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In amphibians, thyrotropin (TSH), corticotropin (ACTH) and prolactin (PRL) are regarded as the major pituitary hormones involved in metamorphosis, their releasing factors being corticotropin-releasing factor (CRF), arginine vasotocin (AVT), and thyrotropin-releasing hormone (TRH), respectively. It is also known that thyrotropes and corticotropes are equipped with CRF type-2 receptor and AVT V1b receptor, respectively. As for PRL cells, information about the type of receptor for TRH (TRHR) through which the action of TRH is mediated to induce the release of PRL is lacking. In order to fill this gap, an attempt was made to characterize the TRHR subtype existing in the PRL cells of the anterior pituitary gland of the bullfrog, Rana catesbeiana. We cloned cDNAs for three types of bullfrog TRHRs, namely TRHR1, TRHR2 and TRHR3, and confirmed that all of them are functional receptors for TRH by means of reporter gene assay. Analyses with semi-quantitative reverse transcription-PCR and in situ hybridization revealed that TRHR3 mRNA is expressed in the anterior lobe and that the signals reside mostly in the PRL cells. It was also noted that the expression levels of TRHR3 mRNA in the anterior pituitary as well as in the PRL cells of metamorphosing tadpoles elevate as metamorphosis progresses. Since the pattern of changes in TRHR3 mRNA levels in the larval pituitary is almost similar to that previously observed in the pituitary PRL mRNA and plasma PRL levels, we provide a view that TRHR3 mediates the action of TRH on the PRL cells to induce the release of PRL that is prerequisite for growth and metamorphosis in amphibians.

Thyrotropin-releasing hormone (TRH) was the first trophic “factor” from the hypothalamus to be isolated and identified chemically. It was known to have a stimulatory effect on thyroid-stimulating hormone (TSH) production and secretion by cells within the adenohypothesis, and its structure was revealed to be a modified tripeptide: (pyro)Glu-His-Pro-NH2. Intravenous or intramuscular injection of TRH to horses results in an immediate rise in plasma TSH concentrations, as would be expected. However, it is now known that TRH has consistent effects on four of the other five cell types in the equine adenohypophysis as well. Administration of TRH to horses stimulates not only plasma TSH concentrations, but also plasma prolactin, adrenocorticotropic hormone, and melanocyte-stimulating hormone concentrations. In contrast, administration of TRH simultaneously or within 1 hour before administration of secretagogues for growth hormone (GH) greatly reduces the GH response to the secretagogues. To date, TRH has not been reported to have positive or inhibitory effects on the release of luteinizing hormone or follicle-stimulating hormone from gonadotropes. Whether the noted effects of TRH on cells other than the thyrotropes are physiologic or pharmacologic is not clear. Regardless, a significant clinical utility has developed for the use of TRH in diagnosing thyroid gland disease as well as pituitary pars intermedia dysfunction in horses.

Shenrong Wuzi Pill (SWP) is a traditional Chinese medicine used for hypothyroidism treatment. It contains fourteen herbs. However, little is known about the active components and the mechanism of SWP. To reveal the chemical components and drug targets and to systematically dissect the underlying mechanism of SWP as a treatment for hypothyroidism, a network pharmacology approach was used in this study. Four networks were set up, including a hypothyroidism gene network, a pathway of hypothyroidism network, a compound-compound target network, and a compound-compound target-hypothyroidism gene network. Moreover, further analysis focused on overlapping genes between compound targets and hypothyroidism genes. As a result, 14 compounds, 29 overlapping genes and 3 pathways were selected. It was found that SWP and lauric acid from Plantago asiatica L., both upregulated thyrotropin-releasing hormone receptor (TRHR) at mRNA and protein levels in NG108-15 cells. Additionally, lauric acid and atropine from Lycium barbarum L. both upregulated growth hormone secretagogue receptor (GHSR) at the mRNA level; adenine from Poria cocos (Schw.) Wolf. and 17α-estradiol from Cornu Cervi Pantotrichum upregulated adenylyl cyclase 10 (ADCY10) and follicle ¬stimulating hormone receptor (FSHR) at mRNA levels, respectively. However, mRNA levels of GHSR, ADCY10 and FSHR were not affected significantly by SWP treatment. Furthermore, SWP, lauric acid, adenine and atropine all upregulated cellular cyclic adenosine monophosphate (cAMP) concentration, which is important for triiodothyronine (T3) and thyroxine (T4) production. Taken together, it was concluded SWP affects the pathway of thyroid hormone synthesis by targeting TRHR and altering cAMP production.

PCBs and DDT cause the disturbance of thyroid hormone (TH) homeostasis in humans and animals. To test the hypothesis that the PI3K/Akt and MAPK pathways would play significant roles in TH imbalance caused by PCBs and DDT, Sprague-Dawley rats were dosed with PCB153 and p,p'-DDE intraperitoneally for 5 consecutive days, and human thyroid follicular epithelial (Nthy-ori 3-1 cell line) were treated with PCB153 and p,p'-DDE for different time. Results showed that serum total thyroxine (TT4), free thyroxine (FT4), total triiodothyronine (TT3) and thyroid stimulating hormone (TSH) were decreased, whereas serum free triiodothyronine (FT3) and thyrotropin releasing hormone (TRH) were not changed. The PI3K/Akt and ERK pathways were activated in vivo and in vitro after the treatment with PCB153 and p,p'-DDE. Moreover, TH receptor β1 (TRβ1) was elevated after the activation of the PI3K/Akt pathway and was depressed after the inhibition of the PI3K/Akt pathway; TRH receptor (TRHr) was increased after the activation of the ERK pathway and was decreased after the inhibition of the ERK pathway. Though TH receptor α1 (TRα1) level was increased in the hypothalamus, TRα1 and TSHr were not influenced by the status of signaling pathways in in vitro study. Taken together, after exposure to PCB153 and p,p'-DDE, activated PI3K/Akt and ERK pathways disrupt the hypothalamic-pituitary-thyroid (HPT) axis via TRβ1 and TRHr and then decrease TH levels, and that would be a potential mechanism by which PCBs and DDT disturb TH homeostasis.

