TRH Binding Research Articles

Molecular cloning, molecular evolution and gene expression of cDNAs encoding thyrotropin-releasing hormone receptor subtypes in a teleost, the sockeye salmon ( Oncorhynchus nerka)

Molecular cloning of thyrotropin-releasing hormone receptors (TRHR) was performed in a teleost, the sockeye salmon ( Oncorhynchus nerka). Four different TRHR cDNAs were cloned and named TRHR1, TRHR2a, TRHR2b and TRHR3 based on their similarity to known TRHR subtypes in vertebrates. Important residues for TRH binding were conserved in deduced amino acid sequences of the three TRHR subtypes except for the TRHR2b. Seven transmembrane domains were predicted for TRHR1, TRHR2a and TRHR3 proteins but only five for TRHR2b which appears to be truncated. In silico database analysis identified putative TRHR sequences including invertebrate TRHR and reptilian, avian and mammalian TRHR3. Phylogenetic analyses predicted the molecular evolution of TRHR in vertebrates: from the common ancestral TRHR (i.e. invertebrate TRHR), the TRHR2 subtype diverged first and then TRHR1 and TRHR3 diverged. Reverse transcription-polymerase chain reaction analyses revealed TRHR1 transcripts in the brain (hypothalamus), retina, pituitary gland and large intestine; TRHR2a in the brain (telencephalon and hypothalamus); and TRHR3 in the brain (olfactory bulbs) and retina.

A Developmentally Regulated Two-Step Process Generates a Noncentrosomal Microtubule Network in Drosophila Tracheal Cells

Microtubules (MTs) are essential for many cell features, such as polarity, motility, shape, and vesicle trafficking. Therefore, in a multicellular organism, their organization differs between cell types and during development; however, the control of this process remains elusive. Here, we show that during Drosophila tracheal morphogenesis, MT reorganization is coupled to relocalization of the microtubule organizing centers (MTOC) components from the centrosome to the apical cell domain from where MTs then grow. We reveal that this process is controlled by the trachealess patterning gene in a two-step mechanism. MTOC components are first released from the centrosome by the activity of the MT-severing protein Spastin, and then anchored apically through the transmembrane protein Piopio. We further show that these changes are essential for tracheal development, thus stressing the functional relevance of MT reorganization for morphogenesis.

Valproate modulates TRH receptor, TRH and TRH-like peptide levels in rat brain

We have tested our hypothesis that alterations in the levels of TRH receptors, and the synthesis and release of tripeptide TRH, and other neurotropic TRH-like peptides mediate some of the mood stabilizing effects of valproate (Valp). We have directly compared the effect of 1 week of feeding two major mood stabilizers, Valp and lithium chloride (LiCl) on TRH binding in limbic and extra-limbic regions of male WKY rats. Valp increased TRH receptor levels in nucleus accumbens and frontal cortex. Li increased TRH receptor binding in amygdala, posterior cortex and cerebellum. The acute, chronic and withdrawal effects of Valp on brain levels of TRH (pGlu-His-Pro-NH 2, His-TRH) and five other TRH-like peptides, Glu-TRH, Val-TRH, Tyr-TRH, Leu-TRH and Phe-TRH were measured by combined HPLC and RIA. Acute treatment increased TRH and TRH-like peptide levels within most brain regions, most strikingly in pyriform cortex. The fold increases (in parentheses) were: Val-TRH (58), Phe-TRH (54), Tyr-TRH (25), TRH (9), Glu-TRH (4) and Leu-TRH (3). We conclude that the mood stabilizing effects of Valp may be due, at least in part, to its ability to alter TRH and TRH-like peptide, and TRH receptor levels in the limbic system and other brain regions implicated in mood regulation and behavior.

