Search

Activation of thyrotropin-releasing hormone receptor (TRH-R) signaling has an irreplaceable role in a number of cellular processes. Thyroliberin (TRH) plays an important role in the regulation of secretion of other hormones and there are also mentions of its possible antiapoptotic and neuroprotective effects. On the other hand, TRH is quickly degraded and also its other properties, such as cardiac and endocrine side effects and low lipophilicity, disadvantage the therapeutic applications of this hormone compared to its various analogues. Due to their effects on the central nervous systém, TRH and its analogues represent a potential possibility for the treatment of various neurological diseasses, including neurodegenerative disorders. The molecular mechanisms responsible for the beneficial effects of TRH and its analogues have not yet been fully elucidated. So far, little is known about the involvement of TRH, or its analogues, in the regulation of energy metabolism or impact on the cellular phosphoproteome. In the first part of the thesis, we focused on the study of the effect of TRH and interacting partners of TRH-R on the lateral mobility of this receptor in the membrane of the TRY-1 cell line stably expressing TRH-R labeled with yellow fluorescent protein (YFP) at the C-terminus. The results of...

The physiological roles of thyrotropin-releasing hormone (TRH) are proposed to be mediated by TRH receptors (TRHR), which have been divided into 3 subtypes, namely, TRHR1, TRHR2, and TRHR3, in vertebrates. Although 2 TRH receptors (TRHR1 and TRHR3) have been predicted to exist in birds, it remains unclear whether TRHR3 is a functional TRH receptor similar to TRHR1. Here, we reported the functionality and tissue expression of TRHR3 in chickens. The cloned chicken TRHR3 (cTRHR3) encodes a receptor of 387 amino acids, which shares high-amino-acid identities (63–80%) to TRHR3 of parrots, lizards, Xenopus tropicalis, and tilapia and comparatively lower sequence identities to chicken TRHR1 or mouse TRHR2. Using cell-based luciferase reporter assays and Western blot, we demonstrated that similar to chicken TRHR1 (cTRHR1), cTRHR3 expressed in HEK 293 cells can be potently activated by TRH and that its activation stimulates multiple signaling pathways, indicating both TRH receptors are functional. Quantitative real-time PCR revealed that cTRHR1 and cTRHR3 are widely, but differentially, expressed in chicken tissues, and their expression is likely controlled by promoters located upstream of exon 1, which display strong promoter activities in cultured DF-1 cells. cTRHR1 is highly expressed in the anterior pituitary and testes, while cTRHR3 is highly expressed in the muscle, testes, fat, pituitary, spinal cord, and many brain regions (including hypothalamus). These findings indicate that TRH actions are likely mediated by 2 TRH receptors in chickens. In conclusion, our data provide the first piece of evidence that both cTRHR3 and cTRHR1 are functional TRH receptors, which helps to elucidate the physiological roles of TRH in birds.

After we identified pGlu-βGlu-Pro-NH2 as the first functional antagonist of the cholinergic central actions of the thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2), we became interested in finding the receptor-associated mechanism responsible for this antagonism. By utilizing a human TRH receptor (hTRH-R) homology model, we first refined the active binding site within the transmembrane bundle of this receptor to enhance TRH’s binding affinity. However, this binding site did not accommodate the TRH antagonist. This directed us to consider a potential allosteric binding site in the extracellular domain (ECD). Searches for ECD binding pockets prompted the remodeling of the extracellular loops and the N-terminus. We found that different trajectories of ECDs produced novel binding cavities that were then systematically probed with TRH, as well as its antagonist. This led us to establish not only a surface-recognition binding site for TRH, but also an allosteric site that exhibited a selective and high-affinity binding for pGlu-βGlu-Pro-NH2. The allosteric binding of this TRH antagonist is more robust than TRH’s binding to its own active site. The findings reported here may shed light on the mechanisms and the multimodal roles by which the ECD of a TRH receptor is involved in agonist and/or antagonist actions.

