Thyrotropin-releasing Hormone Binding Research Articles

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins

Objectives: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins.Methods: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP).Results: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of Giα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling.Conclusion: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.

Arrestin Binds to Different Phosphorylated Regions of the Thyrotropin-Releasing Hormone Receptor with Distinct Functional Consequences

Arrestin binding to agonist-occupied phosphorylated G protein-coupled receptors typically increases the affinity of agonist binding, increases resistance of receptor-bound agonist to removal with high acid/salt buffer, and leads to receptor desensitization and internalization. We tested whether thyrotropin-releasing hormone (TRH) receptors lacking phosphosites in the C-terminal tail could form stable and functional complexes with arrestin. Fibroblasts from mice lacking arrestins 2 and 3 were used to distinguish between arrestin-dependent and -independent effects. Arrestin did not promote internalization or desensitization of a receptor that had key Ser/Thr phosphosites mutated to Ala (4Ala receptor). Nevertheless, arrestin greatly increased acid/salt resistance and the affinity of 4Ala receptor for TRH. Truncation of 4Ala receptor just distal to the key phosphosites (4AlaStop receptor) abolished arrestin-dependent acid/salt resistance but not the effect of arrestin on agonist affinity. Arrestin formed stable complexes with activated wild-type and 4Ala receptors but not with 4AlaStop receptor, as measured by translocation of arrestin-green fluorescent protein to the plasma membrane or chemical cross-linking. An arrestin mutant that does not interact with clathrin and AP2 did not internalize receptor but still promoted high affinity TRH binding, acid/salt resistance, and desensitization. A sterically restricted arrestin mutant did not cause receptor internalization or desensitization but did promote acid/salt resistance and high agonist affinity. The results demonstrate that arrestin binds to proximal or distal phosphosites in the receptor tail. Arrestin binding at either site causes increased agonist affinity and acid/salt resistance, but only the proximal phosphosites evoke the necessary conformational changes in arrestin for receptor desensitization and internalization.

A novel TRH analog, Glp–Asn–Pro– d-Tyr– d-TrpNH 2, binds to [ 3H][3-Me-His 2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2

Glp–Asn–Pro– d-Tyr– d-TrpNH 2 is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all- l counterpart (Glp–Asn–Pro–Tyr–TrpNH 2) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [ 3H][3-Me-His 2]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His 2]TRH potently displaced [ 3H][3-Me-His 2]TRH binding from all tissues/cells investigated. Glp–Asn–Pro– d-Tyr– d–TrpNH 2 in concentrations up to 10 −5 M did not displace [ 3H][3-Me-His 2]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all- l peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC 50 Glp–Asn–Pro– d-Tyr– d-TrpNH 2: 7.7 ± 0.2; pIC 50 Glp–Asn–Pro–Tyr–TrpNH 2: 6.6 ± 0.2), analogous to cortical membranes (pIC 50 Glp–Asn–Pro– d-Tyr– d-TrpNH 2: 7.8 ± 0.2; pIC 50 Glp–Asn–Pro–Tyr–TrpNH 2: 6.6 ± 0.2). Neither peptide, however, displaced [ 3H][3-Me-His 2]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp–Asn–Pro– d-Tyr– d-TrpNH 2 is not displacing [ 3H][3-Me-His 2]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.

Agonist-Induced Conformational Changes in Thyrotropin-Releasing Hormone Receptor Type I: Disulfide Cross-Linking and Molecular Modeling Approaches

The conformational changes at the cytoplasmic ends of transmembrane helices 5 and 6 (TMH5 and TMH6) of thyrotropin-releasing hormone (TRH) receptor type I (TRH-R1) during activation were analyzed by cysteine-scanning mutagenesis followed by disulfide cross-linking and molecular modeling. Sixteen double cysteine mutants were constructed by substitution of one residue at the cytoplasmic end of TMH5 and the other at that of TMH6. The cross-linking experiments indicate that four mutants, Q263C/G212C, Q263C/Y211C, T265C/G212C, and T265C/Y211C, exhibited disulfide bond formation that was sensitive to TRH occupancy. We refined our previous TRH-R1 models by embedding them into a hydrated explicit lipid bilayer. Molecular dynamics simulations of the models, as well as in silico double cysteine models, generated trajectories that were in agreement with experimental results. Our findings suggest that TRH binding induces a separation of the cytoplasmic ends of TMH5 and TMH6 and a rotation of TMH6. These changes likely increase the surface accessible area at the juxtamembrane region of intracellular loop 3 that could promote interactions between G proteins and key residues within the receptor.