Valproate modulates TRH receptor, TRH and TRH-like peptide levels in rat brain

We have tested our hypothesis that alterations in the levels of TRH receptors, and the synthesis and release of tripeptide TRH, and other neurotropic TRH-like peptides mediate some of the mood stabilizing effects of valproate (Valp). We have directly compared the effect of 1 week of feeding two major mood stabilizers, Valp and lithium chloride (LiCl) on TRH binding in limbic and extra-limbic regions of male WKY rats. Valp increased TRH receptor levels in nucleus accumbens and frontal cortex. Li increased TRH receptor binding in amygdala, posterior cortex and cerebellum. The acute, chronic and withdrawal effects of Valp on brain levels of TRH (pGlu-His-Pro-NH 2 , His-TRH) and five other TRH-like peptides, Glu-TRH, Val-TRH, Tyr-TRH, Leu-TRH and Phe-TRH were measured by combined HPLC and RIA. Acute treatment increased TRH and TRH-like peptide levels within most brain regions, most strikingly in pyriform cortex. The fold increases (in parentheses) were: Val-TRH (58), Phe-TRH (54), Tyr-TRH (25), TRH (9), Glu-TRH (4) and Leu-TRH (3). We conclude that the mood stabilizing effects of Valp may be due, at least in part, to its ability to alter TRH and TRH-like peptide, and TRH receptor levels in the limbic system and other brain regions implicated in mood regulation and behavior.

Characterization and functional expression of cDNAs encoding thyrotropin-releasing hormone receptor from Xenopus laevis.

Thyrotropin-releasing hormone receptor (TRHR) has already been cloned in mammals wherethyrotropin-releasing hormone (TRH) is known to act as a powerful stimulator of thyroid-stimulating hormone (TSH) secretion. The TRH receptor of amphibians has not yet been characterized, although TRH is specifically important in the adaptation of skin color to environmental changes via the secretion of alpha-melanocyte-stimulating hormone (alpha-MSH). Using a dege-nerate PCR strategy, we report on the isolation of three distinct cDNA species encoding TRHR from the brain of Xenopus laevis. We have designated these as xTRHR1, xTRHR2 and xTRHR3. Analysis of the predicted amino acid sequences revealed that the three Xenopus TRHRs are only 54-62% identical and contain all the highly conserved residues constituting the TRH binding pocket. Amino acid sequences and phylogenetic analysis revealed that xTRHR1 is a member of TRHR subfamily 1 and xTRHR2 belongs to subfamily 2, while xTRHR3 is a new TRHR subtype awaiting discovery in other animal species. The three Xeno-pus TRHRs have distinct patterns of expression. xTRHR3 was abundant in the brain and much scarcer in the peripheral tissues, whereas xTRHR1 was found mainly in the stomach and xTRHR2 in the heart. The Xenopus TRHR subtype 1 was found specifically in the intestine, lung and urinary bladder. These observations suggest that the three xTRHRs each have specific functions that remain to be elucidated. Expression in Xenopus oocytes and HEK-293 cells indicates that the three Xenopus TRHRs are fully functional and are coupled to the inositol phosphate/calcium pathway. Interestingly, activation of xTRHR3 required larger concentrations of TRH compared with the other two receptors, suggesting marked differences in receptor binding, coupling or regulation.

The coumarin osthol attenuates the binding of thyrotropin-releasing hormone in rat pituitary GH4C1 cells.

The influence of two plant coumarins, osthol and xanthotoxin, on intracellular Ca2+ ([Ca2+]i) transients evoked by TRH were studied in clonal rat pituitary GH4C1 cells. Osthol, but not xanthotoxin, decreased the TRH-induced transient increase in [Ca2+]i in Fluo-3 loaded cells incubated in Ca(2+)-free buffer. Binding experiments with [3H]TRH showed that osthol decreased the binding of TRH to its receptor, whereas the affinity of the receptor for TRH increased. This resulted in a decreased TRH-evoked production of IP3 in cells treated with osthol, and a decreased mobilization of sequestered calcium. Osthol did not inhibit the release of calcium evoked by exogenous IP3 in permeabilized cells. Furthermore, osthol decreased the uptake of 45Ca2+ in response to high K+. Xanthotoxin had no effects in these experiments. The results show that osthol modulates TRH-evoked responses by interacting with the TRH receptor.

Juxtamembrane regions in the third intracellular loop of the thyrotropin-releasing hormone receptor type 1 are important for coupling to Gq.