MicroRNAs (miRNA) are believed to play a crucial role in the cause and treatment of temporal lobe epilepsy (TLE) by controlling gene expression in different stages of the disease. To investigate role of miRNA in the latent stage following status epilepticus, we first compared microRNA expression profiles in mice hippocampus at 1 week after pilocarpine-induced status epilepticus (SE) vs. controls in hippocampal tissues using Exiqon miRCURY LNA™ miRNAs Array. Then, the target genes of altered miRNAs were predicted using both TargetScan 7.1 and miRDB V5, and were further selected by intersecting with another independent mRNA expression profile dataset from the samples at the same time point. We found out 14 common genes as down miRNA target (up-mRNA) and 4 common genes as up miRNA target (down mRNA) in SE mice. miR-669m-3p-TRHR (thyrotropin releasing hormone receptor), miR-669m-3p-B3galt2 (β-1,3-Galactosyltransferase 2), miR-105-PDPN (Podoplanin) and miR-883b-3p-CLEC-2 (C-type-lectin-like-2) were found to be potential molecular mechanisms to modulate the calcium signaling pathway, glycosylation pathways and chemokine mediated inflammatory processes in mice hippocampus at 1 week after pilocarpine-induced SE, respectively. Our results offered potential novel insights into the cellular events in the mice hippocampus mediated by miRNASs-target genes that shape SE-evoked epileptogenesis.

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.

Background/Aims: Triclosan, as an antimicrobial agent and a potential endocrine disruptor, has been used extensively in diverse products, resulting in widespread human exposure. In recent years, studies suggest that triclosan could disturb thyroid functions and decline thyroid hormones (THs). Methods: To verify our hypothesis that the MAPK pathway may function significantly in triclosan-induced hypothyroidism, Sprague-Dawley rats were gavaged with triclosan for 31 consecutive days; Nthy-ori 3-1 cells were treated with triclosan in the presence/absence of NAC, inhibitors (SB203580 and SB202474), or TRHr siRNA. Tissues and/or cells were analyzed by several techniques including transmission electron microscopy, confocal laser scanning microscopy, gene silencing, western blot, and real-time PCR. Results: Triclosan led to histopathologic changes in the thyroid and decreases in triiodothyronine (T3) and thyroxine (T4). Triclosan stimulated ROS production and oxidative stress occurrence, thereby activating the p38 pathway in vivo and in vitro. Thyrotropin releasing hormone receptor (TRHr) was induced when the p38 pathway was activated, and was suppressed when that pathway was inhibited. Moreover, thyroid peroxidase (TPO) was restrained and modulated by the p38/TRHr pathway after triclosan treatment. Furthermore, deiodinase 3 (D3) and hepatic enzymes (Ugt2b1, CYP1a1, CYP1a2, CYP2b1, CYP3a1, and Sult1e1) were also induced by triclosan. Conclusion: Taken together, p38/TRHr-dependent regulation of TPO in thyroid cells contributes to the hypothyroidism of triclosan-treated rats.

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.

ZFP36L2 (L2) destabilizes AU-rich element (ARE)-containing transcripts and has been implicated in female fertility. We have shown that only one of three putative AREs within the 3′ UTR of murine luteinizing hormone receptor mRNA, ARE2197 (UAUUUAU), is capable of interacting with L2. To assess whether structural elements of ARE2197 could explain this unique binding ability, we performed whole-transcript SHAPE-MaP (selective 2′ hydroxyl acylation by primer extension-mutational profiling) of the full-length mLHR mRNA. The data revealed that the functional ARE2197 is located in a hairpin loop structure and most nucleotides are highly reactive. In contrast, each of the nonbinding AREs, 2301 and 2444, contains only a pentamer AUUUA; and in ARE2301 much of the ARE sequence is poorly accessible. Because the functional mARE was also found to be conserved in humans at the sequence level (ARE 2223), we decided to investigate whether binding and structure are also preserved. Similar to mouse, only one ARE in hLHR mRNA is capable of binding to L2; and it is also located in a hairpin structure, based on our SHAPE-MaP data. To investigate the role of secondary structure in the binding, we mutated specific nucleotides in both functional AREs. Mutations in the flexible stem region proximal to the loop that enforce strong base-pairing, drastically reduced L2 binding affinity; this confirms that the structural context is critical for L2 recognition of hARE2223. Collectively, our results suggest that a combination of minimal ARE sequence, placement of the ARE in a hairpin loop, and stem flexibility mediate high-affinity L2 binding to hLHR mRNA.

