The aim of this work was to verify the possibility of interactions between the human TRH receptor (an integral membrane protein which belongs to family 1 of G-protein coupled receptors) and TRH-like peptides presented in the prostate gland. These peptides are characterized by substitution of basic amino acid histidine (related to authentic TRH) for neutral or acidic amino acid, such as glutamic acid, phenylalanine, glutamine or tyrosine. The physiological function of TRH-like peptides in peripheral tissues is not precisely known. However, according to our recent experiments, we assume the existence of a local hormonal network formed by TRH-like peptides and TSH in the prostate gland. The network can be associated with circulating thyroid and steroid hormones, and may represent a new regulatory mechanism influencing the proliferative ability of prostatic tissue. A similar network of authentic TRH and TSH was already found in the gastrointestinal tract. The experimentally determined 3D-structures of human TRH receptor (hTRHr) and TRH-like peptides are not available. From this point of view we used de novo modeling procedures of G-protein coupled receptors on an automated protein modeling server used at the Glaxo Wellcome Experimental Research (Geneva, Switzerland). 3D-structures of TRH-like peptides were determined with a computer program CORINA (written by the team of J. Gasteiger, Computer-Chemie-Centrum and Institute for Organic Chemistry, University of Erlangen-Nurenberg, Germany). The generated PDB files with 3D-coordinates were visualized with Swiss-Pdb Viewer Release 3.51 (Glaxo Wellcome). From recent results it is evident that polar amino acids belonging to the extracellular terminus of hTRHr transmembrane regions can participate in interactions between TRH and hTRHr. There is no direct evidence that TRH-like peptides interact with the presented hTRHr model. On the contrary, with respect to the similar 3D-shape and the identity of terminal amino acids, it appears that these interactions are highly probable as well as the nearly 100 % cross-reactions between TRH or TRH-like peptides and antibody specific against authentic TRH. Closed terminal amino acids (pyroglutamic acid and proline-amide) of TRH or TRH-like peptides are important for these interactions. Desamido-TRH or glutamyl metabolites will be repelled by the negative potential of ASP195 (E: D93) and GLU298 (G: E137).

• MeSH
• hormon uvolňující thyreotropin chemie   genetika   MeSH
• lidé MeSH
• molekulární modely * MeSH
• molekulární sekvence - údaje MeSH
• peptidové fragmenty chemie   genetika   MeSH
• počítačová simulace * MeSH
• receptory thyroliberinu chemie   genetika   MeSH
• sekundární struktura proteinů genetika   MeSH
• sekvence aminokyselin genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hormon uvolňující thyreotropin MeSH
• peptidové fragmenty MeSH
• receptory thyroliberinu MeSH

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

• Klíčová slova
• FRAP, Gq/11 protein, TRH receptor, lateral mobility, phospholipase Cβ1, β-arrestin,
• MeSH
• beta arrestiny chemie   genetika   MeSH
• buněčná membrána účinky léků   MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• hormon uvolňující thyreotropin chemie   metabolismus   MeSH
• kinasa 2 receptorů spřažených s G-proteiny chemie   MeSH
• lidé MeSH
• ligandy MeSH
• midazolam farmakologie   MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go chemie   genetika   MeSH
• receptory thyroliberinu agonisté   antagonisté a inhibitory   chemie   genetika   MeSH
• signální transdukce účinky léků   genetika   MeSH
• vazba proteinů účinky léků   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta arrestiny MeSH
• hormon uvolňující thyreotropin MeSH
• kinasa 2 receptorů spřažených s G-proteiny MeSH
• ligandy MeSH
• midazolam MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go MeSH
• receptory thyroliberinu MeSH

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 Gβ 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.

• MeSH
• buněčná membrána metabolismus   MeSH
• buněčné linie MeSH
• HEK293 buňky MeSH
• hormon uvolňující thyreotropin genetika   metabolismus   MeSH
• lidé MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 chemie   genetika   metabolismus   MeSH
• receptory thyroliberinu chemie   genetika   metabolismus   MeSH
• signální transdukce MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hormon uvolňující thyreotropin MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• receptory thyroliberinu MeSH

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.