Juxtamembrane residues in the putative third intracellular (I3) loops of a number of G protein-coupled receptors (GPCRs) have been shown to be important for coupling to G proteins. According to standard hydropathy analysis, the I3 loop of the mouse TRH receptor type 1 (mTRH-R1) is composed of 51 amino acids from position-213 to position-263. We constructed deletion and site-specific I3 loop TRH-R mutants and studied their binding and TRH-stimulated signaling activities. As expected, the effects of these mutations on TRH binding were small (less than 5-fold decreases in affinity). No effect on TRH-stimulated signaling activity was found in a mutant receptor in which the I3 loop was shortened to 16 amino acids by deleting residues from Asp-226 to Ser-260. In contrast, mutants with deletions from Asp-222 to Ser-260 or from Asp-226 to Gln-263 exhibited reduced TRH-stimulated signaling. In the region near transmembrane helix 6, single site-specific substitution of either Arg-261 or Lys-262 by neutral glutamine had little effect on signaling, but mutant TRH-Rs that were substituted by glutamine at both basic residues exhibited reduced TRH-stimulated activity. The reduced signaling activity of this doubly substituted mutant was reversed by over expressing the a subunit of Gq. These data demonstrate that the juxtamembrane regions in the TRH-R I3 loop are important for coupling to Gq.

Diversity of Thyrotropin-Releasing Hormone Receptors in the Pituitary and Discrete Brain Regions of Rats

In order to analyze the receptor properties of central nervous system (CNS)-stimulant thyrotropin-releasing hormone (L-pyroglutamyl-L-histidyl-L-prolinamide, TRH), we evaluated the binding of TRH and its analog taltirelin hydrate ((—)-N-[(S)-hexahydro-1-methyl-2,6-dioxo-4-pyrimidinylcar-bonyl]-L-histidyl-L-prolinamide tetrahydrate; taltirelin, TA-0910) in rat anterior pituitary and several brain regions. There was a specific binding of [3H]methyl TRH (MeTRH) in the anterior pituitary, hypothalamus, brain stem, cerebral cortex and cerebellum with Kd values of 1.0–1.6 nM. The inhibition of [3H]MeTRH binding by TRH and taltirelin was monophasic in the anterior pituitary, hypothalamus and brain stem with Ki values of 6.3 – 8.0 nM and 145.5 – 170.4 nM for TRH and taltirelin, respectively. In contrast, the biphasic inhibition was revealed in the cerebral cortex and cerebellum. The IQ values for TRH and taltirelin were 4.1 – 4.3 nM and 67.8 – 73.4 nM for the high affinity binding site and 3.6–4.2 μM and 82.3–197.5 μM for the low affinity binding site, respectively. Addition of 100 μM GTP or its analog 5'-guanylylimidodiphos-phate (Gpp[NH]p) affected neither the biphasic inhibition by TRH nor that by taltirelin. Thus the results suggest the presence of distinct high and low affinity TRH receptors in the CNS in contrast to the pituitary.

Real Time Visualization of Agonist-mediated Redistribution and Internalization of a Green Fluorescent Protein-tagged Form of the Thyrotropin-releasing Hormone Receptor

The long isoform of the rat thyrotropin-releasing hormone receptor (TRHR) was modified by the addition of a vesicular stomatitis virus (VSV) epitope tag and green fluorescent protein (GFP). VSV-TRHR-GFP bound TRH with affinity similar to that of the unmodified receptor and stimulated [3H]inositol phosphate production. A clone stably expressing VSV-TRHR-GFP at some 120,000 copies/cell was selected to visualize this receptor during cellular exposure to TRH. Internalization was detected within 3-5 min after treatment with 1 x 10(-7) M TRH, with dramatic reductions in plasma membrane localization achieved within 10-15 min. The TRHR antagonist/inverse agonist chlordiazepoxide competitively inhibited internalization. Hyperosmotic sucrose inhibited internalization of VSV-TRHR-GFP, measured both by intact cell [3H]TRH binding studies and by confocal microscopy. Now TRH caused a redistribution of VSV-TRHR-GFP to highly punctate but plasma membrane-delineated foci. Pretreatment with the microtubule-disrupting agent nocodazole allowed internalization of the VSV-TRHR-GFP construct but only into vesicles that remained in close apposition to the plasma membrane. Covisualization of VSV-TRHR-GFP and Texas Red transferrin initially indicated entirely separate localizations. After exposure to TRH substantial amounts of VSV-TRHR-GFP were present in vesicles overlapping those containing Texas Red transferrin. Such results demonstrate the G protein-coupling capacity and provide real time visualization of the processes of internalization of a TRH-receptor-GFP construct in response to agonist.