Glycoprotein hormones (GpH) interact very specifically with their receptors to mediate hypothalamic-pituitary-peripheral gland endocrine signaling. Vertebrates typically have three functionally distinct GpH endocrine signaling complexes: follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone, and their receptors. Each hormone consists of a common α subunit bound to one of three different β subunits. Individual hormone subunits and receptors are present in genomes of early metazoans, and a subset of hormone subunits and receptors has been recently characterized in sea lamprey. However, it remains unclear when the full complement of hormone and receptor protein families first appeared, and when specificity of interactions between GpH hormones and receptors first evolved. Here we present phylogenetic analyses showing that the elephant shark (Callorhinchus milii) genome contains sequences representing the current diversity of all hormone subunits and receptors in these co-evolving protein families. We examined specificity of hormone and receptor interactions using functional assays testing reporter gene activation by elephant shark follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone receptors. We show highly specific, dose-responsive hormone interactions for all three complexes. Our results suggest that co-evolution of specificity between proteins in these endocrine signaling complexes occurred prior to the divergence of Chondrichthyes from the chordate lineage.

Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of follicular development and maturation. Multiple injections of protein formulations are used during clinical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of assisted reproductive technologies. In order to increase patient convenience and compliance several research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines. We report here the discovery of a series of substituted benzamides as positive allosteric modulators (PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR) and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising in vitro and pharmacokinetic profiles.

Two novel small molecule gonadotropin-releasing hormone (GnRH) receptor antagonists (12 and 13) of the furamide-class were synthesized and evaluated in vitro for their receptor binding affinities for the rat GnRH receptor. Radiolabeling with no carrier added fluorine-18 of the appropriate precursors was investigated in a one-step reaction. Log P (Octanol/PBS pH 7.4) and serum stability of the compounds were investigated. The antagonists showed low nM affinity for the rat GnRH receptor. 18F-radiolabled compounds were obtained in high radiochemical purity (>95%) and specific activity (>75 GBq/μmol). These findings suggest this class of compounds holds promise as potential probes for PET targeting of GnRH-receptor expression.

Background Early studies on the mechanism of action of thyroid hormone (TH) measured changes in enzyme activities following the addition of l -thyroxine (T4) and 3, 3′, 5-triiodothyronine (T3) to tissue extracts and purified enzymes. Scope of review As techniques for isolation of mitochondria, ribosomes, nuclei and chromatin, were increasingly refined, it became possible to study complex cellular processes, such as oxidative phosphorylation, protein synthesis, transcription and chromosomal structure. Uncoupling of oxidative phosphorylation and direct action on protein synthesis as mechanisms of action of TH, proposed in the 1950s and 1960s, were found to be untenable as mechanisms of physiological action because of inappropriate experimental conditions. Major conclusions Several findings in the 1960s and 1970s, mainly 1) that near-physiological doses of T3 stimulated transcription measured in vivo or in nuclei isolated from tissues of rats and frog tadpoles, 2) the inhibition of hormone action by inhibitors of transcription and 3) the rapid and almost identical kinetics of accumulation of labelled hormone and RNA synthesis in target cell nuclei, pointed to the cell nucleus as a major site of its action. The application of technologies of recombinant DNA, gene cloning and DNA sequencing in the mid-1980s allowed the identification and understanding of the structure and function of nuclear receptors of TH. General significance This review traces the road leading to the nuclear receptors of thyroid hormone, thus explaining how the hormone influences gene expression. It also illustrates the importance of how new concepts originate from the progression of technological innovations. This article is part of a Special Issue entitled Thyroid hormone signalling.

Thyrotropin-releasing hormone (TRH), produced in the hypothalamus and stimulating secretion of thyrotropin from the pituitary, is central in regulating the hypothalamic–pituitary–thyroid (HPT) axis. The number of TRH receptors (TRHR) expressed on the surface of target cell is a crucial factor influencing the magnitude of TRH response. The objective of this study was to identify the DNA polymorphisms existing in pig TRH and TRHR gene and to evaluate their associations with porcine growth, carcass and meat quality traits. Six polymorphisms in TRH and seven in TRHR gene in total were identified by re-sequencing. Among the polymorphisms, TRH ss325994920 was a missense mutation at TRH precursor residue 173 causing Val>Met (GTG>ATG) and would generate mutant pFE22/pSE14 peptides which are non-TRH peptides derived from the TRH precursor and have roles in regulating hormone ACTH and PRL. Allele frequencies of the polymorphisms were evaluated in four Western and two Chinese pig breeds (N = 228). Opposite alleles prevailed in Western pigs and Chinese pigs at loci ss325994933, ss325994918, ss325994923, ss325994924 and ss325994925, and existence of high linkage disequilibrium blocks in all six breeds were observed. A resource population (N = 409) based on crosses of Pietrain and Jinhua pigs were used for the association tests. TRH gene was significantly (P

Ghrelin is a 28-amino acid peptide hormone produced in the stomach. It binds to the growth hormone secretagogue receptor 1a (GHS-R1a), a class A G-protein-coupled receptor. In the present study, we describe the design, synthesis and characterization of a truncated, 18-amino acid analog of ghrelin conjugated to a fluorescent molecule, fluorocein isothiocyanate (FITC), through the addition of a lysine at its C terminus ([Dpr(octanoyl) 3 , Lys(fluorescein) 19 ]ghrelin(1–19)). Receptor binding affinity of this novel fluorescein-ghrelin(1–18) was similar to that of wild-type ghrelin and a synthetic GHS-R1a ligand, hexarelin. Live cell imaging in CHO/GHS-R1a cells demonstrated cell surface receptor labeling and internalization, and agonist activity of fluorescein-ghrelin(1–18) was confirmed by increased phosphorylation of ERK1/2. We also show that GHS-R1a protein is expressed primarily in the heart when compared to all other organs, suggesting high receptor density in the left ventricle. Finally, we demonstrate that fluorescein-ghrelin(1–18) binds specifically to heart tissue in situ, and its binding is displaced by both wt ghrelin and hexarelin. We have therefore developed a novel imaging probe, fluorescein-ghrelin(1–18), that can be used to image GHS-R1a in situ, for the purposes of investigating mechanisms of receptor trafficking or pharmacological agents that target GHS-R1a.