• Klíčová slova
• Cholesterol, DPH fluorescence, FRAP, G protein coupling, RICS, TRH-R-eGFP mobility,
• MeSH
• algoritmy MeSH
• buněčná membrána chemie   metabolismus   MeSH
• cholesterol metabolismus   MeSH
• difenylhexatrien chemie   metabolismus   MeSH
• difuze MeSH
• fluorescenční polarizace MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus   MeSH
• receptory thyroliberinu chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• zelené fluorescenční proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• difenylhexatrien MeSH
• enhanced green fluorescent protein MeSH Prohlížeč
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• receptory thyroliberinu MeSH
• zelené fluorescenční proteiny MeSH

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 β-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/β-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 β-arrestin2 resulted in a reduced number of alterations in phosphorylation, highlighting the critical role of β-arrestin2 in the signal transduction from TRH-R in the plasma membrane to transcription factors in the nucleus.

• Klíčová slova
• Cell cycle, Phosphosignaling, Taltirelin, Thyrotropin-releasing hormone, β-arrestin2,
• MeSH
• beta-katenin * MeSH
• buněčný cyklus MeSH
• fosfoproteiny MeSH
• hormon uvolňující thyreotropin metabolismus   MeSH
• krysa rodu Rattus MeSH
• receptory thyroliberinu * genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-katenin * MeSH
• fosfoproteiny MeSH
• hormon uvolňující thyreotropin MeSH
• receptory thyroliberinu * MeSH

TRH-like peptides are characterized by substitution of basic amino acid histidine (related to authentic TRH) with neutral or acidic amino acid, like glutamic acid, phenylalanine, glutamine, tyrosine, leucin, valin, aspartic acid and asparagine. The presence of extrahypothalamic TRH-like peptides was reported in peripheral tissues including gastrointestinal tract, placenta, neural tissues, male reproductive system and certain endocrine tissues. Work deals with the biological function of TRH-like peptides in different parts of organisms where various mechanisms may serve for realisation of biological function of TRH-like peptides as negative feedback to the pituitary exerted by the TRH-like peptides, the role of pEEPam such as fertilization-promoting peptide, the mechanism influencing the proliferative ability of prostatic tissues, the neuroprotective and antidepressant function of TRH-like peptides in brain and the regulation of thyroid status by TRH-like peptides.

• MeSH
• centrální nervový systém chemie   MeSH
• hormon uvolňující thyreotropin analogy a deriváty   fyziologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• peptidy chemie   fyziologie   MeSH
• prostata chemie   MeSH
• receptory thyroliberinu fyziologie   MeSH
• štítná žláza chemie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• hormon uvolňující thyreotropin MeSH
• peptidy MeSH
• receptory thyroliberinu MeSH

We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G(q/11)alpha proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G(11)alpha. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G(q/11)alpha was assessed by agonist-stimulated [(35)S]GTPgammaS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca(2+) response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G(q/11)alpha proteins.

• MeSH
• buněčná membrána metabolismus   ultrastruktura   MeSH
• buněčné linie MeSH
• cholesterol fyziologie   MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• hormon uvolňující thyreotropin metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• membránové mikrodomény metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus   MeSH
• receptory thyroliberinu metabolismus   MeSH
• signální transdukce * MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• guanosin 5'-O-(3-thiotrifosfát) MeSH
• hormon uvolňující thyreotropin MeSH
• ligandy MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• receptory thyroliberinu MeSH
• vápník MeSH

Thyrotropin-releasing hormone (TRH) is an important endocrine agent that regulates the function of cells in the anterior pituitary and the central and peripheral nervous systems. By controlling the synthesis and release of thyroid hormones, TRH affects many physiological functions, including energy homeostasis. This hormone exerts its effects through G protein-coupled TRH receptors, which signal primarily through Gq/11 but may also utilize other G protein classes under certain conditions. Because of the potential therapeutic benefit, considerable attention has been devoted to the synthesis of new TRH analogs that may have some advantageous properties compared with TRH. In this context, it may be interesting to consider the phenomenon of biased agonism and signaling at the TRH receptor. This possibility is supported by some recent findings. Although knowledge about the mechanisms of TRH receptor-mediated signaling has increased steadily over the past decades, there are still many unanswered questions, particularly about the molecular details of post-receptor signaling. In this review, we summarize what has been learned to date about TRH receptor-mediated signaling, including some previously undiscussed information, and point to future directions in TRH research that may offer new insights into the molecular mechanisms of TRH receptor-triggered actions and possible ways to modulate TRH receptor-mediated signaling.