A model of inverse agonist action at thyrotropin-releasing hormone receptor type 1: role of a conserved tryptophan in helix 6.

A binding pocket for thyrotropin-releasing hormone (TRH) within the transmembrane helices of the TRH receptor type 1 (TRH-R1) has been identified based on experimental evidence and computer simulations. To determine the binding site for a competitive inverse agonist, midazolam, three of the four residues that directly contact TRH and other residues that restrain TRH-R1 in an inactive conformation were screened by mutagenesis and binding assays. We found that two residues that directly contact TRH, Asn-110 in transmembrane helix 3 (3.37) and Arg-306 in transmembrane helix 7 (7.39), were important for midazolam binding but another, Tyr-282 in transmembrane helix 6 (6.51), was not. A highly conserved residue, Trp-279 in transmembrane helix 6 (6.48), which was reported to be critical in stabilizing TRH-R1 in an inactive state but not for TRH binding, was critical for midazolam binding. We used our previous model of the unoccupied TRH-R1 to generate a model of the TRH-R1/midazolam complex. The experimental results and the molecular model of the complex suggest that midazolam binds to TRH-R1 within a transmembrane helical pocket that partially overlaps the TRH binding pocket. This result is consistent with the competitive antagonism of midazolam binding. We suggest that the mechanism of inverse agonism effected by midazolam involves its direct interaction with Trp-279, which contributes to the stabilization of the inactive conformation of TRH-R1.

Pharmacologically distinct binding sites in rat brain for [ [formula omitted]]thyrotropin-releasing hormone (TRH) and [ [formula omitted]][3-methyl-histidine 2]TRH

We have used a directed peptide library, in which the histidyl residue of thyrotropin-releasing hormone (TRH) was systematically replaced by a series of 24 natural and unnatural amino acids, to characterise TRH binding sites in rat brain cortex. This was achieved by measuring the ability of library peptides to compete with [ 3 H ][3-Me-His 2]TRH or [ 3 H ]TRH binding to rat cortical homogenates. [ 3 H ][3-Me-His 2]TRH was observed to bind to a single population of high-affinity, low-capacity sites ( K d : 4.54±0.62 nM, N=5; B max: 4.38±0.21 fmol/mg wet weight tissue, N=5), consistent with them being central TRH receptors. Displacement studies showed TRH to bind to these sites with an apparent K i of 22 nM. K i values for the library peptides at [ 3 H ][3-Me-His 2]TRH-labelled sites varied from 10 −3 to 10 −9 M; the potency order was: [3- Me- His 2]> His> Thi> Leu, Phe, Asn> Gln , Arg, Thr, Ala, HomoPhe. All other replacements had K i values >10 −4 M. [ 3 H ]TRH was observed to label a single population of low-affinity, high-capacity sites ( K d : 7.55±1.23 μM, N=6; B max: 3.40±0.63 pmol/mg wet weight tissue, N=6). The affinities of the synthetic peptides for [ 3 H ]TRH-labelled sites did not correlate with their affinities for [ 3 H ][3-Me-His 2]TRH-labelled sites ( r=0.33, N=18, P>0.1). They did, however, correlate significantly with previously reported binding affinities for TRH-degrading ectoenzyme ( r=0.72, N=12, P<0.01). These results strongly indicate that the identity of the low-affinity, [ 3 H ]TRH-labelled site is the membrane-bound enzyme, TRH-degrading ectoenzyme, not a subpopulation of TRH receptors. They also provide the first comprehensive description of the influence of the histidyl residue in TRH on binding of TRH to brain receptors.

Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic.