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.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metabolism. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a number of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the association of TRβ with steroid receptor coactivator 2 (SRC2), we identified a novel methylsulfonylnitrobenzoate (MSNB)-containing series that blocks this interaction at micromolar concentrations. Here we have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRβ antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum analysis suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRβ to disrupt SRC2 association.

In this study, the expression time and profiles of thyroid hormone receptor alpha (trα), type I and II deiodinase enzymes (d1 and d2), transthyretin (ttr), sodium iodide symporter (nis), and thyrotropin-releasing hormone receptor (trhr) genes in Chinese rare minnow (Gobiocypris rarus) were determined using real-time PCR. Meanwhile, the changes of these genes were investigated by exogenous T(3) (3.8 nM) in larvae and adult fish. The retardation of swim bladder development and growth inhibition were observed for larvae, and the transcription of trα, d1, d2, nis, and trhr was significantly down-regulated at the end of exposure (21 d). In adults, a down-regulation of trα, d1, nis, and trhr mRNA levels occurred at 7th or 14th day of exposure, but returned back to their normal levels similar to control at the end of exposure. The down-regulation of gene mRNA expression could serve as a compensatory mechanism for the activation of thyroid system.

Thyroid hormone exerts a pleiotropic effect on development and homeostasis. A novel thyroid hormone receptor beta isoform (hereafter referred to as TR beta 4) was cloned using PCR from a human pituitary cDNA library as a template. Analysis of the PCR products revealed a 137-bp insertion, which contains a stop codon in the middle, between the 5th and 6th exons that encode the ligand-binding domain of TR beta. The corresponding sequence of this insertion exists within the 5th intron of the human TR beta gene and consensus splice sequences were found at the junction sites. RACE analysis revealed that TR beta 4 is a carboxyl-terminal splicing variant of TR beta 1. RT-PCR and Northern blot analyses indicate that TR beta 4 mRNA is expressed in various human tissues, and especially abundant in testis and skeletal muscle. The TR beta 4 protein was unable to bind thyroid hormone (T3) and transient transfection assays demonstrate that TR beta 4 construct does not mediate T3-dependent gene regulation. TR beta 4 weakly but significantly inhibited transcription mediated by functional TR. Thus, this novel isoform may modulate hormone action as an endogenous antagonist in the tissue or cellular context.

The follicle stimulating hormone receptor (FSHR), a member of the G-protein coupled receptor family, has a large extracellular domain (ECD) which is responsible for hormone binding specificity. Whether the extracellular loops (ELs) of FSHR which are outward projections of its transmembrane domain have any role in receptor function is not yet well understood. Here, we use antipeptide antibodies to peptides corresponding to the FSHR-ELs to check the surface accessibility of the loops. These antibodies were further used to understand the involvement of the loops in hormone binding and signaling. The results demonstrate that EL1 and EL3 are surface accessible on the mature receptor in spite of the presence of a large ECD. It is observed that the EL1 and EL3 serve as secondary binding sites and they possibly interact with the ECD-bound hormone's alpha subunit which is common to the gonadotropins.

Design and synthesis of a novel 3-hydroxy-cyclobut-3-ene-1,2-dione derivatives are reported and their in vitro thyroid hormone receptor selectivity has been evaluated in the thyroid luciferase receptor assay. The 3-[3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)-phenylamino]-4-hydroxy-cyclobut-3-ene-1,2-dione 21 has shown selectivity towards thyroid hormone receptor beta.

The nongenomic actions of thyroid hormone require a plasma membrane receptor or nuclear receptors located in cytoplasm. The plasma membrane receptor is located on integrin alphaVbeta3 at the Arg-Gly-Asp recognition site important to the binding by the integrin of extracellular matrix proteins. l-Thyroxine (T(4)) is bound with greater affinity at this site than 3,5,3'-triiodo-l-thyronine (T(3)). Mitogen-activated protein kinase (MAPK; ERK1/2) transduces the hormone signal into complex cellular/nuclear events including angiogenesis and tumor cell proliferation. Acting at the integrin receptor and without cell entry, thyroid hormone can foster ERK1/2-dependent serine phosphorylation of nuclear thyroid hormone receptor-beta1 (TRbeta1) and de-repress the latter. The integrin receptor also mediates actions of the hormone on intracellular protein trafficking and on plasma membrane ion pumps, including the sodium/protein antiporter. Tetraiodothyroacetic (tetrac) is a T(4) analog that inhibits binding of iodothyronines to the integrin receptor and is a probe for the participation of this receptor in cellular actions of the hormone. Tetrac blocks thyroid hormone effects on angiogenesis and cancer cell proliferation. Acting on a truncated form of nuclear TRalpha1 (TRDeltaalpha1) located in cytoplasm, T(4) and 3,3',5'-triiodothyronine (reverse T(3)), but not T(3), cause conversion of soluble actin to fibrous (F) actin that is important to cell motility, e.g., in cells such as glia and neurons. Normal development of the central nervous system requires such motility. TRbeta1 in cytoplasm mediates action of T(3) on expression of certain genes via phosphatidylinositol 3-kinase (PI 3-K) and the protein kinase B/Akt pathway. PI 3-K and, possibly, cytoplasmic TRbeta1 are involved in stimulation by T(3) of insertion of Na,K-ATPase in the plasma membrane and of increase in activity of this pump. Because ambient thyroid hormone levels are constant in the euthyroid intact organism, these nongenomic hormone actions are likely to be contributors to basal rate-setting of transcription of certain genes and of complex cellular events such as angiogenesis and cancer cell proliferation.