Thyroid stimulating hormone receptor (TSHR) plays a pivotal role in thyroid hormone metabolism. It is a major controller of thyroid cell function and growth. Mutations in TSHR may lead to several thyroid diseases, most commonly hyperthyroidism. Although its genetic and epigenetic alterations do not directly lead to carcinogenesis, it has a crucial role in tumor growth, which is initiated by several oncogenes. This article will provide a brief review of TSHR and related diseases.Tiroid stimulan hormon reseptörü (TSHR) tiroid hormon metabolizmasında merkezi bir rol oynar. Tiroid hücrelerinin fonksiyonlarının ve büyümesinin kontrolünde yardımcıdır. TSHR mutasyonları bir dizi tiroid hastalığına, en çok hipertiroidizme neden olmaktadır. Her ne kadar bu reseptörün genetik ve epigenetik değişiklikleri direkt olarak karsinogeneze yol açmasa da, farklı onkogenler tarafından başlatılmış tümörün büyümesinde ciddi bir rol oynar. Bu makalede TSHR ve ilişkili hastalıklar özetlenmektedir.

Integrin αVβ3 is a structural protein of the plasma membrane that transduces signals from extracellular matrix proteins and has recently been shown to contain a novel receptor for thyroid hormone. Thyroid hormone signals are converted by αVβ3 into mitogen-activated protein kinase (MAPK) (ERK1/2) activation and downstream intracellular events in the cell nucleus. The latter include post-translational modification of the nuclear thyroid hormone receptor (TRβ1) and complex cellular or tissue responses, such as hormone-induced angiogenesis via basic fibroblast growth factor release. The integrin receptor for thyroid hormone has been shown to mediate proliferative effects of the hormone on certain tumor cell lines, including murine glioma/glioblastoma cells and human breast cancer (MCF-7) cells. More than one mechanism may account for this hormonal action, but in vitro studies indicate a direct hormonal action on cellular proliferation. Other possible mechanisms involve indirect actions via the release of tumor growth factors and effects on cell migration. In the intact organism, support of tumor growth by thyroid hormone is postulated to include angiogenesis. Crosstalk between the integrin thyroid hormone receptor and the epidermal growth factor receptor on the plasma membrane may be another mechanism by which thyroid hormone may modify tumor cell growth. Tetraiodothyroacetic acid (tetrac) is an iodothyronine analog that has no agonist activity at the integrin receptor, but inhibits binding of l-thyroxine and 3,5,3´-triiodo-l-thyronine to the receptor, preventing MAPK activation and consequent actions downstream of MAPK. In vitro studies and a preliminary in vivo experiment indicate that tetrac blocks the action of thyroid hormone on tumor cell proliferation. Both unmodified tetrac and tetrac reformulated as a nanoparticle that does not gain access to the cell interior are under investigation in animal models as anticancer agents. Also under study is the susceptibility of other human cancer cell lines to induction of proliferation by physiological concentrations of thyroid hormone.

Graves' disease is an autoimmune disorder that causes hyperthyroidism because of autoantibodies that bind to the thyroid-stimulating hormone receptor (TSHR) on the thyroid gland, triggering thyroid hormone release. The physiological control of thyroid hormone homeostasis by the feedback loops involving the hypothalamus-pituitary-thyroid axis is disrupted by these stimulating autoantibodies. To reset the endogenous thyrotrophic feedback control, we designed a synthetic mammalian gene circuit that maintains thyroid hormone homeostasis by monitoring thyroid hormone levels and coordinating the expression of a thyroid-stimulating hormone receptor antagonist (TSHAntag), which competitively inhibits the binding of thyroid-stimulating hormone or the human autoantibody to TSHR. This synthetic control device consists of a synthetic thyroid-sensing receptor (TSR), a yeast Gal4 protein/human thyroid receptor-α fusion, which reversibly triggers expression of the TSHAntag gene from TSR-dependent promoters. In hyperthyroid mice, this synthetic circuit sensed pathological thyroid hormone levels and restored the thyrotrophic feedback control of the hypothalamus-pituitary-thyroid axis to euthyroid hormone levels. Therapeutic plug and play gene circuits that restore physiological feedback control in metabolic disorders foster advanced gene- and cell-based therapies.