In some individuals with autosomal dominant isolated growth hormone deficiency, one copy of growth hormone lacks amino acids 32-71 and is severely misfolded. We transfected COS7 cells with either wild-type human growth hormone or Delta 32-71 growth hormone and investigated subcellular localization of growth hormone and other proteins. Delta 32-71 growth hormone was retained in the endoplasmic reticulum, whereas wild-type hormone accumulated in the Golgi apparatus. When cells transfected with wild-type or Delta 32-71 growth hormone were dually stained for growth hormone and the Golgi markers beta-COP, membrin or 58K, wild-type growth hormone was colocalized with the Golgi markers, but beta-COP, membrin and 58K immunoreactivity was highly dispersed or undetectable in cells expressing Delta 32-71 growth hormone. Examination of alpha-tubulin immunostaining showed that the cytoplasmic microtubular arrangement was normal in cells expressing wild-type growth hormone, but microtubule-organizing centers were absent in nearly all cells expressing Delta 32-71 growth hormone. To determine whether Delta 32-71 growth hormone would alter trafficking of a plasma membrane protein, we cotransfected the cells with the thyrotropin-releasing hormone (TRH) receptor and either wild-type or Delta 32-71 growth hormone. Cells expressing Delta 32-71 growth hormone, unlike those expressing wild-type growth hormone, failed to show normal TRH receptor localization or binding. Expression of Delta 32-71 growth hormone also disrupted the trafficking of two secretory proteins, prolactin and secreted alkaline phosphatase. Delta 32-71 growth hormone only weakly elicited the unfolded protein response as indicated by induction of BiP mRNA. Pharmacological induction of the unfolded protein response partially prevented deletion mutant-induced Golgi fragmentation and partially restored normal TRH receptor trafficking. The ability of some misfolded proteins to block endoplasmic reticulum-to-Golgi traffic may explain their toxic effects on host cells and suggests possible strategies for therapeutic interventions.

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.

Thyrotropin-releasing hormone interacts with vasoactive intestinal peptide-specific receptor in guinea pig cecal circular smooth muscle cells

The relationship between thyrotropin-releasing hormone (TRH) binding sites and vasoactive intestinal peptide (VIP) receptors in circular muscle cells obtained from the guinea pig cecum was investigated using antagonists of VIP receptors and a selective receptor protection method. Both VIP10–28, a VIP antagonist, and atrial natriuretic peptide1–11 (ANP1–11), a VIP-specific receptor antagonist, completely inhibited 10 −5 M TRH-induced relaxation in a concentration-dependent manner. The muscle cells where cholecystokinin octapeptide (CCK-8) and TRH binding sites were protected completely preserved the inhibitory responses to TRH and ANP (a VIP-specific receptor agonist), and partially the inhibitory response to VIP. Peptide histidine isoleucine (PHI: a VIP-preferring receptor agonist) had no inhibitory effect on these cells. The muscle cells where CCK-8 and ANP (VIP-specific) receptors were protected completely preserved the inhibitory responses to TRH and ANP and partially the inhibitory response to VIP. PHI had no inhibitory effect on these cells. The muscle cells where CCK-8 and VIP receptors (both VIP-specific and VIP-preferring receptors) were protected preserved completely the inhibitory responses to TRH, VIP, ANP, and PHI. The muscle cells where CCK-8 and PHI (VIP-preferring) receptors were protected completely preserved the inhibitory response to PHI and partially the inhibitory response to VIP. TRH and ANP had no inhibitory effect on these cells. This study first demonstrates that TRH interacts with VIP-specific receptor in guinea pig cecal circular smooth muscle cells.

A hydrophobic cluster between transmembrane helices 5 and 6 constrains the thyrotropin-releasing hormone receptor in an inactive conformation.