Prolactin (PRL) is the most versatile hormone identified with multiple functions from osmoregulation in euryhaline fish to lactation in mammals. However, little is known about the basic physiological control of PRL in stenohaline freshwater fish. In this study, goldfish is used as a model for the study of PRL gene expression in stenohaline fish. We report herein the identification of the goldfish PRL (gfPRL) genomic sequence which possesses five exons with its 5'-flanking gene promoter region characterized in vitro using GH3 and GH4 cell-lines. Dopamine and thyrotropin releasing hormone were found to down-regulate the transcription of this gfPRL gene promoter in vitro. This was further confirmed by the decrease of PRL mRNA levels in vitro (in goldfish pituitary primary cells) and in vivo (intra-peritoneal injection) following the administrations of dopamine and thyrotropin releasing hormone. The Pit-1 binding sites of gfPRL are highly conserved with a consensus DNA sequence of 5'TATNCAT-3', as confirmed with the electrophoretic mobility shift assay using nuclear extract from the GH3 cell-line, may be responsible for the control of gfPRL gene promoter.

The growth hormone receptor has been an archetype for ligand-induced receptor dimerisation in cytokine receptor signalling. However, we now know that it exists as a constitutive dimer and is activated by a reorganisation of receptor subunits as a result of asymmetric placement of two receptor binding sites on the hormone monomer. This review highlights several topics including: current models of receptor activation; recent advances in the understanding of GH signalling demonstrating that ligand-induced signalling activates Src/ERK pathway in parallel to the classical JAK2-STAT5 signalling; and the nuclear localised growth hormone receptor correlates with high proliferation status and carcinogenesis.

The parathyroid hormone 2 (PTH2) receptor is a Family B G-protein coupled receptor most highly expressed within the brain. Current evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) is the PTH2 receptor's endogenous ligand. To facilitate investigation of the physiological function of the PTH2 receptor/TIP39 system, we have developed a novel PTH2 receptor antagonist, by changing several residues within the amino terminal domain of TIP39. Histidine4, tyrosine5, tryptophan6, histidine7-TIP39 binds the PTH2 receptor with high affinity, has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and displays no detectable agonist activity. This ligand should be useful for in vivo investigation of PTH2 receptor function.

A cell surface receptor for thyroid hormone has recently been identified on the extracellular domain of integrin alphavbeta3. In a variety of human and animal cell lines this hormone receptor mediates activation by thyroid hormone of the cellular mitogen-activated protein kinase (MAPK) signal transduction cascade. An arginine-glycine-aspartate (RGD) recognition site on the heterodimeric integrin is essential to the binding of a variety of extracellular matrix proteins. Recent competition data reveal that RGD peptides block hormone-binding by the integrin and consequent MAPK activation, suggesting that the hormone interaction site is located at or near the RGD recognition site on integrin alphavbeta3. A deaminated thyroid hormone (l-thyroxine, T4) analogue, tetraiodothyroacetic acid (tetrac, T4ac), inhibits binding of T4 and 3,5,3'-triiodo-l-thyronine (T3) to alphavbeta3, but does not activate MAPK. Structural data show that the RGD cyclic peptide binds at the interface of the propeller of the alphav and the B domains on the integrin head [Xiong JP, Stehle T, Zhang R, Joachimiack A, Frech M, Goodman SL, et al. Crystal structure of the extracellular segment of integrin alphavbeta3 in complexing with an Arg-Gly-Asp ligand. Science 2002;296:151-5]. To model potential interactions of thyroid hormone analogues with integrin, we mapped T4 and T4ac to the binding site of the RGD peptide. Modeling studies indicate that there is sufficient space in the cavity for the thyroid hormone to bind. Since the hormone is smaller in overall length than the RGD peptide, the hormone does not interact with the Arg recognition site in the propeller domain from alphav. In this model, most of the hormone interactions are with betaA domain of the integrin. Mutagenic studies can be carried out to validate the role of these residues in directing hormone interactions.

This printed version of the Wigglesworth Lecture reviews the evidence that juvenile hormone (JH) acts on the follicular epithelium of the ovary through a membrane receptor to control access of yolk proteins to the oocyte surface. The thyroid hormones mimic this action through the same receptor. Conversely, both JH III and 3,5,3' triiodothyronine (T3) increase the activity of Ca ATPase in isolated erythrocyte membrane preparations from sheep, apparently through the same membrane receptor. These effects are mimicked by exposure of the respective tissues to CO(2). These findings suggest that the hormones arose as biotic signals, originally using existing CO(2) receptors.

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.

A recently identified thyroid hormone cell surface receptor on the extracellular domain of integrin alphaVbeta3 leads in human cell lines to activation of the mitogen-activated protein kinase (MAPK) signal transduction cascade. Examples of MAPK-dependent thyroid hormone actions are plasma membrane ion pump stimulation and specific nuclear events. These events include serine phosphorylation of the nuclear thyroid hormone receptor, leading to coactivator protein recruitment and complex tissue responses, such as thyroid hormone-induced angiogenesis or tumor cell growth. The existence of this cell surface receptor means that the activity of administered hormone could be limited through structural modification of the molecule to reproduce or inhibit only those hormone actions initiated at the cell surface. Examples of such modifications are provided.

The thyrotropin receptor (TSHr) is a member of the glycoprotein hormone receptors (GPHR), themselves, part of the rhodopsin-like G protein-coupled receptor family. The GPHR are characterized by a large aminoterminal extracellular extension responsible for the recognition and binding of their dimeric 30 kDa agonists (glycoprotein hormones thyrotropin (TSH), lutropin/chorionic gonadotropin (LH/CG) or follitropin (FSH)). This ectodomain is composed of a central portion made of nine leucine-rich motifs and two flanking domains containing several disulfide bridges. In addition of encoding the specificity for hormone recognition, the ectodomain has been shown to exert an inhibitory constraint on the constitutionally active serpentine portion of the receptor coupled in priority to Gsα. This conclusion was reached from experiments with receptor constructs harboring truncated ectodomains, which displayed an increase in their constitutive activity. When compared with the wild type receptor maximally stimulated by TSH or the most active mutants of the ectodomain (e.g. Ser281Leu), the truncated constructs showed only partial activation. Interestingly, the “beheaded” receptor could be further activated by mutations affecting the transmembrane helices of the serpentine but not by selected mutations in the exoloops of the serpentine, which are known to be potent activators of the holoreceptor. From these observations, we propose a model for TSHr activation in which binding of the agonist would switch the ectodomain from a tethered inverse agonist into a tethered agonist.