Thyroid hormone (T3) activates (positive regulation) or represses (negative regulation) target genes at the transcriptional level. The molecular mechanism of the former has been elucidated in detail; however, the mechanism for negative regulation has not been established. The best example of the gene that is negatively regulated by T3 is the thyrotropin (thyroid-stimulating hormone) β subunit (TSHβ) gene. Analogous to the T3-responsive element (TRE) in positive regulation, a negative TRE (nTRE) has been postulated in the TSHβ gene. However, TSHβ promoter analysis, performed in the presence of transcription factors Pit1 and GATA2, which are determinants of thyrotroph differentiation in the pituitary, revealed that the nTRE is dispensable for inhibition by T3. We propose a tethering model in which the T3 receptor is tethered to GATA2 via protein-protein interaction and inhibits GATA2-dependent transactivation of the TSHβ gene in a T3-dependent manner.

Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor β isoform (TRβ4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRβ4 from a molecular basis. Temporal expression of TRβ4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRβ1 is potentially stable. TRβ1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRβ4 in a dose-dependent manner. Thus, TRβ4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRβ1 and TRβ4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRβ4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRβ1. In contrast, TRβ4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRβ1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRβ4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRβs, we found that both TRβ1 and TRβ4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRβs within the nucleus.

The type-1 parathyroid hormone receptor (PTHR1) is a family B G protein-coupled receptor (GPCR) that mediates the actions of two polypeptide ligands; parathyroid hormone (PTH), an endocrine hormone that regulates the levels of calcium and inorganic phosphate in the blood by acting on bone and kidney, and PTH-related protein (PTHrP), a paracrine-factor that regulates cell differentiation and proliferation programs in developing bone and other tissues. The type-2 parathyroid hormone receptor (PTHR2) binds a peptide ligand, called tuberoinfundibular peptide-39 (TIP39), and while the biologic role of the PTHR2/TIP39 system is not as defined as that of the PTHR1, it likely plays a role in the central nervous system as well as in spermatogenesis. Mechanisms of action at these receptors have been explored through a variety of pharmacological and biochemical approaches, and the data obtained support a basic "two-site" mode of ligand binding now thought to be used by each of the family B peptide hormone GPCRs. Recent crystallographic studies on the family B GPCRs are providing new insights that help to further refine the specifics of the overall receptor architecture and modes of ligand docking. One intriguing pharmacological finding for the PTHR1 is that it can form surprisingly stable complexes with certain PTH/PTHrP ligand analogs and thereby mediate markedly prolonged cell signaling responses that persist even when the bulk of the complexes are found in internalized vesicles. The PTHR1 thus appears to be able to activate the Gα(s)/cAMP pathway not only from the plasma membrane but also from the endosomal domain. The cumulative findings could have an impact on efforts to develop new drug therapies for the PTH receptors.

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.

Ghrelin is an orexigenic peptide hormone that primarily regulates growth hormone secretion, food intake, and energy homeostasis. It has been shown to also play a role in numerous higher brain functions, such as the regulation of inflammation and cell proliferation. Ghrelin is the endogenous ligand of the growth hormone secretagogue receptor (GHSR), a G-protein-coupled receptor highly expressed in brain and detectable in some peripheral tissues. The wide distribution of ghrelin receptor and the number of tissues and cell types known to respond to ghrelin suggest that a number of systems may be affected by treatment with this hormone or its analogues. In this study, we characterized a new GHSR specific monoclonal antibody recognizing specifically the ghrelin receptor. This could be a useful tool for immunoassays aimed at obtaining insights into the physiological and pathological significance of the GHSR/ghrelin system.

