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Graeme Milligan - One of the best experts on this subject based on the ideXlab platform.
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analysis of the c terminal tail of the rat thyrotropin releasing Hormone Receptor 1 in interactions and cointernalization with beta arrestin 1 green fluorescent protein
Molecular Pharmacology, 2001Co-Authors: D A Groarke, Tomas Drmota, D S Bahia, N A Evans, Shelagh Wilson, Graeme MilliganAbstract:Coexpression of the rat thyrotropin releasing Hormone Receptor-1 with beta-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the Receptor. Truncations of the Receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the Receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the Receptor or its cointernalization with beta-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta-arrestin 1-GFP and a 10-fold reduction in internalization of the Receptor. Despite this, the Receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing Hormone Receptor-1 C-terminal tail required for effective contacts with beta-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the Receptor.
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analysis of the c terminal tail of the rat thyrotropin releasing Hormone Receptor 1 in interactions and cointernalization with beta arrestin 1 green fluorescent protein
Molecular Pharmacology, 2001Co-Authors: D A Groarke, Tomas Drmota, D S Bahia, N A Evans, Shelagh Wilson, Graeme MilliganAbstract:Coexpression of the rat thyrotropin releasing Hormone Receptor-1 with β-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the Receptor. Truncations of the Receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the Receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the Receptor or its cointernalization with β-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with β-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with β-arrestin 1-GFP and a 10-fold reduction in internalization of the Receptor. Despite this, the Receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing Hormone Receptor-1 C-terminal tail required for effective contacts with β-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the Receptor.
Karin A Eidne - One of the best experts on this subject based on the ideXlab platform.
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demonstration of improvements to the bioluminescence resonance energy transfer bret technology for the monitoring of g protein coupled Receptors in live cells
Journal of Biomolecular Screening, 2008Co-Authors: Martina Kocan, Karin A Eidne, Ruth M Seeber, Heng B See, Kevin D G PflegerAbstract:The bioluminescence resonance energy transfer (BRET) technique has become extremely popular for studying protein-protein interactions in living cells and real time. Of particular interest is the ability to monitor interactions between G protein-coupled Receptors, such as the Thyrotropin-Releasing Hormone Receptor (TRHR), and proteins critical for regulating their function, such as beta-arrestin. Using TRHR/beta-arrestin interactions, we have demonstrated improvements to all 3 generations of BRET (BRET(1), BRET(2), and eBRET) by using the novel forms of luciferase, Rluc2 and Rluc8, developed by the Gambhir laboratory. Furthermore, for the 1st time it was possible to use the BRET2 system to detect ligand-induced G protein-coupled Receptor/beta-arrestin interactions over prolonged periods (on the scale of hours rather than seconds) with a very stable signal. As demonstrated by our Z'-factor data, these luciferases increase the sensitivity of BRET to such an extent that they substantially increase the potential applicability of this technology for effective drug discovery high-throughput screening.
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constitutive and agonist dependent homo oligomerization of the thyrotropin releasing Hormone Receptor detection in living cells using bioluminescence resonance energy transfer
Journal of Biological Chemistry, 2001Co-Authors: Karen M Kroeger, Aylin C Hanyaloglu, Ruth M Seeber, Lauren E C Miles, Karin A EidneAbstract:The ability of G-protein-coupled Receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the Thyrotropin-Releasing Hormone Receptor (TRHR) forms constitutive homo-oligomers. This formation of TRHR homo-oligomers in the absence of ligand was shown by demonstration of an energy transfer between TRHR molecules fused to either donor, Renilla luciferase (Rluc) or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This interaction was shown to be specific, since energy transfer was not detected between co-expressed tagged TRHRs and either complementary tagged gonadotropin-releasing Hormone (GnRH) or β2-adrenergic Receptors. Furthermore, generation of a BRET signal between the TRHRs could only be inhibited by co-expression of the wild-type TRHR and not by other GPCRs. Agonist stimulation led to a time- and dose-dependent increase in the amount of energy transfer. Inhibition of Receptor internalization by co-expression of dynamin mutant K44A did not affect the interaction between TRHRs, suggesting that clustering of Receptors within clathrin-coated pits is not sufficient for energy transfer to occur. BRET also provided evidence for the agonist-induced oligomerization of another GPCR, the GnRH Receptor (GnRHR), and the presence of an agonist-induced interaction of the adaptor protein, β-arrestin, with TRHR and the absence of an interaction of β-arrestin with GnRHR. This study supports the usefulness of BRET as a powerful tool for studying GPCR aggregations and Receptor/protein interactions in general and presents evidence that the functioning unit of TRHRs exists as homomeric complexes.