Thyrotropin or thyroid-stimulating hormone (TSH) secretion in the chicken is controlled by several hypothalamic hormones. It is stimulated by thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), whereas somatostatin (SRIH) exerts an inhibitory effect. In order to determine the mechanism by which these hypothalamic hormones modulate chicken TSH release, we examined the cellular localization of TRH receptors (TRH-R), CRH receptors type 1 (CRH-R1) and somatostatin subtype 2 receptors (SSTR2) in the chicken pars distalis by in situ hybridization (ISH), combined with immunological staining of thyrotropes. We show that thyrotropes express TRH-Rs and SSTR2s, allowing a direct action of TRH and SRIH at the level of the thyrotropes. CRH-R1 expression is virtually confined to corticotropes, suggesting that CRH-induced adrenocorticotropin release is the result of a direct stimulation of corticotropes, whereas CRH-stimulated TSH release is not directly mediated by the known chicken CRH-R1. Possibly CRH-induced TSH secretion is mediated by a yet unknown type of CRH-R in the chicken. Alternatively, a pro-opiomelanocortin (POMC)-derived peptide, secreted by the corticotropes following CRH stimulation, could act as an activator of TSH secretion in a paracrine way.

To assess the effect of histone H3 on pituitary hormone secretion, rat anterior pituitary (AP) cells were used and growth hormone, prolactin, thyrotropin, luteinizing hormone and follicle stimulating hormone measured by radioimmunoassay. Incubation of cells with H3 (1, 6, and 30 microM) stimulated the release of all five hormones in a dose-dependent manner. This effect was blocked by preincubation of H3 with an anti-H3 antibody. Incubation of AP cells with 6 microM H3 in the presence of specific AP hormone secretagogues (GRP-6, thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH)) showed additive effects on hormone secretion. Pharmacological experiments suggested that calcium- and diacylglycerol- (DAG) associated pathways, but not cAMP, participate in the hypophysiotropic activity of H3. Our results confirm previous evidence that histones may act as hypophysiotropic signals.

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of approximately 330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of (580)KVPLITVSKAK(590) was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH alpha but not FSH beta. This is in contrast to photoaffinity labeling of FSH beta by the peptide mimic of the N-terminal region of the receptor. Leu(583) and Ile(584) are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu(583) and Ile(584) side chains appear to project in opposite directions. Ile(584) appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu(583) is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu(583) improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile(584) and Lys(590) abolished the cAMP and IP induction. Our results open a logical question whether Leu(583), Ile(584), and Lys(590) interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.

The dopamine D2 receptor (D2) is involved in the regulation of pituitary hormone secretion. Two isoforms of the D2 receptor, termed D2L and D2S, have been identified. We previously generated D2L knockout mice (D2L-/-), which still express D2S. The present study examined the role of D2S and D2L in spontaneous and drug-induced pituitary hormone secretion. We found that D2L-/- mice had normal serum levels of prolactin and growth hormone. In addition, the antipsychotic drugs haloperidol and clozapine induced a similar dose-dependent increase in serum prolactin in both D2L-/- and wild-type mice. These results suggest that D2S can substitute for the function of D2L in the regulation of pituitary hormone secretion, and that the function of D2S is not dependent on the formation of a receptor heterodimer with D2L.

Luteinizing hormone receptor is a G protein-coupled receptor and consists of two halves: the N-terminal extracellular half (exodomain) and C-terminal membrane-associated half (endodomain). Hormone binds to the exodomain, and the resulting hormone-exodomain complex modulates the endodomain to generate signals. There are mutations that impair either hormone binding or signal generation. We report that the coexpression of a binding defective mutant and a signal-defective mutant rescues signal generation to produce cAMP. This rescue requires both types of mutant receptors and is dependent on the human chorionic gonadotropin dose, the surface concentration of mutant receptors, and the amino acid position of mutations. Furthermore, random collisions among mutant receptors are not involved in the rescue. Our observations provide new insights into the mechanisms of the functional and structural relationship of the exo- and endodomain, signal transduction, and receptor genetics, in particular for defective heterozygotes.

A PCR approach was used to clone thyrotropin-releasing hormone receptors (TRH-R) from the brain and anterior pituitary of the teleost Catostomus commersoni (cc), the white sucker. Two distinct TRH-R, designated ccTRH-R1 and ccTRH-R2, were identified. ccTRH-R1 was similar to mammalian TRH-R of the subtype 1, whereas ccTRH-R2 exhibited the highest identity (61% at the amino acid level) with the recently discovered rat TRH-R2. It is postulated that ccTRH-R2 and rat TRH-R2 are members of the same TRH-R subfamily 2. Functional expression of ccTRH receptors in human embryonic kidney cells and in Xenopus laevis oocytes demonstrated that both ccTRH receptors were fully functional in both systems. Oocytes expressing either receptor responded to the application of TRH by an induction of membrane chloride currents, indicating that ccTRH-R of both subtypes are coupled to the inositol phosphate/calcium pathway. The analysis of genomic clones revealed, for the first time, both similarities and differences in the structure of TRH-R subtype genes. Both ccTRH-R genes contained an intron within the coding region at the beginning of transmembrane domain (TM) 6. The position of this intron is highly conserved, as it was found at an identical position in the human TRH-R1 gene. The ccTRH-R2 gene contained an additional intron at the end of TM 3 that was not found in any of the TRH-R1 genes identified so far. The analysis of the gene structure of ccTRH-R and the amino acid sequence comparisons of mammalian and teleost TRH-R of both subtypes suggest that TRH receptors have been highly conserved during the course of vertebrate evolution. A common ancestral TRH receptor gene that could be found much earlier in evolution, possibly in invertebrates, might be the origin of ccTRH-R genes.