This study quantified the agonist-induced endocytotic and recycling events of the mammalian gonadotropin releasing hormone receptor (GnRH-R) and investigated the role of the intracellular carboxyl (C)-terminal tail in regulating agonist-induced receptor internalization kinetics. The rate of internalization for the rat GnRH-R was found to be exceptionally low when compared with G-protein coupled receptors (GPCRs) which possess a cytoplasmic C-terminal tail (thyrotropin-releasing hormone receptor (TRH-R), catfish GnRH-R (cfGnRH-R) and GnRH/TRH-R chimeric receptor). These data provide evidence that the presence of a functional intracellular cytoplasmic C-terminal tail is essential for rapid internalization of the studied GPCRs.

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.

The primary response to a stressful event is activation of the hypothalamic–pituitary–adrenal axis. The stress response results in the synthesis and release of corticotropin-releasing hormone (CRH) from the hypothalamus, followed by an increased release of proopiomelanocortin peptides (adrenocorticotropic hormone, melanocyte-stimulating hormones, and endorphins) from the pituitary gland. Adrenocorticotropic hormone (ACTH) acts on the adrenal gland to cause release of glucocorticoids. The initial isolation of CRH, a 41-amino acid peptide, was based on its function as the major secretagogue of ACTH. Three more mammalian CRH-related ligands, urocortin (Ucn), urocortin II (Ucn 2), and urocortin III (Ucn 3), have now been cloned. The importance of the CRH system as a major integrator of behavioral, autonomic and endocrine responses to stress is underscored by its effects on learning and memory, Alzheimer's and Parkinson disease, appetite and weight control, addiction, cancer and a possible role in affective disorders such as melancholic depression. The diverse functions of the CRH peptides in physiology and disease is attributed to the downstream signaling pathways they modulate in a diverse number of cell types and tissues and disease states.

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.

We recently reported that the novel thyroid hormone receptor β (TRβ) selective agonists SKL-12846 and SKL-13784 reduce blood cholesterol levels without affecting thyroid-stimulating hormone (TSH) in cholesterol-fed rats. Our aim in this study was to elucidate what sets apart these SKL-compounds as TRβ agonists with no effect on TSH. To this end, we determined SKL-compounds pharmacokinetics and tissue distribution in normal rats and compared them to those of GC-1, a liver-selective TRβ agonist with concomitant effect on TSH. The present study explains why SKL-12846 and SKL-13784 have beneficial effects on lowering lipids without affecting heart rate and TSH production at the therapeutic dose in cholesterol-fed rats. In addition, we found that SKL-13784 shows no sign of escape phenomenon in fructose-fed rats. These results demonstrate the advantages of extremely high liver specificity to TRβ agonists. However, SKL-13784 has been found significantly to reduce endogenous T4 levels at doses lower than its lipid-lowering dose, which may raise concerns over this compound's ability to alter thyroid hormone metabolism in the liver. While the mechanism by which SKL-13784 reduces endogenous T4 levels is still unclear, our results would help design better liver-selective TRβ modulators.

The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain.

