in striatum, consistent with an increase in neuronal release of Met-enkephalin together with increases in synthesis and processing of proenkephalin A in this brain region. This regimen of nicotine also decreased levels of native Met-enkephalin and of both native and cryptic Leu-enkephalin in neurointermediate lobe, consistent with nicotine-induced release of both proenkephalin A- and prodynorphin-derived peptides from neurointermediate lobe.     

Identification, characterization and localization of thyrotropin-releasing hormone precursor peptides in perinatal rat pancreas

The sequence of rat hypothalamic prepro TRH, deduced from its complementary DNA, contains five TRH progenitor sequences and six cryptic sequences separated by paired basic amino acid residues. We have utilised antisera against two synthetic peptides corresponding to sequences within proTRH, [Tyr53] preproTRH (53-74), part of the amino terminal leader sequence of proTRH and [Cys 74,83] preproTRH-(75-82), representing a TRH progenitor sequence flanked by cysteine residues (pCC10) in radioimmunoassays (RIA) to identify and chromatographically characterize proTRH derived peptides in extracts of rat perinatal pancreas and to localize these peptides immunohistochemically. Two forms of immunoreactive pYT22 (ipYT22) were observed, similar in size to ipYT22 seen in extracts of adult rat brain. By RIA immunoreactive pCC10 was detectable in neonatal but not fetal pancreas. However, immunohistochemical double staining of both fetal and neonatal rat pancreas colocalized both ipYT22 and ipCC10 with immunoreactive insulin in the B-cell of the developing Islets of Langerhans. These findings indicate that the B-cell of the perinatal pancreas synthesizes TRH from a prohormone encoded by a mRNA similar to that present in adult rat hypothalamus.     

The mode of conversion of proparathormone to parathormone by a particulate converting enzymic activity of the parathyroid gland

The cleavage products from the conversion of proparathormone to parathormone by a bovine and porcine parathyroid microsomal converting activity have been analyzed. In the conversion reaction, the first 6 amino acid residues of the prohormone (Lys-Ser-Val-Lys-Lys-Arg-) are released as an intact hexapeptide. This is rapidly converted to a pentapeptide by removal of the NH2-terminal lysine and then to a tetrapeptide by removal of the COOH-terminal arginine. In order to test for the presence of a postulated COOH-terminal extension of the parathormone sequence in proparathormone, mixtures of 14C-proparathormone and 3H-parathormone were subjected to digestion by trypsin or Staphylococcus aureus protease. The resulting radioactive peptides from the hormone and its precursor were compared. There was no evidence that any fragments different from those from the hormone were released from the prohormone except those accounted for by the NH2-terminal hexapeptide adduct on proparathormone. Thus, the conversion of the prohormone to the hormone catalyzed by the microsomal membrane activity requires only the cleavage of this hexapeptide.     

Deficiencies in pro-thyrotropin-releasing hormone processing and abnormalities in thermoregulation in Cpefat/fat mice

Cpe(fat/fat) mice are obese, diabetic, and infertile. They have a mutation in carboxypeptidase E (CPE), an enzyme that converts prohormone intermediates to bioactive peptides. The Cpe(fat) mutation leads to rapid degradation of the enzyme. To test whether pro-thyrotropin-releasing hormone (TRH) conversion to TRH involves CPE, processing was examined in the Cpe(fat/fat) mouse. Hypothalamic TRH is depressed by at least 75% compared with wild-type controls. Concentrations of pro-TRH forms are increased in homozygotes. TRH-[Gly(4)-Lys(5)-Arg(6)] and TRH-[Gly(4)-Lys(5)] represent approximately 45% of the total TRH-like immunoreactivity in Cpe(fat/fat) mice; they constitute approximately 1% in controls. Levels of TRH-[Gly(4)] were depressed in homozygotes. Because the hypothalamus contains some TRH, another carboxypeptidase must be responsible for processing. Immunocytochemical studies indicate that TRH neurons contain CPE- and carboxypeptidase D-like immunoreactivity. Recombinant CPE or carboxypeptidase D can convert synthetic TRH-[Gly(4)-Lys(5)] and TRH-[Gly(4)-Lys(5)-Arg(6)] to TRH-[Gly(4)]. When Cpe(fat/fat) mice are exposed to cold, they cannot maintain their body temperatures, and this loss is associated with hypothalamic TRH depletion and reduction in thyroid hormone. These findings demonstrate that the Cpe(fat) mutation can affect not only carboxypeptidase activity but also endoproteolysis. Because Cpe(fat/fat) mice cannot sustain a cold challenge, and because alterations in the hypothalamic-pituitary-thyroid axis can affect metabolism, deficits in pro-TRH processing may contribute to the obese and diabetic phenotype in these mice.     