Postnatal ontogeny of the thyrotropin-releasing hormone receptor messenger ribonucleic acids in the rat forebrain

Postnatal developmental change of the thyrotropin-releasing hormone receptors (TRHR) in the rat forebrain was investigated using TRH binding assays and Northern blot analyses from postnatal day 8 to the age of 2 years. TRH binding assays, with [ 3H]MeHisTRH as the radioactive ligand, demonstrated that the binding capacity in the forebrain was lowest at postnatal day 8 and increased to a maximum level at postnatal day 20. The TRH binding significantly decreased to adult levels between days 20 and 35, and no significant change was observed thereafter. Northern blot analysis, with a 32P-labeled TRHR cRNA probe, revealed that expression of the TRHR gene in the forebrain was not detectable on day 8 after birth, whereas apparent gene expression could be detected in the anterior pituitary. In contrast to the binding capacities, TRHR mRNA levels were very low until postnatal day 20, and increased significantly between days 20 and 35. No significant alteration in mRNA levels was observed after day 35. These results indicated that: (1) TRH binding capacities in the forebrain increased to a maximum level between the second and third postnatal week and thereafter decreased to adult level, (2) the levels of TRHR mRNA and the TRH-binding capacities did not correlate in that period, suggesting that the TRHR number in the immature forebrain might be regulated by a posttranscriptional mechanism, and (3) expression of the TRHR gene in the forebrain and pituitary seemed to be regulated differentially during development.

Cis-unsaturated free fatty acids block growth hormone and prolactin secretion in thyrotropin-releasing hormone-stimulated GH3 cells by perturbing the function of plasma membrane integral proteins.

In vivo FFA block basal and stimulated GH secretion and have been implicated in the pathogenesis of the altered GH secretion present in obesity and Cushing's syndrome. Although a direct action on the somatotroph cell has been postulated, the FFA mechanism of action is unknown. The main biological target for FFA action is the cellular membrane, and it has been shown that these metabolites can block the activity of a number of plasma membrane pumps, channels, and receptor systems. In the present work, it was observed using different types of pituitary cells (GH3, GH4C1, and rat pituitary primary cultures) that cis-unsaturated fatty acids, such as oleic, 1) do not perturb TRH binding or the homologous desensitization of the TRH receptor; 2) inhibit TRH-induced inositol 1,4,5-trisphosphate/diacylglycerol generation, probably by a direct perturbation of phospholipase C; 3) reduce the TRH-induced intracellular Ca2+ redistribution and the ensuing changes in membrane potential; 4) completely inhibit the [Ca2+]i rise due to the TRH-induced opening of voltage-gated Ca2+ channels; and 5) abolish the TRH-induced Ca2+ efflux through plasma membrane Ca2+ pumps. These results suggest that cis-unsaturated FFA such as oleic acid selectively perturb the function of integral membrane proteins such as enzymes, channels, and pumps without perturbing the binding of ligands to receptors.

A disulfide bonding interaction role for cysteines in the extracellular domain of the thyrotropin-releasing hormone receptor