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gonadotropin releasing Hormone Receptors with intracellular carboxyl terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics
Journal of Biological Chemistry, 1998Co-Authors: Anders Heding, Milka Vrecl, Jan Bogerd, Alison Mcgregor, Robin Sellar, P L Taylor, Karin A EidneAbstract:Abstract The mammalian gonadotropin-releasing Hormone Receptor (GnRH-R) is the only G-protein-coupled Receptor (GPCR) in which the intracellular C-terminal tail is completely absent. In contrast to other GPCRs, the GnRH-R does not show rapid desensitization of total inositol (IP) production, and the rates of internalization are exceptionally slow. We investigated whether the incorporation of a cytoplasmic tail into the C terminus of the GnRH-R affects desensitization events and Receptor internalization rates. A GnRH-R/TRH-R chimera was created where the intracellular tail of the rat Thyrotropin-Releasing Hormone Receptor (TRH-R) was engineered into the C terminus of the rat GnRH-R. Three different rat GnRH-R cDNA stop codon mutations (one for each reading frame) were also made. The GnRH-stimulated IP production of the wild-type rat GnRH-R expressed in either COS-7 or HEK 293 cells did not desensitize even after prolonged stimulation with GnRH. In contrast, the catfish GnRH-R (which does possess an intracellular tail) and the TRH-R rapidly (<10 min) desensitized following agonist stimulation. The GnRH-R/TRH-R chimera also desensitized following treatment with GnRH, resembling the pattern shown by the TRH-R and the catfish GnRH-R. Two of the stop codon mutants did not show desensitization of IP production, and the third mutant with the longest tail was not functional. Internalization experiments showed that the rat GnRH-R had the slowest endocytosis and recycling rates compared with the TRH-R, the catfish GnRH-R, and the chimeric GnRH/TRH-R. This study demonstrates that the addition of a functional intracellular C-terminal tail to the GnRH-R produces rapid desensitization of IP production and significantly increases internalization rates.
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a disulfide bonding interaction role for cysteines in the extracellular domain of the thyrotropin releasing Hormone Receptor
Endocrinology, 1996Co-Authors: J V Cook, A Mcgregor, T Lee, G Milligan, Karin A EidneAbstract:The roles of disulfide and sulfhydryl groups in the specific binding of TRH to its Receptor have been examined. In all TRH Receptors (TRH-Rs) isolated from different species so far, there are only two extracellular cysteine residues (Cys98 in the extracellular loop between transmembrane helices 2 and 3 and Cys179 in the extracellular loop between transmembrane helices 4 and 5) that are in positions homologous to cysteine residues in other G protein-coupled Receptors. Another Cys (Cys100) is located in close proximity to Cys98 at the interface between the first extracellular loop and third transmembrane domain. To assess the role of these TRH-R Cys residues in disulfide bonding interactions, they were mutated to either Ser or Ala. Six mutant Receptors (Cys98Ser, Cys98Ala, Cys179Ser, Cys179Ala, Cys100Ser, and Cys100Ala) were expressed in COS-1 cells and tested for their ability to bind TRH and to activate total inositol phosphate (IP) formation. TRH-R mutants Cys100Ser and Cys100Ala showed TRH binding affin...
Marvin C. Gershengorn - One of the best experts on this subject based on the ideXlab platform.
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taltirelin is a superagonist at the human thyrotropin releasing Hormone Receptor
Frontiers in Endocrinology, 2012Co-Authors: Nanthakumar Thirunarayanan, B M Raaka, Marvin C. GershengornAbstract:Taltirelin (TAL) is a Thyrotropin-Releasing Hormone (TRH) analog that is approved for use in humans in Japan. In this study, we characterized TAL binding to and signaling by the human TRH Receptor (TRH-R) in a model cell system. We found that TAL exhibited lower binding affinities than TRH and lower signaling potency via the inositol-1,4,5-trisphosphate/calcium pathway than TRH. However, TAL exhibited higher intrinsic efficacy than TRH in stimulating inositol-1,4,5-trisphosphate second messenger generation. This is the first study that elucidates the pharmacology of TAL at TRH-R and shows that TAL is a superagonist at TRH-R. We suggest the superagonism exhibited by TAL may in part explain its higher activity in mediating CNS effects in humans compared to TRH.
