P38/TRHr-Dependent Regulation of TPO in Thyroid Cells Contributes to the Hypothyroidism of Triclosan-Treated Rats.

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.

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

OBJECTIVES: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins. METHODS: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP). RESULTS: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gß, ß-arrestin2 and phospholipase Cß1, but not of Giα1, ß-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and ß-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling. CONCLUSION: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.

Possible involvement of thyrotropin-releasing hormone receptor 3 in the release of prolactin in the metamorphosing bullfrog larvae.

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.

Contributions of Rod and Cone Pathways to Retinal Direction Selectivity Through Development.

UNLABELLED: Direction selectivity is a robust computation across a broad stimulus space that is mediated by activity of both rod and cone photoreceptors through the ON and OFF pathways. However, rods, S-cones, and M-cones activate the ON and OFF circuits via distinct pathways and the relative contribution of each to direction selectivity is unknown. Using a variety of stimulation paradigms, pharmacological agents, and knockout mice that lack rod transduction, we found that inputs from the ON pathway were critical for strong direction-selective (DS) tuning in the OFF pathway. For UV light stimulation, the ON pathway inputs to the OFF pathway originated with rod signaling, whereas for visible stimulation, the ON pathway inputs to the OFF pathway originated with both rod and M-cone signaling. Whole-cell voltage-clamp recordings revealed that blocking the ON pathway reduced directional tuning in the OFF pathway via a reduction in null-side inhibition, which is provided by OFF starburst amacrine cells (SACs). Consistent with this, our recordings from OFF SACs confirmed that signals originating in the ON pathway contribute to their excitation. Finally, we observed that, for UV stimulation, ON contributions to OFF DS tuning matured earlier than direct signaling via the OFF pathway. These data indicate that the retina uses multiple strategies for computing DS responses across different colors and stages of development. SIGNIFICANCE STATEMENT: The retina uses parallel pathways to encode different features of the visual scene. In some cases, these distinct pathways converge on circuits that mediate a distinct computation. For example, rod and cone pathways enable direction-selective (DS) ganglion cells to encode motion over a wide range of light intensities. Here, we show that although direction selectivity is robust across light intensities, motion discrimination for OFF signals is dependent upon ON signaling. At eye opening, ON directional tuning is mature, whereas OFF DS tuning is significantly reduced due to a delayed maturation of S-cone to OFF cone bipolar signaling. These results provide evidence that the retina uses multiple strategies for computing DS responses across different stimulus conditions.

Taltirelin alleviates fatigue-like behavior in mouse models of cancer-related fatigue.

Fatigue affects most cancer patients and has numerous potential causes, including cancer itself and cancer treatment. Cancer-related fatigue (CRF) is not relieved by rest, can decrease quality of life, and has no FDA-approved therapy. Thyrotropin-releasing hormone (TRH) has been proposed as a potential novel treatment for CRF, but its efficacy against CRF remains largely untested. Thus, we tested the TRH analog, taltirelin (TAL), in mouse models of CRF. To model fatigue, we used a mouse model of chemotherapy, a mouse model of radiation therapy, and mice bearing colon 26 carcinoma tumors. We used the treadmill fatigue test to assess fatigue-like behavior after treatment with TAL. Additionally, we used wild-type and TRH receptor knockout mice to determine which TRH receptor was necessary for the actions of TAL. Tumor-bearing mice displayed muscle wasting and all models caused fatigue-like behavior, with mice running a shorter distance in the treadmill fatigue test than controls. TAL reversed fatigue-like behavior in all three models and the mouse TRH1 receptor was necessary for the effects of TAL. These data suggest that TAL may be useful in alleviating fatigue in all cancer patients and provide further support for evaluating TAL as a potential therapy for CRF in humans.

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP.

UNLABELLED: Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. IN CONCLUSION: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling.

Exploring the larval transcriptome of the common sole (Solea solea L.).