The roles of disulfide and sulfhydryl groups in the specific binding of TRH to its receptor have been examined. In all TRH receptors (TRH-Rs) isolated from different species so far, there are only two extracellular cysteine residues (Cys98 in the extracellular loop between transmembrane helices 2 and 3 and Cys179 in the extracellular loop between transmembrane helices 4 and 5) that are in positions homologous to cysteine residues in other G protein-coupled receptors. Another Cys (Cys100) is located in close proximity to Cys98 at the interface between the first extracellular loop and third transmembrane domain. To assess the role of these TRH-R Cys residues in disulfide bonding interactions, they were mutated to either Ser or Ala. Six mutant receptors (Cys98Ser, Cys98Ala, Cys179Ser, Cys179Ala, Cys100Ser, and Cys100Ala) were expressed in COS-1 cells and tested for their ability to bind TRH and to activate total inositol phosphate (IP) formation. TRH-R mutants Cys100Ser and Cys100Ala showed TRH binding affinities and IP activation similar to the wild-type (WT). In contrast, mutants Cys98Ser, Cys98Ala, Cys179Ser, and Cys179Ala showed no high affinity TRH binding. The potencies of Cys98Ala and Cysl79Ala as measured by IP stimulation were decreased by four orders of magnitude when compared with WT. Cys98Ser potency decreased by five orders of magnitude, whereas Cys179Ser showed no IP production. Northern blotting confirmed expression of all the mutant TRH-Rs at the messenger RNA (mRNA) level. An epitope tag derived from the Haemophilus influenza hemagglutinin protein was incorporated at the NH2 termini of the TRH-R WT and TRH-R Cys mutants to allow the independent assessment of cell surface expression of receptor protein. TRH-R mutants that failed to show receptor binding (Cys98Ser, Cys98Ala, Cys179Ala) showed WT levels of cell surface receptor expression, indicating that loss of receptor binding in these mutants is not attributable to loss of receptor expression. In contrast, cell surface expression of Cysl79Ser, which showed no ligand induced IP stimulation, could not be detected. Dithiothreitol, a disulfide bond reducing agent, and p-chloromercuribenzoic acid (p-CMB), a sulfhydryl blocking compound, reduced specific TRH binding in a dose-dependent manner. The inhibition of binding by dithiothreitol implies that the integrity of a disulfide bond is important for TRH binding to its receptor. The dramatic inhibition of TRH binding by p-CMB indicates that free sulfhydryl groups are also associated with the binding of the ligand to its receptor. This study presents evidence that a disulfide bond exists between Cys98 and Cys179 which is essential for maintaining the receptor in the correct conformation for ligand binding. Cys100 is not thought to have a disulfide bonding interaction role. Results obtained after chemical modification have shown that free sulfhydryl groups within the TRH-R may also have a role in ligand interactions.

A disulfide bonding interaction role for cysteines in the extracellular domain of the thyrotropin-releasing hormone receptor.

The roles of disulfide and sulfhydryl groups in the specific binding of TRH to its receptor have been examined. In all TRH receptors (TRH-Rs) isolated from different species so far, there are only two extracellular cysteine residues (Cys98 in the extracellular loop between transmembrane helices 2 and 3 and Cys179 in the extracellular loop between transmembrane helices 4 and 5) that are in positions homologous to cysteine residues in other G protein-coupled receptors. Another Cys (Cys100) is located in close proximity to Cys98 at the interface between the first extracellular loop and third transmembrane domain. To assess the role of these TRH-R Cys residues in disulfide bonding interactions, they were mutated to either Ser or Ala. Six mutant receptors (Cys98Ser, Cys98Ala, Cys179Ser, Cys179Ala, Cys100Ser, and Cys100Ala) were expressed in COS-1 cells and tested for their ability to bind TRH and to activate total inositol phosphate (IP) formation. TRH-R mutants Cys100Ser and Cys100Ala showed TRH binding affinities and IP activation similar to the wild-type (WT). In contrast, mutants Cys98Ser, Cys98Ala, Cys179Ser, and Cys179Ala showed no high affinity TRH binding. The potencies of Cys98Ala and Cysl79Ala as measured by IP stimulation were decreased by four orders of magnitude when compared with WT. Cys98Ser potency decreased by five orders of magnitude, whereas Cys179Ser showed no IP production. Northern blotting confirmed expression of all the mutant TRH-Rs at the messenger RNA (mRNA) level. An epitope tag derived from the Haemophilus influenza hemagglutinin protein was incorporated at the NH2 termini of the TRH-R WT and TRH-R Cys mutants to allow the independent assessment of cell surface expression of receptor protein. TRH-R mutants that failed to show receptor binding (Cys98Ser, Cys98Ala, Cys179Ala) showed WT levels of cell surface receptor expression, indicating that loss of receptor binding in these mutants is not attributable to loss of receptor expression. In contrast, cell surface expression of Cysl79Ser, which showed no ligand induced IP stimulation, could not be detected. Dithiothreitol, a disulfide bond reducing agent, and p-chloromercuribenzoic acid (p-CMB), a sulfhydryl blocking compound, reduced specific TRH binding in a dose-dependent manner. The inhibition of binding by dithiothreitol implies that the integrity of a disulfide bond is important for TRH binding to its receptor. The dramatic inhibition of TRH binding by p-CMB indicates that free sulfhydryl groups are also associated with the binding of the ligand to its receptor. This study presents evidence that a disulfide bond exists between Cys98 and Cys179 which is essential for maintaining the receptor in the correct conformation for ligand binding. Cys100 is not thought to have a disulfide bonding interaction role. Results obtained after chemical modification have shown that free sulfhydryl groups within the TRH-R may also have a role in ligand interactions.