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taltirelin is a superagonist at the human thyrotropin releasing Hormone Receptor
Frontiers in Endocrinology, 2012Co-Authors: Nanthakumar Thirunarayanan, B M Raaka, Marvin C. GershengornAbstract:Taltirelin (TAL) is a Thyrotropin-Releasing Hormone (TRH) analog that is approved for use in humans in Japan. In this study, we characterized TAL binding to and signaling by the human TRH Receptor (TRH-R) in a model cell system. We found that TAL exhibited lower binding affinities than TRH and lower signaling potency via the inositol-1,4,5-trisphosphate/calcium pathway than TRH. However, TAL exhibited higher intrinsic efficacy than TRH in stimulating inositol-1,4,5-trisphosphate second messenger generation. This is the first study that elucidates the pharmacology of TAL at TRH-R and shows that TAL is a superagonist at TRH-R. We suggest the superagonism exhibited by TAL may in part explain its higher activity in mediating central nervous system effects in humans compared to TRH.
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high levels of thyrotropin releasing Hormone Receptors activate programmed cell death in human pancreatic precursors
Pancreas, 2009Co-Authors: Christopher M Mulla, B M Raaka, Elizabeth Gerasraaka, Marvin C. GershengornAbstract:Objectives: Thyrotropin-Releasing Hormone (TRH) is expressed in rodent and human adult pancreata and in mouse pancreas during embryonic development. However, expression of TRH Receptors (TRHRs) in the pancreas is controversial. We sought to provide evidence that the TRH/TRHR system might play a role in fetal development. Methods: We used quantitative reverse transcription-polymerase chain reaction to measure TRH and TRHR messenger RNA (mRNA). To study the effects of TRHR expression in a pancreatic progenitor population, we expressed TRHRs in human islet-derived precursor cells (hIPCs) by infection with adenoviral vector AdCMVmTRHR. Thyrotropin-Releasing Hormone Receptor signaling was measured as inositol phosphate production and intracellular calcium transients. Thyrotropin-Releasing Hormone Receptor expression was measured by [3H]methyl-TRH binding. Apoptosis was monitored by release of cytochrome c from mitochondria. Results: We show that TRH mRNA is expressed in human fetal and adult pancreata, and that TRHR mRNA is expressed in fetal human pancreas but not in adult human pancreas. Thyrotropin-Releasing Hormone Receptors expressed in hIPCs were shown to signal normally. Most importantly, TRH treatment for several days stimulated apoptosis in hIPCs expressing approximately 400,000 TRHRs per cell. Conclusions These findings suggest a possible role for TRH/TRHR signaling in pancreatic precursors to promote programmed cell death, a normal constituent of morphogenesis during embryonic development in humans.
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role of the extracellular loops of the thyrotropin releasing Hormone Receptor evidence for an initial interaction with thyrotropin releasing Hormone
Biochemistry, 1997Co-Authors: Jeffrey H Perlman, Annyodile Colson, Rahul Jain, Bryan K Czyzewski, Louis A Cohen, Roman Osman, Marvin C. GershengornAbstract:Thyrotropin-Releasing Hormone (TRH), like most small ligands, appears to bind within the seven transmembrane-spanning helices (TMs) of its G protein-coupled Receptor (TRH-R). A role for the extracellular loops (ECLs) of TRH-R has not been established. We substituted residues in the ECLs of TRH-R and show that Tyr-181 is important for high-affinity binding because its substitution leads to a 3700-fold lowering of the estimated affinity compared to wild-type TRH-R. Using TRH analogues, we provide evidence that there is a specific interaction between Tyr-181 in ECL-2 and the pyroGlu moiety of TRH. It was previously suggested that the pyroGlu of TRH may interact with Asn-110 in TM-3 and with Asn-289 in ECL-3; N110A and N289A TRH-Rs exhibit similar apparent affinities that are only 20-30-fold lower than wild-type TRH-R. To better understand these findings, we analyzed a computer-generated model which predicts that the ECLs form an entry channel into the TRH-R TM bundle, that Tyr-181 projects into this channel and that the pyroGlu of TRH cannot simultaneously interact with residues in the TMs and ECLs. Kinetic analysis showed that the association rate of [Ntau-methyl-His]TRH with N289A TRH-R is slower than with wild-type TRH-R and largely accounts for the lower apparent affinity; the association rate with N110A TRH-R is similar to that of wild-type TRH-R. These data are consistent with the idea that there are initial interactions between TRH and the residues of a putative entry channel of TRH-R. We suggest that a role of the ECLs in all G protein-coupled Receptors for small ligands may be to initially contact the ligand and allow entry into a TM binding pocket.