Mammalian gonadotropin-releasing hormone (GnRH I) is a hypothalamic decapeptide that stimulates gonadotropic hormone secretion upon interaction with its membrane receptors (type I) on pituitary cells, thereby governing reproductive processes. A second releasing hormone (GnRH II) expressed in mammals was shown earlier to be expressed in nonmammals and to have its own receptor. Here we demonstrate that a second receptor (type II) gene is present in the human genome, and report the cloning and characterization of its cDNA from monkeys. The cDNA encodes a G-protein-coupled/7 transmembrane receptor having a C-terminal cytoplasmic tail; it resembles more closely the type II receptors of amphibians and fish (approximately 55% identity) than it does the type I receptor of humans (approximately 39%). The GnRH type II receptor proved to be experimentally functional with, and specific for, GnRH II. GnRH receptor type II RNA is expressed ubiquitously in human tissues. This is the first report of a GnRH type II receptor in mammals. Its identification will permit exploration of its role in regulating gonadotropin secretion, female sexual behavior, and tumor cell growth.

The ability of G-protein-coupled receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the thyrotropin-releasing hormone receptor (TRHR) forms constitutive homo-oligomers. This formation of TRHR homo-oligomers in the absence of ligand was shown by demonstration of an energy transfer between TRHR molecules fused to either donor, Renilla luciferase (Rluc) or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This interaction was shown to be specific, since energy transfer was not detected between co-expressed tagged TRHRs and either complementary tagged gonadotropin-releasing hormone (GnRH) or β2-adrenergic receptors. Furthermore, generation of a BRET signal between the TRHRs could only be inhibited by co-expression of the wild-type TRHR and not by other GPCRs. Agonist stimulation led to a time- and dose-dependent increase in the amount of energy transfer. Inhibition of receptor internalization by co-expression of dynamin mutant K44A did not affect the interaction between TRHRs, suggesting that clustering of receptors within clathrin-coated pits is not sufficient for energy transfer to occur. BRET also provided evidence for the agonist-induced oligomerization of another GPCR, the GnRH receptor (GnRHR), and the presence of an agonist-induced interaction of the adaptor protein, β-arrestin, with TRHR and the absence of an interaction of β-arrestin with GnRHR. This study supports the usefulness of BRET as a powerful tool for studying GPCR aggregations and receptor/protein interactions in general and presents evidence that the functioning unit of TRHRs exists as homomeric complexes.

We have investigated with histochemical techniques the expression of peptides and other neurochemical markers in the hypothalamus and olfactory bulb of male mice, in which the genes encoding the alpha and beta thyroid hormone receptors (TRalpha1, TRbeta1 and TRbeta2) have been deleted. Thyrotropin-releasing hormone messenger RNA levels were increased in the hypothalamic paraventricular nucleus and in the medullary raphe nuclei of mutant mice lacking the thyroid hormone receptors alpha1 and beta (alpha1(-/-)beta(-/-)), as compared to wild-type mice. In contrast, galanin messenger RNA levels were lower in the hypothalamic paraventricular nucleus of mutant animals, as was galanin-like immunoreactivity in the internal layer of the median eminence. Substance P messenger RNA levels were unchanged in the medullary raphe nuclei. Thyrotropin-releasing hormone receptor messenger RNA levels were increased in motoneurons, unchanged in the subiculum, and lower in the amygdala of mutant animals. Galanin messenger RNA levels were unchanged in the hypothalamic dorsomedial and arcuate nuclei of the thyroid hormone receptor alpha1(-/-)beta(-/-) mice, as was the immunocytochemistry for oxytocin and for vasopressin in the hypothalamic paraventricular nucleus. A reduction in tyrosine hydroxylase messenger RNA levels was found in the arcuate nucleus of mutant mice. In the olfactory bulb, immunohistochemistry for calbindin and for tyrosine hydroxylase revealed a reduction in the intensity of labeling of nerve processes in the glomerular layer of thyroid hormone receptor alpha1(-/-)beta(-/-) mice. The tyrosine hydroxylase messenger RNA levels were also slightly reduced. In contrast, the levels of galanin and neuropeptide Y messenger RNA in this region were unchanged in thyroid hormone receptor alpha1(-/-)beta(-/-) mice as compared to wild-type mice. Together these studies reveal many regional and neurochemically selective alterations in neuronal phenotype of mice devoid of all known thyroid hormone receptors.

The parathyroid hormone (PTH) family currently includes three peptides and three receptors. PTH regulates calcium homeostasis through bone and kidney PTH1 receptors. PTH-related peptide, probably also through PTH1 receptors, regulates skeletal, pancreatic, epidermal, and mammary gland differentiation and bladder and vascular smooth muscle relaxation and has a CNS role that is under investigation. Tuberoinfundibular peptide of 39 residues (TIP39) was recently purified from bovine hypothalamus based on selective PTH2 receptor activation. PTH2 receptor expression is greatest in the CNS, where it is concentrated in limbic, hypothalamic, and sensory areas, especially hypothalamic periventricular neurons, nerve terminals in the median eminence, superficial layers of the spinal cord dorsal horn, and the caudal part of the sensory trigeminal nucleus. It is also present in a number of endocrine cells. Thus TIP39 and PTH2 receptor-influenced functions may range from pituitary and pancreatic hormone release to pain perception. A third PTH-recognizing receptor has been found in zebrafish.

Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (LeuValMetIle) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in G(s) activation.

Anti-Müllerian hormone induces the regression of fetal Müllerian ducts and inhibits the transcription of gonadal steroidogenic enzymes. It belongs to the transforming growth factor-beta family whose members signal through a pair of serine/threonine kinase receptors and Smad effectors. Only the anti-Müllerian hormone type II receptor has been identified. Our goal was to determine whether anti-Müllerian hormone could share a type I receptor with another family member. Co-immunoprecipitation of known type I receptors with anti-Müllerian hormone type II receptor clearly showed that the bone morphogenetic protein type IB receptor was the only cloned type I receptor interacting in a ligand-dependent manner with this type II receptor. Anti-Müllerian hormone also activates the bone morphogenetic protein-specific Smad1 pathway and the XVent2 reporter gene, an anti-Müllerian hormone type II receptor-dependent effect abrogated by a dominant negative version of bone morphogenetic protein type IB receptor. Reverse amplification experiments showed that bone morphogenetic protein type IB receptor is co-expressed with anti-Müllerian hormone type II receptor in most anti-Müllerian hormone target tissues. Our data support a model in which a ligand, anti-Müllerian hormone, gains access to a shared type I receptor and Smad1 system through a highly restricted type II receptor.