Tryptic digestion of peptides corresponding to modified fragments of human growth hormone-releasing hormone

The objective of this study was to examine the degradation of short peptides corresponding to modified fragments of human growth hormone-releasing hormone by trypsin. Six analogues of pentapeptide 9-13 of human growth hormone-releasing hormone containing homoarginine, ornithine, glutamic acid, glycine, leucine or phenylalanine residue in position 11, two analogues of hexapeptide 8-13 of human growth hormone-releasing hormone and two analogues of heptapeptide 7-13 of human growth hormone-releasing hormone containing homoarginine or glycine residue in position 11 were obtained. The peptides were subjected to digestion by trypsin and the course of reaction was monitored using HPLC. It was found that the rate of hydrolysis of the Lys(12)-Val(13) peptide bond depends on the amino-acid residue preceding Lys(12). The extension of the peptide chain towards the N-terminus by introduction of consecutive amino-acid residues corresponding to the human growth hormone-releasing hormone sequence accelerates the hydrolysis process. These results may be of assistance in designing new analogues of human growth hormone-releasing hormone, more resistant to the activity of proteolytic enzymes.     

The distribution of galanin message-associated peptide-like immunoreactivity in the pig.

Using a radioimmunoassay (RIA) developed to the N-terminal part of the predicted sequence of porcine galanin message-associated peptide (GMAP), we have confirmed the existence of GMAP-like immunoreactivity (-LI) in normal porcine tissues. GMAP-LI was found to parallel the distribution of galanin-immunoreactivity (-IR), although consistently the concentrations detected were, on a molar ratio, significantly less than those measured for galanin throughout the gastrointestinal tract, brain, spinal cord, adrenal and pituitary gland. As cleavage of the prohormone would be expected to produce galanin and GMAP on an equimolar basis, it is possible that the endogenous, intact GMAP peptide does not fully cross-react with the antibody raised to the N-terminal GMAP sequence. Gel chromatography of tissue extracts revealed a single molecular form of galanin-IR in the gut and four distinct molecular forms in the adrenal gland. GMAP-LI eluted as a single immunoreactive component in the gut, and in the adrenal gland there were two major molecular forms, one of which was apparently also detected by the galanin assay, and a small amount of N-terminal fragment. This molecular heterogeneity seems likely to be a result of the various possible prohormone cleavage products and/or posttranslational processing modifications. Further analysis of the galanin gene products needs to be undertaken in order to confirm this.     

Isolation and identification of two neutral thyrotropin releasing hormone-like peptides, pyroglutamylphenylalanineproline amide and pyroglutamylglutamineproline amide, from human seminal fluid.