We have studied the role of a highly conserved tryptophan and other aromatic residues of the thyrotropin-releasing hormone (TRH) receptor (TRH-R) that are predicted by computer modeling to form a hydrophobic cluster between transmembrane helix (TM)5 and TM6. The affinity of a mutant TRH-R, in which Trp279 was substituted by alanine (W279A TRH-R), for most tested agonists was higher than that of wild-type (WT) TRH-R, whereas its affinity for inverse agonists was diminished, suggesting that W279A TRH-R is constitutively active. We found that W279A TRH-R exhibited 3.9-fold more signaling activity than WT TRH-R in the absence of agonist. This increased basal activity was inhibited by the inverse agonist midazolam, confirming that the mutant receptor is constitutively active. Computer-simulated models of the unoccupied WT TRH-R, the TRH-occupied WT TRH-R, and various TRH-R mutants predict that a hydrophobic cluster of residues, including Trp279 (TM6), Tyr282, and Phe199 (TM5), constrains the receptor in an inactive conformation. In support of this model, we found that substitution of Phe199 by alanine or of Tyr282 by alanine or phenylalanine, but not of Tyr200 (by alanine or phenylalanine), resulted in a constitutively active receptor. We propose that a hydrophobic cluster including residues in TM5 and TM6 constrains the TRH-R in an inactive conformation via interhelical interactions. Disruption of these constraints by TRH binding or by mutation leads to changes in the relative positions of TM5 and TM6 and to the formation of an active form of TRH-R.

Cloning and Characterization of a cDNA Encoding a Novel Subtype of Rat Thyrotropin-releasing Hormone Receptor

A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.

Receptors for thyrotropin-releasing hormone on rat lactotropes and thyrotropes.

Primary cultures of rat pituitary cells were stained with an antibody to the native thyrotropin-releasing hormone (TRH) receptor and with a bioactive, fluorescent analogue of TRH, Rhod-TRH. Rhod-TRH specifically stained 86% of lactotropes and 21% of nonlactotropes from primary pituitary cell cultures. Lactotropes and thyrotropes accounted for 90% of cells that stained with Rhod-TRH, but there were occasional lactotropes and thyrotropes that did not show detectable staining with antireceptor antibodies or with Rhod-TRH. The intensity of staining was generally higher in the GH3 line of tumor cells than in normal pituicytes, and 100% of the tumor cells stained with Rhod-TRH. To determine whether the TRH receptor undergoes ligand-directed endocytosis in normal cells, TRH receptor immunocytochemistry was performed before and after TRH binding. TRH receptors were localized on the surface of cells prior to TRH exposure, and Rhod-TRH fluorescence was confined to the plasma membrane when TRH binding was performed at 0 degrees C, where endocytosis is blocked. When cells were incubated with TRH at 37 degrees C, receptors were found in intracellular vesicles in both lactotropes and thyrotropes, and Rhod-TRH was rapidly internalized into endosomes at elevated temperatures. Internalization of Rhod-TRH was inhibited by hypertonic sucrose, indicating that it occurs through clathrin-coated pits. These findings show that some of the heterogeneity in the secretory and calcium responses of pituicytes to TRH occurs at the level of the TRH receptor.

Cloning and Characterization of a New Subtype of Thyrotropin-Releasing Hormone Receptors

A new subfamily member of thyrotropin releasing hormone (TRH) receptor gene,TRHR2, was isolated from rat brain cDNAs. The deduced amino acid sequence ofTRHR2is 51 % identical to that of rat TRH receptor gene which was reported previously. Northern blot analysis withTRHR2probe revealed brain-specific expression of a 9.5 kb mRNA. In a binding experiment using the TRHR2-expressing COS cells, specific binding of TRH to TRHR2 was observed with Kd value of 9 nM which was equivalent to the Kd value (= 13 nM) of TRH binding to the TRH receptor previously reported. The active metabolite of TRH, histidyl-proline diketopiperazine, or cyclo(His-Pro), showed no specific binding activity. These results suggest that TRHR2 is a novel subtype of TRH receptor.