The luteinizing, follicle-stimulating, and thyroid-stimulating hormone receptors belong to the huge family of G-protein-coupled receptors. Identification of either activating or inactivating mutations of these receptors has led to a fundamental improvement in our understanding of glycoprotein hormone/receptor interaction. Furthermore, clinical phenotypes such as precocious puberty, follicle-stimulating hormone (FSH) insensitivity syndrome, and congenital hypthyroidism are now being explained by mutated glycoprotein hormone receptors. Since there is an ongoing worldwide search for certain clinical phenotypes that might be caused by mutations of these receptors, there is a demand for strategies and techniques that can be used to screen patients in a effective and reliable way. This article focuses, therefore, on patient selection and techniques for the detection of mutations of glycoprotein hormone receptors, and compiles useful laboratory protocols to conduct such studies.

The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is, at present, the only G-protein-coupled receptor that activates phospholipase C and lacks a C-terminal tail. We have previously demonstrated that this unique structural feature is associated with resistance to rapid desensitization of phosphoinositide signaling in COS-7 and HEK-293 cells (Heding, A., Vrecl, M., Bogerd, J., McGregor, A., Sellar, R., Taylor, P. L., and Eidne, K. A. (1998) J. Biol. Chem. 273, 11472-11477). Using receptors tagged with a nonapeptide of the influenza hemagglutinin protein to enable immunoprecipitation, we now demonstrate that the mammalian GnRH-R is not phosphorylated in an agonist-dependent manner. In contrast, the mammalian thyrotropin-releasing hormone receptor and the African catfish GnRH-R, both of which have a C-terminal tail, are phosphorylated in response to agonist challenge. Furthermore, chimeras of the mammalian GnRH-R with the C-terminal tail of either the mammalian thyrotropin-releasing hormone receptor or the catfish GnRH-R are also phosphorylated in an agonist-dependent manner. Only those receptors having C-terminal tails showed desensitization of phosphoinositide responses within 5-10 min of agonist challenge. We also show that the internalization of all these receptors when expressed transiently in COS-7 cells is similar. This dissociates receptor internalization from rapid desensitization and demonstrates that the lack of a C-terminal tail in the mammalian GnRH-R results in an inability of the receptor to undergo agonist-dependent phosphorylation and that this results directly in a resistance to rapid desensitization.

The role of brain corticotropin-releasing hormone receptors in modulating hypothalamic-pituitary-adrenal and sympathoadrenal responses to acute immobilization stress was studied in conscious rats under central corticotropin-releasing hormone receptor blockade by intracerebroventricular injection of a peptide corticotropin-releasing hormone receptor antagonist. Blood for catecholamines, adrenocorticotropic hormone and corticosterone levels was collected through vascular catheters, and brains were removed at 3 h for in situ hybridization for tyrosine hydroxylase messenger RNA in the locus coeruleus, and corticotropin-releasing hormone and corticotropin-releasing hormone receptor messenger RNA in the hypothalamic paraventricular nucleus. Central corticotropin-releasing hormone receptor blockade reduced the early increases in plasma epinephrine and dopamine, but not norepinephrine, during stress. Immobilization stress increased tyrosine hydroxylase messenger RNA levels in the locus coeruleus by 36% in controls, but not in corticotropin-releasing hormone antagonist-injected rats. In control rats, corticotropin-releasing hormone messenger RNA and type 1 corticotropin-releasing hormone receptor messenger RNA in the paraventricular nucleus increased after stress (P

Somatostatin (SST) regulates growth hormone (GH) secretion from pituitary somatotrophs by interacting with members of the SST family of G-protein-coupled receptors (sst1-5). We have used potent, nonpeptidyl SST agonists with sst2 and sst5 selectivity to determine whether these receptor subtypes are involved in regulating growth hormone releasing hormone (GHRH) stimulated secretion. GHRH stimulated GH release from pituitary cells in a dose-dependent manner, and this secretion was inhibited by Tyr(11)-SST-14, a nonselective SST analog. A sst2 selective agonist, L-779,976, potently inhibited GHRH-stimulated GH release. In addition, L-817, 818, a potent sst5 receptor selective agonist, also inhibited GH secretion, but was approximately 10-fold less potent (P0.01, ANOVA) in inhibiting GH release than either Tyr(11)-SST-14 or L-779, 976. These results show that both sst2 and sst5 receptor subtypes regulate GHRH-stimulated GH release from rat pituitary cells.

A fusion protein (beta-arrestin-1-green fluorescent protein (GFP)) was constructed between beta-arrestin-1 and a modified form of the green fluorescent protein from Aequorea victoria. Expression in HEK293 cells allowed immunological detection of an 82-kDa cytosolic polypeptide with antisera to both beta-arrestin-1 and GFP. Transient expression of this construct in HEK293 cells stably transfected to express the rat thyrotropin-releasing hormone receptor-1 (TRHR-1) followed by confocal microscopy allowed its visualization evenly distributed throughout the cytoplasm. Addition of thyrotropin-releasing hormone (TRH) caused a profound and rapid redistribution of beta-arrestin-1-GFP to the plasma membrane followed by internalization of beta-arrestin-1-GFP into distinct, punctate, intracellular vesicles. TRH did not alter the cellular distribution of GFP transiently transfected into these cells nor the distribution of beta-arrestin-1-GFP following expression in HEK293 cells lacking the receptor. To detect potential co-localization of the receptor and beta-arrestin-1 in response to agonist treatment, beta-arrestin-1-GFP was expressed stably in HEK293 cells. A vesicular stomatitis virus (VSV)-tagged TRHR-1 was then introduced transiently. Initially, the two proteins were fully resolved. Short term exposure to TRH resulted in their plasma membrane co-localization, and sustained exposure to TRH resulted in their co-localization in punctate, intracellular vesicles. In contrast, beta-arrestin-1-GFP did not relocate or adopt a punctate appearance in cells that did not express VSV-TRHR-1. Reciprocal experiments were performed, with equivalent results, following transient expression of beta-arrestin-1 into cells stably expressing VSVTRHR-1-GFP. These results demonstrate the capacity of beta-arrestin-1-GFP to interact with the rat TRHR-1 and directly visualizes their recruitment from cytoplasm and plasma membrane respectively into overlapping, intracellular vesicles in an agonist-dependent manner.