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 receptor type 2 (TRH-R2), not TRH-R1, has been proposed to mediate the CNS effects of TRH and its more effective analog taltirelin (TAL). Consistent with this idea, TAL exhibited higher binding affinity and signaling potency at mouse TRH-R2 than TRH-R1 in a model cell system. We used TRH-R1 knockout (R1ko), R2ko and R1/R2ko mice to determine which receptor mediates the CNS effects of TAL. There was no TRH-R1 mRNA in R1ko and R1/R2ko mice and no TRH-R2 mRNA in R2ko and R1/R2ko mice. Specific [(3)H]MeTRH binding to whole brain membranes was 5% of wild type (WT) for R1ko mice, 100% for R2ko mice and 0% for R1/R2ko mice, indicating TRH-R1 is the predominant receptor expressed in the brain. In arousal assays, TAL shortened sleep time with pentobarbital sedation in WT and R2ko mice by 44 and 49% and with ketamine/xylazine sedation by 66 and 55%, but had no effect in R1ko and R1/R2ko mice. In a tail flick assay of nociception, TAL increased response latency by 65 and 70% in WT and R2ko mice, but had no effect in R1ko and R1/R2ko mice. In a tail suspension test of depression-like behavior, TAL increased mobility time by 49 and 37% in WT and R2ko mice, but had no effect in R1ko and R1/R2ko mice. Thus, in contrast to the generally accepted view that the CNS effects of TAL are mediated by TRH-R2, these effects are mediated primarily if not exclusively by TRH-R1 in mice.

Arginine is one of the most common natural amino acids that takes part to the structure of the messenger ribonucleic acid (mRNA). In mammals l-arginine is a semiessential or an essential amino acid depending on age.

The gut-derived hormone ghrelin regulates growth hormone release, appetite, metabolism, and immune function through its receptor, the growth hormone secretagogue receptor 1a (GHSR1a). In this issue of Science Signaling, Chebani et al decode GHSR1a signaling by using transgenic rats with a mutation in GHSR1a that prevents its interaction with β-arrestin. These mutant rats are more responsive to endogenous ghrelin, resulting in increased fat deposition and insulin resistance without affecting food intake.

The patent claims peptidic/nonpeptidic inhibitors of the ghrelin receptor, the growth hormone secretagogue receptor (GHSR) 1A. Among these compounds, it was disclosed that the addition in some compounds of a GlyMetAla tripeptide at the N-terminus of the ghrelin peptide agonists converts them into ghrelin receptor antagonists. One of these peptides, among the few that have been studied in vivo, was shown to be able to reduce food intake and body weight gain in rats.

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

Aiming to apply the multivalency concept to melanoma imaging, we have assessed the in vivo melanocortin type 1 receptor (MC1R)-targeting properties of 99mTc(I)-labeled homobivalent peptide conjugates which contain copies of the α-melanocyte-stimulating hormone (α-MSH) analog [Ac-Nle4, Asp5, d-Phe7, Lys11]α-MSH4–11 separated by linkers of different length (L 2 nine atoms and L 3 14 atoms). The MC1R-binding affinity of L 2 and L 3 is significantly higher than that of the monovalent conjugate L 1 . Metallation of these conjugates yielded the complexes fac-[M(CO)3(k3-L)]+ (M is 99mTc/Re; 1/1a, L is L 1 ; 2/2a, L is L 2 ; 3/3a, L is L 3 ), with IC50 values in the subnanomolar and nanomolar range. The MC1R-mediated internalization of 2 and 3 is higher than that of 1 in B16F1 melanoma cells. Biodistribution studies in melanoma-bearing mice have shown low nonspecific accumulation with a tumor uptake that correlates with IC50 values. However, no correlation between tumor uptake and valency was found. Nevertheless, 2 displayed the highest tumor retention, and the best tumor to nontarget organ ratios. M (I)-Homobivalent α-MSH analogs for melanocortin-1 receptor targeting (M = 99mTc, Re) have been synthesized and biologically evaluated.

Clonidine is an α2-adrenergic agonist, historically known to treat high blood pressure. Further studies showed that it could be used in the treatment of neuropsychiatric disorders. Afterwards, it has been reported that clonidine stimulated growth hormone (GH) release in many species including man. Using a transnasal surgery technique in awake sheep that allowed accessing hypothalamopituitary portal vessels, our laboratory previously reported that the injection of clonidine in sheep induced a significant, immediate and short-lasting increase in peripheral GH and portal GH-releasing hormone (GHRH) levels. In this study, we show that the clonidine-induced peripheral GH and portal GHRH increase in sheep appears to be mediated by the tachykinin NK2 receptor.