BACKGROUND: The common sole (Solea solea) is a promising candidate for European aquaculture; however, the limited knowledge of the physiological mechanisms underlying larval development in this species has hampered the establishment of successful flatfish aquaculture. Although the fact that genomic tools and resources are available for some flatfish species, common sole genomics remains a mostly unexplored field. Here, we report, for the first time, the sequencing and characterisation of the transcriptome of S. solea and its application for the study of molecular mechanisms underlying physiological and morphological changes during larval-to-juvenile transition. RESULTS: The S. solea transcriptome was generated from whole larvae and adult tissues using the Roche 454 platform. The assembly process produced a set of 22,223 Isotigs with an average size of 726 nt, 29 contigs and a total of 203,692 singletons. Of the assembled sequences, 75.2% were annotated with at least one known transcript/protein; these transcripts were then used to develop a custom oligo-DNA microarray. A total of 14,674 oligonucleotide probes (60 nt), representing 12,836 transcripts, were in situ synthesised onto the array using Agilent non-contact ink-jet technology. The microarray platform was used to investigate the gene expression profiles of sole larvae from hatching to the juvenile form. Genes involved in the ontogenesis of the visual system are up-regulated during the early stages of larval development, while muscle development and anaerobic energy pathways increase in expression over time. The gene expression profiles of key transcripts of the thyroid hormones (TH) cascade and the temporal regulation of the GH/IGF1 (growth hormone/insulin-like growth factor I) system suggest a pivotal role of these pathways in fish growth and initiation of metamorphosis. Pre-metamorphic larvae display a distinctive transcriptomic landscape compared to previous and later stages. Our findings highlighted the up-regulation of gene pathways involved in the development of the gastrointestinal system as well as biological processes related to folic acid and retinol metabolism. Additional evidence led to the formation of the hypothesis that molecular mechanisms of cell motility and ECM adhesion may play a role in tissue rearrangement during common sole metamorphosis. CONCLUSIONS: Next-generation sequencing provided a good representation of the sole transcriptome, and the combination of different approaches led to the annotation of a high number of transcripts. The construction of a microarray platform for the characterisation of the larval sole transcriptome permitted the definition of the main processes involved in organogenesis and larval growth.

Persistent signaling by thyrotropin-releasing hormone receptors correlates with G-protein and receptor levels.

G-protein-coupled receptors with dissociable agonists for thyrotropin, parathyroid hormone, and sphingosine-1-phosphate were found to signal persistently hours after agonist withdrawal. Here we show that mouse thyrotropin-releasing hormone (TRH) receptors, subtypes 2 and 1(TRH-R2 and TRH-R1), can signal persistently in HEK-EM293 cells under appropriate conditions, but TRH-R2 exhibits higher persistent signaling activity. Both receptors couple primarily to Gα(q/11). To gain insight into the mechanism of persistent signaling, we compared proximal steps of inositolmonophosphate (IP1) signaling by TRH-Rs. Persistent signaling was not caused by slower dissociation of TRH from TRH-R2 (t(1/2)=77 ± 8.1 min) compared with TRH-R1 (t(1/2)=82 ± 12 min) and was independent of internalization, as inhibition of internalization did not affect persistent signaling (115% of control), but required continuously activated receptors, as an inverse agonist decreased persistent signaling by 60%. Gα(q/11) knockdown decreased persistent signaling by TRH-R2 by 82%, and overexpression of Gα(q/11) induced persistent signaling in cells expressing TRH-R1. Lastly, persistent signaling was induced in cells expressing high levels of TRH-R1. We suggest that persistent signaling by TRHRs is exhibited when sufficient levels of agonist/receptor/G-protein complexes are established and maintained and that TRH-R2 forms and maintains these complexes more efficiently than TRH-R1.

Ectopic expression of thyrotropin releasing hormone (TRH) receptors in liver modulates organ function to regulate blood glucose by TRH.

Maintenance of blood glucose by the liver is normally initiated by extracellular regulatory molecules such as glucagon and vasopressin triggering specific hepatocyte receptors to activate the cAMP or phosphoinositide signal transduction pathways, respectively. We now show that the normal ligand-receptor regulators of blood glucose in the liver can be bypassed using an adenovirus vector expressing the mouse pituitary thyrotropin releasing hormone receptor (TRHR) cDNA ectopically in rat liver in vivo. The ectopically expressed TRHR links to the phosphoinositide pathway, providing a means to regulate liver function with TRH, an extracellular ligand that does not normally affect hepatic function. Administration of TRH to these animals activates the phosphoinositide pathway, resulting in a sustained rise in blood glucose. It should be possible to use this general strategy to modulate the differentiated functions of target organs in a wide variety of pathologic states.

Regulation of phosphosignaling pathways involved in transcription of cell cycle target genes by TRH receptor activation in GH1 cells.