The thyrotropin-releasing hormone (TRH) receptor was expressed in embryonic fibroblasts from mice lacking the alpha subunits of Gq and G11 (Fq/11 cells) to determine whether G protein coupling is necessary for agonist-dependent receptor internalization. Neither TRH nor agonists acting on endogenous receptors increased intracellular calcium unless the cells were co-transfected with the alpha subunit of Gq. In contrast, temperature-dependent internalization of [3H]MeTRH in Fq/11 cells was the same whether Gqalpha was expressed or not. A rhodamine-labeled TRH analog and fluorescein-labeled transferrin co-localized in endocytic vesicles in Fq/11 cells, indicating that endocytosis took place via the normal clathrin pathway. Cotransfection with beta-arrestin or V53D beta-arrestin increased TRH-dependent receptor sequestration. Fq/11 cells were co-transfected with the TRH receptor and a green fluorescent protein (GFP)-beta-arrestin conjugate. GFP-beta-arrestin was uniformly distributed in the cytoplasm of untreated cells and quickly translocated to the periphery of the cells when TRH was added. A truncated TRH receptor that lacks potential phosphorylation sites in the cytoplasmic carboxyl terminus signaled but did not internalize or cause membrane localization of GFP-beta-arrestin. These results prove that calcium signaling by the TRH receptor requires coupling to a G protein in the Gq family, but TRH-dependent binding of beta-arrestin and sequestration do not.

We tested the endogenous tripeptide TRH (thyrotropin releasing hormone) ability to bind to MC (melanocortin) receptor subtypes. We discovered that TRH binds to the human MCI receptor expressed in COS cells and to murine B16 melanoma cells with 5790+/-1010 nM and 6370+/-1260 nM Ki's, respectively. TRH did not bind to the human MC3, MC4 or MC5 receptor subtypes. Moreover, TRH also stimulated cAMP production in murine B16 melanoma cells reaching the same maximum level of cAMP as found for alpha-MSH. However, several analogues of TRH, including TRH-OH, TRH-Gly-NH2 and other analogues, where each of the three amino acid residues in TRH had been exchanged by a related residue, did not bind to any of the MC receptors tested in this study. C(alpha) atoms of molecular models of TRH and the core of a MSH peptide were aligned with r.m.s. of 0.01 A. Moreover, TRH could be docked into a binding pocket of a molecular model of the MC1 receptor at only a little higher energy than a short cyclic MSH peptide. The data indicate a similarity in the mode of TRH and MSH activation of the MCI receptor.

Upon hormone binding, the activated glucocorticoid receptor (GR) functions as a transcription factor via different modes of action to control gene expression. Recent gene-targeting studies in mice provide new insight into the role of GR in vivo and are helping decipher the molecular mechanisms underlying its actions.

It has been proposed that, after agonist binding, the thyrotropin-releasing hormone receptor (TRHR) becomes internalised associated with Gq, as part of a TRH-TRHR-Gq ternary complex [13]. We tested this hypothesis directly by examining the intracellular distribution of the TRHR and Gq/11 after agonist binding. The localisation of the TRH-TRHR complex and Gq/11alpha was studied by the biochemical isolation of clathrin-coated vesicles (CCVs). The internalised TRH-TRHR complex was localised in CCVs. The CCVs, which had internalised [3H]MeTRH, contained 4-fold higher levels of radiolabelled ligand than did CCVs from cells incubated with [3H]MeTRH at 4 degrees C. Like the receptor-ligand (RL) complex, G11alpha also translocated to these endocytic vesicles. For example, CCVs from cells with internalised TRH-TRHR complexes contained G11alpha, whereas CCVs from cells without internalised RL complexes lacked G11alpha. We conclude that, after agonist-induced TRHR-G11alpha coupling, both the TRH-TRHR complex and G11alpha are internalised in CCVs.

The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends on the potency of the agonist used. Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive. Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated. Our results suggest that yeast cells would be a suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process. (C) 1998 Elsevier Science Ltd. All rights reserved.

Background: Thyroid hormones regulate many different physiological processes in different tissues in vertebrates. Most of the actions of thyroid hormones are mediated by the thyroid hormone receptor (TR), which is a member of the nuclear receptor superfamily of ligand-activated transcription regulators. There are two different genes that encode two different TRs, TRa and TRβ, and these two TRs are often co-expressed at different levels in different tissues. Most thyroid hormones do not discriminate between the two TRs and bind both with similar affinities. Results: We have designed and synthesized a thyroid hormone analog that has high affinity for the TRs and is selective in both binding and activation functions for TRβ over TRα. The compound, GC-1, was initially designed to solve synthetic problems that limit thyroid hormone analog preparation, and contains several structural changes with respect to the natural hormone 3,5,3′-triiodo-l-thyronine (T 3 ). These changes include replacement of the three iodines with methyl and isopropyl groups, replacement of the biaryl ether linkage with a methylene linkage, and replacement of the amino-acid sidechain with an oxyacetic-acid sidechain. Conclusions: The results of this study show that GC-1 is a member of a new class of thyromimetic compounds that are more synthetically accessible than traditional thyromimetics and have potentially useful receptor binding and activation properties. The TRβ selectivity of GC-1 is particularly interesting and suggests that GC-1 might be a useful in vivo probe for studying the physiological roles of the different thyroid hormone receptor isoforms.