Importance of extracellular domains for ligand binding in the thyrotropin-releasing hormone receptor.

The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 Nu Mu [3H]pGlu-N(tau)-MeHis-Pro-NH2 ([3H] MeTRH) was measured. When binding was not detected, signal transduction at 10 microM MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [3H]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr93 to Ala resulted in more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr181 to Phe, Tyr188 to Ala, and Phe199 to Ala resulted in a large ( > 100-fold) decrease in affinity for MeTRH, and conversion of Tyr 188 to Phe and Phe196 to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn289 to Ala and of Ser290 to Ala caused a large ( > 100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extra-cellular domain of its receptor.

A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist.

A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R). Chlordiazepoxide (CDE), a known competitive inhibitor of TRH binding to wild-type (WT) TRH-Rs, is shown to compete for binding to C335Stop TRH-Rs also. More importantly, CDE is shown to be a negative antagonist of C335Stop TRH-Rs. CDE rapidly caused the basal rate of inositol phosphate second messenger (IP) formation to decrease in AtT-20 pituitary cells stably expressing C335Stop TRH-Rs (AtT-C335Stop cells), but not in cells expressing WT TRH-Rs (AtT-WT cells). Similar observations were made in HeLa cells transiently expressing C335Stop or WT TRH-Rs. CDE inhibition of IP formation was shown to be specific for TRH-Rs using GH4C1 cells expressing both TRH-Rs and receptors for bombesin. In these cells, CDE inhibited TRH-stimulated IP formation, but had no effect on bombesin-stimulated IP formation. The effects of chronic administration of CDE were studied. Preincubation of AtT-C335Stop cells, but not AtT-WT cells, with CDE for several hours caused an increase in cell surface receptor number (up-regulation) that led to increased TRH stimulation of inositol phosphate formation and elevation of intracellular free Ca2+. Preincubation with CDE did not affect methyl-TRH binding affinity or TRH potency in cells expressing AtT-C335Stop or in AtT-WT cells. We conclude that CDE is a negative antagonist of C335Stop TRH-Rs and that constitutively active C335Stop TRH-Rs are down-regulated in AtT-20 pituitary cells in the absence of agonist.

Transcriptional down-regulation by epidermal growth factor of TRH receptor mRNA in rat pituitary cells

To gain insight into the mechanism underlying the epidermal growth factor (EGF)-induced changes in responsiveness to TRH and in the numbers of TRH receptors (TRH-Rs) in the pituitary, we investigated the transcriptional regulation by EGF of the TRH-R gene in GH4C1 cells. Northern blot analyses and binding studies revealed that EGF reduced both TRH binding and TRH-R mRNA levels in a dose- and time-dependent manner, while no significant changes were observed in beta-actin mRNA levels. Addition of actinomycin D caused an acute increase in the basal TRH-R mRNA level, and the rate of decrease of the TRH-R mRNA was identical in control and EGF-treated groups, suggesting that the stability of the TRH-R mRNA was not significantly affected in EGF-treated cells. Incubation with cycloheximide also induced an increase in the basal TRH-R mRNA level and completely reversed the EGF-induced reduction of TRH-R mRNA levels. Furthermore, a nuclear run-on assay demonstrated that the rate of transcription of the TRH-R gene was significantly inhibited in cells treated with EGF. We conclude that (1) EGF decreases the expression of the TRH-R mRNA largely by reducing its rate of transcription, and this action requires the synthesis of new proteins, and (2) inhibitors of protein and RNA synthesis cause a significant increase in the basal TRH-R mRNA level, suggesting that there may be a short-lived protein suppressing the TRH-R mRNA level in the pituitary.