Purpose of review This review summarizes our current understanding of the role of gonadotropin-releasing hormone (GnRH)/GnRH receptor (GnRHR) signaling at the maternal-fetal interface. Recent findings Several isoforms of GnRH and GnRHR are described. The hypothalamic decapeptide, GnRH-I, binds to the anterior pituitary and induces the synthesis and secretion of luteinizing hormone and follicle-stimulating hormone. It is also found in extrahypothalamic sites. A second isoform, GnRH-II, acts both in the hypothalamus and other organ systems, including placenta, breast, endometrium, and ovary. Although several putative isoforms of GnRHR have been identified, it is clear that, in humans, both GnRH-I and GnRH-II signal through a single receptor, GnRHR-I. GnRH-I, GnRH-II, and GnRHR-I mRNA and protein have been identified in placenta and regulate the β-subunit of human chorionic gonadotropin production, which is essential for the maintenance of early pregnancy. They may also play a role in the autocrine/paracrine regulation of trophoblast invasion through extracellular matrix remodeling. Summary GnRH-I and GnRH-II have multiple extrapituitary roles. In placenta, they bind to GnRHR-I to stimulate the production of β-subunit of human chorionic gonadotropin. They may also play a role in trophoblast invasion. A better understanding of the molecular mechanisms involved in GnRH/GnRHR signaling at the maternal-fetal interface may identify novel roles for GnRH agonists/antagonists in the prevention or treatment of hormonally mediated diseases.

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.

Gonadotropin-releasing hormone (GnRH) receptors are not only detected in the central nervous system but also in tissues such as ovary, endometrium, breast, gastrointestinal system, placenta and malignant tumors of ovary and breast. The direct role of GnRH-antagonists in ovarian function, implantation, cancer pathogenesis and treatment is under extensive investigation. This study reviews the biochemistry and molecular and cellular biology of GnRH-antagonists as well as GnRH types and their receptors.The best clinical evidence with GnRH-antagonists has accumulated in controlled ovarian hyperstimulation protocols for prevention of premature luteinizing hormone surge (cetrorelix, ganirelix) and in the treatment of advanced-stage prostate cancer (abarelix and degarelix). GnRH-GnRH receptor pathways may have a role in the embryo implantation. The controversy still exists whether GnRH antagonist protocols result in slightly decreased clinical pregnancy rates compared with the GnRH agonist protocols. GnRH-antagonists could be used in the near future to treat some cancer types that express GnRH receptors.GnRH-antagonists have various clinical applications in gynecology, reproductive medicine, urology and oncology. The emergence of well tolerated, orally active GnRH-antagonists may provide an alternative to long-term injections and is likely to have a major impact on the utility of GnRH analogues in the treatment of human diseases.

The SSFA-GPHR (Sequence-Structure-Function-Analysis of Glycoprotein Hormone Receptors) database provides a comprehensive set of mutation data for the glycoprotein hormone receptors (covering the lutropin, the FSH, and the TSH receptors). Moreover, it provides a platform for comparison and investigation of these homologous receptors and helps in understanding protein malfunctions associated with several diseases. Besides extending the data set (> 1100 mutations), the database has been completely redesigned and several novel features and analysis tools have been added to the web site. These tools allow the focused extraction of semiquantitative mutant data from the GPHR subtypes and different experimental approaches. Functional and structural data of the GPHRs are now linked interactively at the web interface, and new tools for data visualization (on three-dimensional protein structures) are provided. The interpretation of functional findings is supported by receptor morphings simulating intramolecular changes during the activation process, which thus help to trace the potential function of each amino acid and provide clues to the local structural environment, including potentially relocated spatial counterpart residues. Furthermore, double and triple mutations are newly included to allow the analysis of their functional effects related to their spatial interrelationship in structures or homology models. A new important feature is the search option and data visualization by interactive and user-defined snake-plots. These new tools allow fast and easy searches for specific functional data and thereby give deeper insights in the mechanisms of hormone binding, signal transduction, and signaling regulation. The web application “Sequence-Structure-Function-Analysis of GPHRs” is accessible on the internet at http://www.ssfa-gphr.de/.