Thyrotropin-releasing hormone (TRH) is known to activate several cellular signaling pathway, but the activation of the TRH receptor (TRH-R) has not been reported to regulate gene transcription. The aim of this study was to identify phosphosignaling pathways and phosphoprotein complexes associated with gene transcription in GH1 pituitary cells treated with TRH or its analog, taltirelin (TAL), using label-free bottom-up mass spectrometry-based proteomics. Our detailed analysis provided insight into the mechanism through which TRH-R activation may regulate the transcription of genes related to the cell cycle and proliferation. It involves control of the signaling pathways for ß-catenin/Tcf, Notch/RBPJ, p53/p21/Rbl2/E2F, Myc, and YY1/Rb1/E2F through phosphorylation and dephosphorylation of their key components. In many instances, the phosphorylation patterns of differentially phosphorylated phosphoproteins in TRH- or TAL-treated cells were identical or displayed a similar trend in phosphorylation. However, some phosphoproteins, especially components of the Wnt/ß-catenin/Tcf and YY1/Rb1/E2F pathways, exhibited different phosphorylation patterns in TRH- and TAL-treated cells. This supports the notion that TRH and TAL may act, at least in part, as biased agonists. Additionally, the deficiency of ß-arrestin2 resulted in a reduced number of alterations in phosphorylation, highlighting the critical role of ß-arrestin2 in the signal transduction from TRH-R in the plasma membrane to transcription factors in the nucleus.

Transgenic mice reveal unexpected diversity of on-off direction-selective retinal ganglion cell subtypes and brain structures involved in motion processing.

On-Off direction-selective retinal ganglion cells (DSGCs) encode the axis of visual motion. They respond strongly to an object moving in a preferred direction and weakly to an object moving in the opposite, "null," direction. Historically, On-Off DSGCs were classified into four subtypes according to their directional preference (anterior, posterior, superior, or inferior). Here, we compare two genetically identified populations of On-Off DSGCs: dopamine receptor 4 (DRD4)-DSGCs and thyrotropin-releasing hormone receptor (TRHR)-DSGCs. We find that although both populations are tuned for posterior motion, they can be distinguished by a variety of physiological and anatomical criteria. First, the directional tuning of TRHR-DSGCs is broader than that of DRD4-DSGCs. Second, whereas both populations project similarly to the dorsal lateral geniculate nucleus, they project differently to the ventral lateral geniculate nucleus and the superior colliculus. Moreover, TRHR-DSGCs, but not DRD4-DSGCs, also project to the zona incerta, a thalamic area not previously known to receive direction-tuned visual information. Our findings reveal unexpected diversity among mouse On-Off DSGC subtypes that uniquely process and convey image motion to the brain.

Identification of a preassembled TRH receptor-G(q/11) protein complex in HEK293 cells.

Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gß proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.

Genome-wide association and replication studies identified TRHR as an important gene for lean body mass.

Low lean body mass (LBM) is related to a series of health problems, such as osteoporotic fracture and sarcopenia. Here we report a genome-wide association (GWA) study on LBM variation, by using Affymetrix 500K single-nucleotide polymorphism (SNP) arrays. In the GWA scan, we tested 379,319 eligible SNPs in 1,000 unrelated US whites and found that two SNPs, rs16892496 (p = 7.55 x 10(-8)) and rs7832552 (p = 7.58 x 10(-8)), within the thyrotropin-releasing hormone receptor (TRHR) gene were significantly associated with LBM. Subjects carrying unfavorable genotypes at rs16892496 and rs7832552 had, on average, 2.70 and 2.55 kg lower LBM, respectively, compared to those with alternative genotypes. We replicated the significant associations in three independent samples: (1) 1488 unrelated US whites, (2) 2955 Chinese unrelated subjects, and (3) 593 nuclear families comprising 1972 US whites. Meta-analyses of the GWA scan and the replication studies yielded p values of 5.53 x 10(-9) for rs16892496 and 3.88 x 10(-10) for rs7832552. In addition, we found significant interactions between rs16892496 and polymorphisms of several other genes involved in the hypothalamic-pituitary-thyroid and the growth hormone-insulin-like growth factor-I axes. Results of this study, together with the functional relevance of TRHR in muscle metabolism, support the TRHR gene as an important gene for LBM variation.

Pyroglutamyl peptidase II inhibition enhances the analeptic effect of thyrotropin-releasing hormone in the rat medial septum.

Thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH(2)) has multiple, but transient, homeostatic functions in the brain. It is hydrolyzed in vitro by pyroglutamyl peptidase II (PPII), a narrow specificity ectoenzyme with a preferential localization in the brain, but evidence that PPII controls TRH communication in the brain in vivo is scarce. We therefore studied in male Wistar rats the distribution of PPII mRNA in the septum and the consequence of PPII inhibition on the analeptic effect of TRH injected into the medial septum. Twelve to 14% of cell profiles expressed PPII mRNA in the medial septum-diagonal band of Broca; in this region the specific activity of PPII was relatively high. Twenty to 35% of PPII mRNA-labeled profiles were positive for TRH-receptor 1 (TRH-R1) mRNA. The intramedial septum injection of TRH reduced, in a dose-dependent manner, the duration of ethanol-induced loss of righting reflex (LORR). Injection of the PPII inhibitor pGlu-Asn-Pro-7-amido-4-methylcoumarin into the medial septum enhanced the effect of TRH. The injection of a phosphinic TRH analog, a higher-affinity inhibitor of PPII, diminished the duration of LORR by itself. In contrast, the intraseptal injection of pGlu-Asp-Pro-NH(2), a peptide that did not inhibit PPII activity, or an inhibitor of prolyl oligopeptidase did not change the duration of LORR. We conclude that in the medial septum PPII activity may limit TRH action, presumably by reducing the concentration of TRH in the extracellular fluid around cells coexpressing PPII and TRH-R1.

Sex-dependent and -independent regulation of thyrotropin-releasing hormone expression in the hypothalamic dorsomedial nucleus by negative energy balance, exercise, and chronic stress.

The dorsomedial nucleus of the hypothalamus (DMH) is part of the brain circuits that modulate organism responses to the circadian cycle, energy balance, and psychological stress. A large group of thyrotropin-releasing hormone (Trh) neurons is localized in the DMH; they comprise about one third of the DMH neurons that project to the lateral hypothalamus area (LH). We tested their response to various paradigms. In male Wistar rats, food restriction during adulthood, or chronic variable stress (CVS) during adolescence down-regulated adult DMH Trh mRNA levels compared to those in sedentary animals fed ad libitum; two weeks of voluntary wheel running during adulthood enhanced DMH Trh mRNA levels compared to pair-fed rats. Except for their magnitude, female responses to exercise were like those in male rats; in contrast, in female rats CVS did not change DMH Trh mRNA levels. A very strong negative correlation between DMH Trh mRNA levels and serum corticosterone concentration in rats of either sex was lost in CVS rats. CVS canceled the response to food restriction, but not that to exercise in either sex. TRH receptor 1 (Trhr) cells were numerous along the rostro-caudal extent of the medial LH. In either sex, fasting during adulthood reduced DMH Trh mRNA levels, and increased LH Trhr mRNA levels, suggesting fasting may inhibit the activity of TRHDMH->LH neurons. Thus, in Wistar rats DMH Trh mRNA levels are regulated by negative energy balance, exercise and chronic variable stress through sex-dependent and -independent pathways.

IGSF1 Deficiency Leads to Reduced TSH Production Independent of Alterations in Thyroid Hormone Action in Male Mice.

Loss of function mutations in IGSF1/Igsf1 cause central hypothyroidism. Igsf1 knockout mice have reduced pituitary thyrotropin-releasing hormone receptor, Trhr, expression, perhaps contributing to the phenotype. Because thyroid hormones negatively regulate Trhr, we hypothesized that IGSF1 might affect thyroid hormone availability in pituitary thyrotropes. Consistent with this idea, IGSF1 coimmunoprecipitated with the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in transfected cells. This association was impaired with IGSF1 bearing patient-derived mutations. Wild-type IGSF1 did not, however, alter MCT8-mediated thyroid hormone import into heterologous cells. IGSF1 and MCT8 are both expressed in the apical membrane of the choroid plexus. However, MCT8 protein levels and localization in the choroid plexus were unaltered in Igsf1 knockout mice, ruling out a necessary chaperone function for IGSF1. MCT8 expression was low in the pituitary and was similarly unaffected in Igsf1 knockouts. We next assessed whether IGSF1 affects thyroid hormone transport or action, by MCT8 or otherwise, in vivo. To this end, we treated hypothyroid wild-type and Igsf1 knockout mice with exogenous thyroid hormones. T4 and T3 inhibited TSH release and regulated pituitary and forebrain gene expression similarly in both genotypes. Interestingly, pituitary TSH beta subunit (Tshb) expression was consistently reduced in Igsf1 knockouts relative to wild-type regardless of experimental condition, whereas Trhr was more variably affected. Although IGSF1 and MCT8 can interact in heterologous cells, the physiological relevance of their association is not clear. Nevertheless, the results suggest that IGSF1 loss can impair TSH production independently of alterations in TRHR levels or thyroid hormone action.

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

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

Molecular cloning, gene structure, molecular evolution and expression analyses of thyrotropin-releasing hormone receptors from medaka (Oryzias latipes).