Two tripeptide amides with stuctures similar to thyrotropin releasing hormone were isolated from human seminal fluid and their amino acid sequences determined. The peptides were purified by gel exclusion from Sephadex G50 and were detected by radioimmunoassay with thyrotropin releasing hormone antibody; in addition, N-terminally extended forms were demonstrated by radioimmunoassay after trypsin digestion. Further purification of the tripeptides was by chromatography on SP-Sephadex C25 and by high performance liquid chromatography on C18 Microbondapak using an HCl/acetonitrile gradient. After exclusion from mini-columns of SP-Sephadex C25 and DEAE-Sephadex A25, two neutral peptides were obtained in homogeneous form by high performance liquid chromatography with an HCl/methanol gradient. Amino acid analysis gave the following compositions: Glu, 0.74, Phe, 1.0, Pro, 1.0; and Glu, 1.72, Pro, 1.0. Both peptides possessed a blocked N terminus, but after opening the pyroglutamyl ring the sequences Glu-Phe-Pro and Glu-Glx-Pro were demonstrated. The chromatographic properties of the endogenous peptides were identical to the properties of the corresponding synthetic peptides. The structure of pGlu-Phe-Pro (where p-indicates pyro-) amide was confirmed by fast atom bombardment mass spectrometry. The presence in human semen of three structurally related peptides, pGlu-Phe-Pro amide, pGlu-Gln-Pro amide, and the previously reported pGlu-Glu-Pro amide (Cockle, S. M., Aitken, A., Beg, F., and Smyth, D. G. (1989) J. Biol. Chem. 264, 7788-7791), suggests that this series of peptides may have evolved to fulfil complementary biological roles.     

ProSAAS-Derived Peptides Are Differentially Processed and Sorted in Mouse Brain and AtT-20 Cells

ProSAAS is the precursor for some of the most abundant peptides found in mouse brain and other tissues, including peptides named SAAS, PEN, and LEN. Both SAAS and LEN are found in big and little forms due to differential processing. Initial processing of proSAAS is mediated by furin (and/or furin-like enzymes) and carboxypeptidase D, while the smaller forms are generated by secretory granule prohormone convertases and carboxypeptidase E. In mouse hypothalamus, PEN and big LEN colocalize with neuropeptide Y. In the present study, little LEN and SAAS were detected in mouse hypothalamus but not in cell bodies of neuropeptide Y-expressing neurons. PEN and big LEN show substantial colocalization in hypothalamus, but big LEN and little LEN do not. An antiserum to SAAS that detects both big and little forms of this peptide did not show substantial colocalization with PEN or big LEN. To further study this, the AtT-20 cells mouse pituitary corticotrophic cell line was transfected with rat proSAAS and the distribution of peptides examined. As found in mouse hypothalamus, only some of the proSAAS-derived peptides colocalized with each other in AtT-20 cells. The two sites within proSAAS that are known to be efficiently cleaved by furin were altered by site-directed mutagenesis to convert the P4 Arg into Lys; this change converts the sequences from furin consensus sites into prohormone convertase consensus sites. Upon expression of the mutated form of proSAAS in AtT-20 cells, there was significantly more colocalization of proSAAS-derived peptides PEN and SAAS. Taken together, these results indicate that proSAAS is initially cleaved in the Golgi or trans-Golgi network by furin and/or furin-like enzymes and the resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides.     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Regional distribution of in vitro release of thyrotropin releasing hormone in rat brain

To increase our knowledge of the TRH functions in brain and the processes of TRH compartmentalization and release, we studied the in vitro release of endogenous TRH in different brain areas. We also determined the correlation between TRH levels and release under both basal and stimulated conditions. TRH concentration was measured in tissues and media by specific radioimmunoassay. TRH-like material detected in olfactory bulb and hypothalamic incubates (basal or K+ stimulated) were shown to be chromatographically identical to synthetic TRH. Different brain regions showed high variability in the basal release of TRH (1-20% of tissue content). This suggests the existence of different pools. The response to depolarizing stimulus (56 mM K+) was significant only in the following regions: median eminence, total hypothalamus, preoptic area, nucleus accumbens-lateral septum, amygdala, mesencephalon, medulla oblongata and the cervical region of the spinal cord. These regions have been shown to contain a high number of receptors, a high concentration of TRH nerve endings and are susceptible to TRH effects. These results support the hypothesis that TRH functions as neuromodulator in these areas.     