Static and Dynamic Roles of Extracellular Loops in G-Protein-Coupled Receptors: A Mechanism for Sequential Binding of Thyrotropin-Releasing Hormone to Its Receptor

Small ligands generally bind within the seven transmembrane-spanning helices of G-protein-coupled receptors, but their access to the binding pocket through the closely packed loops has not been elucidated. In this work, a model of the extracellular loops of the thyrotropin-releasing hormone (TRH) receptor (TRHR) was constructed, and molecular dynamics simulations and quasi-harmonic analysis have been performed to study the static and dynamic roles of the extracellular domain. The static analysis based on curvature and electrostatic potential on the surface of TRHR suggests the formation of an initial recognition site between TRH and the surface of its receptor. These results are supported by experimental evidence. A quasi-harmonic analysis of the vibrations of the extracellular loops suggest that the low-frequency motions of the loops will aid the ligand to access its transmembrane binding pocket. We suggest that all small ligands may bind sequentially to the transmembrane pocket by first interacting with the surface binding site and then may be guided into the transmembrane binding pocket by fluctuations in the extracellular loops.

Autoradiographic localization of neurotransmitter binding sites in the hypoglossal and motor trigeminal nuclei of the rat.

The hypoglossal and motor trigeminal nuclei contain somatic motoneurons innervating the tongue, jaw, and palate. These two cranial motor nuclei are myotopically organized and contain neurotransmitter binding sites for thyrotropin-releasing hormone, substance P, and serotonin. Quantitative autoradiography was used to localize thyrotropin-releasing hormone, substance P, and serotonin-1A and serotonin-1B binding sites in the hypoglossal and motor trigeminal nuclei and to relate the relative distributions of these binding sites to the myotopic organizations of the two nuclei. In the hypoglossal nucleus, high-to-moderate concentrations of all four binding sites were present in the dorsal and ventromedial subnuclei, whereas low concentrations were noted in the ventrolateral subnucleus. In the motor trigeminal nucleus, high concentrations of serotonin-1B, moderate densities of thyrotropin-releasing hormone, and low levels of substance P and serotonin-1A binding sites were present in both the ventromedial and dorsolateral subnuclei. These observations demonstrate that neurotransmitter binding sites in the hypoglossal and motor trigeminal nuclei are heterogeneously localized and that their distributions correspond to the previously described myotopic organizations of each nucleus.

Effect of cell type on the subcellular localization of the thyrotropin-releasing hormone receptor.

The localization of an epitope-tagged receptor for thyrotropin-releasing hormone (TRH) expressed in different cell contexts was studied with immunofluorescence microscopy. In pituitary lactotrophs, which normally express TRH receptors, and in AtT20 pituitary corticotrophs, TRH receptor immunoreactivity was primarily confined to the plasma membrane. In HEK 293 and COS7 cells, TRH receptors were predominantly intracellular. In transiently transfected COS7 cells, the TRH receptor colocalized with endoplasmic reticulum and Golgi markers. The pattern of TRH receptor immunofluorescence was the same over a wide range of receptor expression in transiently transfected COS7 cells, and all cell lines bound similar amounts of 3H- and rhodamine-labeled TRH analogs, suggesting that cell-specific differences in TRH receptor localization were not simply the result of overexpression. In all cell contexts, TRH receptors on the plasma membrane underwent extensive ligand-driven endocytosis. Inhibitors of glycosylation did not alter the subcellular distribution of receptors. In HEK 293 cells expressing the transfected TRH receptor, protein synthesis inhibitors caused translocation of intracellular receptors to the cell surface, as shown by a marked increase in cell surface immunofluorescence and [3H][N3-methyl-His2]TRH binding. These results demonstrate that the subcellular localization of the TRH receptor depends on the cell context in which it is expressed and that intracellular receptors are capable of translocation to the plasma membrane.

1,25-Dihydroxyvitamin D3-induced upregulation of the thyrotropin-releasing hormone receptor in clonal rat pituitary GH3 cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)2D3 on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH3) cells. Compared with vehicle-treated cells, 1,25-(OH)2D3 (10 nmol/l) increased specific [3H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose-response experiments, specific [3H]MeTRH binding increased with 1,25-(OH)2D3 concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0.5 nmol 1,25-(OH)2D3/l. The vitamin D metabolite, 25-OH D3, increased [3H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)2D3. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)2D3/l for 48 h increased the maximum binding from 67.4 +/- 8.8 fmol/mg protein in vehicle-treated cells to 96.7 +/- 12.4 fmol/mg protein in treated cells. There was no difference in apparent Kd (1.08 +/- 0.10 nmol/l for 1,25-(OH)2D3-treated and 0.97 +/- 0.11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)2D3/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 micrograms/ml, 6 h) abrogated the 1,25-(OH)2D3-induced increase in [3H]MeTRH binding. Cortisol also increased [3H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)2D3. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)2D3. We conclude that the active vitamin D metabolite, 1,25-(OH)2D3, caused a time- and dose-dependent increase in [3H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding.