Vinblastine and nocodazole inhibit basal and thyrotropin-releasing hormone-stimulated prolactin secretion in GH3 cells

To investigate the efficacy of vinblastine as a possible therapeutic agent in prolactinomas, we have examined the effects of vinblastine on GH(3) cell function. The effects of vinblastine were compared to another anti-microtubule drug, nocodazole. At 24 h, prolactin (PRL) secretion was 737±63 ng/ml in control cells. In cells treated with 0.1, 1 and 10μM: nocodazole for 24 h, PRL secretion was reduced to 200±30 ng/ml. After a 24 h incubation with the drugs, cells were washed with drug-free medium and challenged with 100NM: TRH for 10 min. TRH-stimulated PRL secretion was 35±7 ng/ml in control cells, 14±0.5 ng/ml in vinblastine-treated cells and 8.8±0.1 ng/ml in nocodazole-treated cells. [(3)H]TRH binding to GH(3) cell membrane was inhibited by about 15% by vinblastine and nocodazole. In vinblastine and nocodazole treated cells, polymerized tubulin levels decreased by 46 and 55%, respectively. These observations that vinblastine suppresses PRL secretion by GH(3) cells suggest that this drug might be useful as a therapeutic agent for prolactinomas.

Production and characterization of polyclonal anti-idiotypic anti-TRH antibodies: application to the study of pituitary TRH receptor.

cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteins < or = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells.

Thyrotropin-releasing hormone binding to the mouse pituitary receptor does not involve ionic interactions. A model for neutral peptide binding to G protein-coupled receptors.

Thyrotropin-releasing hormone, TRH (< Glu-His-Proamide), and [N tau-Me-His]TRH (MeTRH) are present as neutral and positively charged forms at physiologic pH, and it was possible that they bind to the TRH receptor (TRH-R) as charged (protonated) species. Binding affinities of TRH and MeTRH to endogenous rat TRH-Rs and to transfected wild type mouse TRH-Rs decreased below pH 7.1. Half-maximal decreases in binding occurred at the approximate pK alpha values of these ligands. Asp to Ala mutations in extracellular loop 1, TM-4, and TM-5 did not decrease binding affinity, but an Asp to Ala mutation in TM-2 caused the affinity to decrease 8-fold. The pH dependences of binding of MeTRH, however, were similar in wild type and all mutant receptors and were consistent with the protonated form of MeTRH binding less well. Thus, the binding of TRH to its receptor does not involve ionic interactions and may be a prototype for binding of neutral peptide ligands to G protein-coupled receptors.

Synthesis and characterization of a high-affinity photoactivatable analogue of thyrotropin-releasing hormone.

An analogue of thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2), i.e. pGlu-His-ProNH-(CH2)6-(4-azidosalicylamide) (TRH-ASA), has been synthesized and, in a radioiodinated form (TRH-IASA), characterized and used as a photoaffinity reagent to label the TRH receptor on rat pituitary GH4C1 cells. TRH-IASA bound to GH4C1 cells with high affinity (Kd = 8 nM), comparable with that of TRH binding. The binding of TRH-IASA was competitive with binding of TRH, two TRH analogues and a TRH receptor antagonist, chlordiazepoxide. TRH-IASA did not bind to or label GH12C1 cells, which lack functional TRH receptors. Labelling of GH4C1 cells with TRH-IASA followed by SDS/PAGE and autoradiography of membrane proteins demonstrated labelling of a single polypeptide which ran as a diffuse band between 71 and 91 kDa, centred at 76 kDa. No change in this labelling pattern was observed as a function of the length of time (between 5 min and 2 h) that GH4C1 cells were incubated with 3 nM-TRH-IASA. Using either a very short (5 s) photolysis interval or low TRH-IASA concentrations, only the 76 kDa band was labelled. Minor bands appeared only after extended photolysis and use of high TRH-IASA concentrations. We conclude that the TRH receptor from rat pituitary GH4C1 cells is a single peptide with an apparent molecular mass of 76 kDa. Details of the chemical synthesis of TRH-ASA are given in Supplementary Publication SUP 50167 (5 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1992) 281, 5.