Prothyrotropin-releasing hormone targets its processing products to different vesicles of the secretory pathway

Prothyrotropin-releasing hormone (pro-TRH) is initially cleaved by the prohormone convertase-1/3 (PC1/3) in the trans-Golgi network generating N- and C-terminal intermediate forms that are then packed into secretory vesicles. However, it is not known whether these peptides are differentially sorted within the secretory pathway. This is of key importance because the processing products of several prohormones fulfill different biological functions. Using AtT20 cells stably transfected with prepro-TRH cDNA, we found that two specific N- and C-terminal peptides were located in different vesicles. Furthermore, the C-terminal pro-TRH-derived peptides were more efficiently released in response to KCl and norepinephrine, a natural secretagogue of TRH. Similar sorting and secretion of N- and C-terminal peptides occurs in vivo. When we blocked the initial proteolytic processing by a mutagenic approach, the differential sorting and secretion of these peptides were prevented. In summary, our data show that pro-TRH-derived peptides are differentially sorted within the secretory pathway and that the initial cleavage in the trans-Golgi network is key to this process. This could be a common mechanism used by neuroendocrine cells to regulate independently the secretion of different bioactive peptides derived from the same gene product.     

Effect of immobilization stress on neuropeptides and their receptors in rat central nervous system

The effect of immobilization stress (IM-stress) on the concentration and the receptor binding of substance P (SP), methionine-enkephalin (ME) and thyrotropin-releasing hormone (TRH) was determined in eight brain regions and the spinal cord. The concentration of SP was decreased in the septum, striatum and hippocampus, and SP receptor binding was decreased in the septum, amygdala + pyriform cortex and hypothalamus. Scatchard analysis indicated that the decrease in the SP binding is mainly due to the decrease in the number of receptors. The concentration of ME was not changed, but ME receptor binding was decreased in the septum. The concentration of TRH was decreased in the frontal cortex, septum, amygdala + pyriform cortex and pons + medulla oblongata, but increased in the spinal cord. TRH receptor binding was decreased in the septum, amygdala + pyriform cortex and hypothalamus. Scatchard analysis indicated that the decrease in TRH binding is due to the decrease in the number of receptors. These results show that IM-stress affects the neuropeptide receptor as well as neuropeptide concentration, and that the septum is a very important region under IM-stress.     

Evidence for thyrotropin-releasing hormone (TRH) biosynthesis in rat prostate

TRH occurs in very high concentration in rat prostate. A species specific protein with repetitive -Gln-His-Pro-Gly- sequences, which are flanked on the N- and C-terminus by paired basic residues, has been shown to be the source of TRH in frog skin and rat hypothalamus. Following cleavage by trypsin-like enzymes, the peptide fragments with N-terminal Gln spontaneously cyclize to pGlu while Gly within the C-terminally extended peptides serves as the -NH2 donor for the alpha-amidation of the proline residue. Because this last step in the biosynthesis of TRH is rate limiting for pGlu-His-Pro-Gly, we have combined several chromatographic and radioimmunoassay techniques to identify this TRH precursor in rat prostate.     

Biosynthesis of thyrotropin-releasing hormone by a rat medullary thyroid carcinoma cell line

Prepro-thyrotropin-releasing hormone (TRH) messenger RNA was detected in the rat medullary thyroid carcinoma cell line CA77. The RNA of 1.6 kilobases comigrated with that found in rat hypothalamus. Using three radioimmunoassays specific for pro-TRH-derived peptides, we demonstrated that CA77 cells synthesize high levels of immunoreactive TRH and all of the other pro-TRH-derived peptides identified in hypothalamic tissue. The relative levels of the pro-TRH-derived peptides also indicate that CA77 cells process the TRH precursor in a manner similar to hypothalamic tissue. CA77 cells provide a promising model system for further studies of prepro-TRH gene regulation and post-translational maturation.