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ectopic expression of thyrotropin releasing Hormone trh Receptors in liver modulates organ function to regulate blood glucose by trh
Nature Genetics, 1996Co-Authors: Gerhard Wolff, Marvin C. Gershengorn, Andrea Mastrangeli, Marcos Heinflink, Erik Falckpedersen, Ronald G CrystalAbstract: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.
D A Groarke - One of the best experts on this subject based on the ideXlab platform.
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analysis of the c terminal tail of the rat thyrotropin releasing Hormone Receptor 1 in interactions and cointernalization with beta arrestin 1 green fluorescent protein
Molecular Pharmacology, 2001Co-Authors: D A Groarke, Tomas Drmota, D S Bahia, N A Evans, Shelagh Wilson, Graeme MilliganAbstract:Coexpression of the rat thyrotropin releasing Hormone Receptor-1 with beta-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the Receptor. Truncations of the Receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the Receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the Receptor or its cointernalization with beta-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta-arrestin 1-GFP and a 10-fold reduction in internalization of the Receptor. Despite this, the Receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing Hormone Receptor-1 C-terminal tail required for effective contacts with beta-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the Receptor.
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analysis of the c terminal tail of the rat thyrotropin releasing Hormone Receptor 1 in interactions and cointernalization with beta arrestin 1 green fluorescent protein
Molecular Pharmacology, 2001Co-Authors: D A Groarke, Tomas Drmota, D S Bahia, N A Evans, Shelagh Wilson, Graeme MilliganAbstract:Coexpression of the rat thyrotropin releasing Hormone Receptor-1 with β-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the Receptor. Truncations of the Receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the Receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the Receptor or its cointernalization with β-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with β-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with β-arrestin 1-GFP and a 10-fold reduction in internalization of the Receptor. Despite this, the Receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing Hormone Receptor-1 C-terminal tail required for effective contacts with β-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the Receptor.
D S Bahia - One of the best experts on this subject based on the ideXlab platform.
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analysis of the c terminal tail of the rat thyrotropin releasing Hormone Receptor 1 in interactions and cointernalization with beta arrestin 1 green fluorescent protein
Molecular Pharmacology, 2001Co-Authors: D A Groarke, Tomas Drmota, D S Bahia, N A Evans, Shelagh Wilson, Graeme MilliganAbstract:Coexpression of the rat thyrotropin releasing Hormone Receptor-1 with beta-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the Receptor. Truncations of the Receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the Receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the Receptor or its cointernalization with beta-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta-arrestin 1-GFP and a 10-fold reduction in internalization of the Receptor. Despite this, the Receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing Hormone Receptor-1 C-terminal tail required for effective contacts with beta-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the Receptor.
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analysis of the c terminal tail of the rat thyrotropin releasing Hormone Receptor 1 in interactions and cointernalization with beta arrestin 1 green fluorescent protein
Molecular Pharmacology, 2001Co-Authors: D A Groarke, Tomas Drmota, D S Bahia, N A Evans, Shelagh Wilson, Graeme MilliganAbstract:Coexpression of the rat thyrotropin releasing Hormone Receptor-1 with β-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the Receptor. Truncations of the Receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the Receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the Receptor or its cointernalization with β-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with β-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with β-arrestin 1-GFP and a 10-fold reduction in internalization of the Receptor. Despite this, the Receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing Hormone Receptor-1 C-terminal tail required for effective contacts with β-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the Receptor.