Identification of Asn289 as a ligand binding site in the rat thyrotropin-releasing hormone (THR) receptor as determined by complementary modifications in the ligand and receptor: a new model for THR binding.

To test the hypothesis that pGlu of the thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2) binds to Asn289 in the third extracellular loop (EL3) of its receptor through a hydrogen bonding interaction, we converted Asn289 to Asp (N289D mutant) and measured the potencies of TRH and Pro1TRH for the wild-type and mutant receptors. TRH was 100 times less potent for the N289D receptor than for the wild-type. In contrast, Pro1TRH, which has a protonated proline in place of the pGlu of TRH, was 10 times more potent for the N289D receptor than for the wild-type. A similar result was obtained when Asn289 was converted to Glu, while the potency of Pro1TRH did not change when Asn289 was converted to Ala, confirming that the increased potency of Pro1TRH for the N289D receptor was due to a charge interaction between Pro1TRH and the mutant receptor. These findings are inconsistent with a previous model indicating a direct interaction of the pGlu of TRH with Asn110 in the third transmembrane helix of the receptor (Perlman et al. (1994) J. Biol. Chem. 269, 23383-23386). When Asn110 was converted to Asp (N110D mutant), unlike the N289D receptor, the potency of Pro1TRH for the N110D receptor was decreased by > 10-fold rather than increased. Therefore, a direct interaction of Asn110 with the pGlu of TRH could not be supported by our experiments. We propose a new model in which the pGlu of TRH binds to Asn289 in EL3 and conclude that, unlike catecholamines which bind completely within the transmembrane domain of their receptors, this tripeptide binds, at least in part, to the extracellular domain of its receptor.

Caffeine Inhibits the Binding of Thyrotropin-Releasing Hormone in GH4C1 Pituitary Cells

Caffeine can modulate intracellular Ca2+ concentration ([Ca2+]i) by triggering the mobilization of Ca2+ from intracellular Ca2+ stores. In the present study we show that in Fura 2 loaded GH4C1 cells, caffeine inhibited, in a dose-dependent manner, the Ca2+ response induced by a submaximally effective dose (3 nM) of thyrotropin-releasing hormone (TRH). We also show that caffeine decreased the specific binding of [3H]TRH. Equilibrium binding studies with [3H]TRH and Scatchard analysis of the binding data showed that caffeine increased the dissociation constant (Kd) from 8 ± 1 nM to 26 ± 3 nM, while the maximum amount of [3H]TRH bound to the cells was increased by 32%. Thus, caffeine inhibited the TRH-evoked increase in [Ca2+]i by inhibiting the binding of TRH to its receptor.

A novel analog of TRH, YM14673, causes a decrease in brain TRH receptors in vitro.

Biochemical mechanisms by which analogs of thyrotropin-releasing hormone (TRH) produce their potent neuropharmacological actions on the brain remain ill-defined. We tested effects of YM14673, a novel analog of TRH, on TRH receptors in rat brains in vitro. No significant binding of [3H]YM14673 to brain plasma membranes occurred. In contrast, preincubation of membranes with YM14673 caused dose-dependent decreases in TRH binding. This was not due to competition for TRH binding sites or existence of metabolites of YM14673. Preincubation with DN1417 (an another TRH analog), cyclo(His-Pro) or methionine-enkephalin did not affect the binding. Affections of YM14673 on TRH binding were observed when cerebral cortical membranes were studied; those were not seen in membranes prepared from hypothalamus, striatum, midbrain, hippocampus, or pons-medulla. The present data indicate that YM14673 exerts its characteristic neuro-pharmacological functions through interacting with TRH binding sites in the brain.