Molecular cloning of thyrotropin-releasing hormone receptors (TRHR) was performed in a model teleost fish, medaka (Oryzias latipes). Four subtypes of TRHR were cloned and named them as TRHR1a, TRHR1b, TRHR2 and TRHR3 based on their similarity to known TRHR subtypes in vertebrates. TRHR1a, TRHR1b, TRHR2, and TRHR3 of medaka encode 416, 398, 451, and 386 amino acid residues, respectively. Comparison of cDNA sequences of medaka TRHR subtypes with respective genomic DNA sequences revealed gene structures: TRHR1a, TRHR1b and TRHR3genes consist of two exons while the TRH2 gene consists of five exons. Molecular phylogenetic analyses depicted the molecular evolution of TRHR in vertebrates: From the ancestral molecule, TRHR2 diverged first and then TRHR1 and TRHR3. Reverse transcription-polymerase chain reaction analyses revealed the sites of TRHR expression: Expression of TRHR1, TRHR1b and TRHR2 subtypes has been confirmed in the brain, pineal organ, retina and pituitary gland. In addition, TRHR1b is expressed in spleen, digestive tract and skin, and TRHR2 in testis, ovary and gill. TRHR3 is widely expressed in various tissues. These results indicate that in medaka, TRH might exert multiple functions mediated by different TRHR subtypes expressed in each tissue.

Importance of regions outside the cytoplasmic tail of G-protein-coupled receptors for phosphorylation and dephosphorylation.

Two GPCRs (G-protein-coupled receptors), TRHR (thyrotropin-releasing hormone receptor) and beta(2)AR (beta(2)-adrenergic receptor), are regulated in distinct manners. Following agonist binding, TRHR undergoes rapid phosphorylation attributable to GRKs (GPCR kinases); beta(2)AR is phosphorylated by both second messenger-activated PKA (protein kinase A) and GRKs with slower kinetics. TRHR co-internalizes with arrestin, whereas beta(2)AR recruits arrestin, but internalizes without it. Both receptors are dephosphorylated following agonist removal, but TRHR is dephosphorylated much more rapidly while it remains at the plasma membrane. We generated chimaeras swapping the C-terminal domains of these receptors to clarify the role of different receptor regions in phosphorylation, internalization and dephosphorylation. beta(2)AR with a TRHR cytoplasmic tail (beta(2)AR-TRHR) and TRHR with a beta(2)AR tail (TRHR-beta(2)AR) signalled to G-proteins normally. beta(2)AR-TRHR was phosphorylated well at the PKA site in the third intracellular loop, but poorly at GRK sites in the tail, whereas TRHR-beta(2)AR was phosphorylated strongly at GRK sites in the tail (Ser(355)/Ser(356) of the beta(2)AR). Both chimaeric receptors exhibited prolonged, but weak, association with arrestin at the plasma membrane, but high-affinity arrestin interactions and extensive co-internalization of receptor with arrestin required a phosphorylated TRHR tail. In contrast, swapping C-terminal domains did not change the rates of phosphorylation and dephosphorylation or the dependence of TRHR dephosphorylation on the length of agonist exposure. Thus the interactions of GPCRs with GRKs and phosphatases are determined not simply by the amino acid sequences of the substrates, but by regions outside the cytoplasmic tails.

Mapping, cDNA cloning and tissue expression of the porcine thyrotropin-releasing hormone receptor gene.

Thyrotropin-releasing hormone receptor (TRHR) is a G-protein-coupled receptor that plays a crucial role in regulating the hypothalamic-pituitary-thyroid axis by conveying the action of the hypothalamic tripeptide TRH, which is the primary central activator of this hormonal cascade. In the present study, the porcine TRHR (pTRHR) gene was localized to chromosome 4 by Radiation hybrid mapping. Quantitative trait loci affecting average backfat thickness, daily gain, and carcass and meat quality traits have been mapped to the region containing this gene. Further, the full-length cDNA of pTRHR was cloned and sequenced. pTRHR contains an open reading frame encoding 398 amino acids and shares 96.2% amino acid identity to human TRHR. Real-time quantitative RT-PCR showed that the mRNA of pTRHR is expressed in a variety of tissues, with high expression in the brain, hypothalamus, pituitary, testis, and fat tissue. The considerable expression level of TRHR mRNA found in fat tissue indicates potential direct action of TRH on lipocyte might exist. Additionally, two alternative spliced transcript variants of pTRHR were also isolated in this study. Our data provided basic molecular information which will be useful for further investigation on pTRHR gene.