• Klíčová slova
• G protein, TRH receptors, signaling, thyrotropin-releasing hormone, β-arrestin,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Some G protein-coupled receptors might be spacially targetted to discrete domains within the plasma membrane. Here we assessed the localization in membrane domains of the epitope-tagged, fluorescent version of thyrotropin-releasing hormone receptor (VSV-TRH-R-GFP) expressed in HEK293 cells. Our comparison of three different methods of cell fractionation (detergent extraction, alkaline treatment/sonication and mechanical homogenization) indicated that the dominant portion of plasma membrane pool of the receptor was totally solubilized by Triton X-100 and its distribution was similar to that of transmembrane plasma membrane proteins (glycosylated and non-glycosylated forms of CD147, MHCI, CD29, CD44, transmembrane form of CD58, Tapa1 and Na,K-ATPase). As expected, caveolin and GPI-bound proteins CD55, CD59 and GPI-bound form of CD58 were preferentially localized in detergent-resistant membrane domains (DRMs). Trimeric G proteins G(q)alpha/G(11)alpha, G(i)alpha1/G(i)alpha2, G(s)alphaL/G(s)alphaS and Gbeta were distributed almost equally between detergent-resistant and detergent-solubilized pools. In contrast, VSV-TRH-R-GFP, Galpha, Gbeta and caveolin were localized massively only in low-density membrane fragments of plasma membranes, which were generated by alkaline treatment/sonication or by mechanical homogenization of cells. These data indicate that VSV-TRH-R-GFP as well as other transmembrane markers of plasma membranes are excluded from TX-100-resistant, caveolin-enriched membrane domains. Trimeric G protein G(q)alpha/G(11)alpha occurs in both DRMs and in the bulk of plasma membranes, which is totally solubilized by TX-100.

• MeSH
• buněčná membrána chemie   MeSH
• buněčné kultury MeSH
• centrifugace - gradient hustoty MeSH
• detergenty MeSH
• fluorescenční spektrometrie MeSH
• imunoblotting MeSH
• kaveolin 1 MeSH
• kaveoliny chemie   MeSH
• lidé MeSH
• membránové mikrodomény chemie   MeSH
• oktoxynol MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 analýza   chemie   MeSH
• radioligandová zkouška MeSH
• receptory thyroliberinu analýza   chemie   MeSH
• rozpustnost MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CAV1 protein, human MeSH Prohlížeč
• detergenty MeSH
• kaveolin 1 MeSH
• kaveoliny MeSH
• oktoxynol MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• receptory thyroliberinu MeSH

Production of thyroid hormone is precisely regulated in a negative feed-back mechanism that depends critically on thyroid hormone receptor β (TRβ). This mechanism decreases production of thyrotropin- releasing hormone (TRH) and thyrotropin (TSH) in the hypothalamus and pituitary gland in response to high levels of circulating thyroid hormones (TH). Despite the wealth of accumulated knowledge, it is still not clear how exactly this negative regulation is executed. The syndrome of resistance to thyroid hormone (RTH), in which the levels of TH are not properly sensed, represents naturally occurring situations in which molecular components of this regulation are displayed and may be uncovered. TRβ, which is central to this regulation, is in the majority of RTH cases mutated in a way that preserves some functions of the receptor. Approximately 150 different mutations in TRβ have been identified to date. Here, we hypothesized that additional pathogenic mutations in TRβ are likely to exist in human population and analysed clinical cases with suspected RTH. In keeping with our prediction, analysis of 17 patients from nine families led to identification of four presumed pathogenic mutations of TRβ, including a previously unknown mutation, T273R. This suggests that threonine 273 is likely to be critical for the normal function of TRβ, possibly due to its role in helix 12 mobility and interaction with coactivators, and thus supports the concept that TRβ-dependent trans-activating function is necessary for the inhibition of TRH and TSH expression in response to elevated levels of TH.

• MeSH
• hormon uvolňující thyreotropin metabolismus   MeSH
• hormony štítné žlázy metabolismus   MeSH
• hypothalamus metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• thyreotropin metabolismus   MeSH
• tyreoidální hormony, receptory beta genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hormon uvolňující thyreotropin MeSH
• hormony štítné žlázy MeSH
• thyreotropin MeSH
• tyreoidální hormony, receptory beta MeSH