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We have studied the role of a highly conserved tryptophan and other aromatic residues of the thyrotropin-releasing hormone (TRH) receptor (TRH-R) that are predicted by computer modeling to form a hydrophobic cluster between transmembrane helix (TM)5 and TM6. The affinity of a mutant TRH-R, in which Trp279 was substituted by alanine (W279A TRH-R), for most tested agonists was higher than that of wild-type (WT) TRH-R, whereas its affinity for inverse agonists was diminished, suggesting that W279A TRH-R is constitutively active. We found that W279A TRH-R exhibited 3.9-fold more signaling activity than WT TRH-R in the absence of agonist. This increased basal activity was inhibited by the inverse agonist midazolam, confirming that the mutant receptor is constitutively active. Computer-simulated models of the unoccupied WT TRH-R, the TRH-occupied WT TRH-R, and various TRH-R mutants predict that a hydrophobic cluster of residues, including Trp279 (TM6), Tyr282, and Phe199 (TM5), constrains the receptor in an inactive conformation. In support of this model, we found that substitution of Phe199 by alanine or of Tyr282 by alanine or phenylalanine, but not of Tyr200 (by alanine or phenylalanine), resulted in a constitutively active receptor. We propose that a hydrophobic cluster including residues in TM5 and TM6 constrains the TRH-R in an inactive conformation via interhelical interactions. Disruption of these constraints by TRH binding or by mutation leads to changes in the relative positions of TM5 and TM6 and to the formation of an active form of TRH-R.

Small ligands generally bind within the seven transmembrane-spanning helices of G-protein-coupled receptors, but their access to the binding pocket through the closely packed loops has not been elucidated. In this work, a model of the extracellular loops of the thyrotropin-releasing hormone (TRH) receptor (TRHR) was constructed, and molecular dynamics simulations and quasi-harmonic analysis have been performed to study the static and dynamic roles of the extracellular domain. The static analysis based on curvature and electrostatic potential on the surface of TRHR suggests the formation of an initial recognition site between TRH and the surface of its receptor. These results are supported by experimental evidence. A quasi-harmonic analysis of the vibrations of the extracellular loops suggest that the low-frequency motions of the loops will aid the ligand to access its transmembrane binding pocket. We suggest that all small ligands may bind sequentially to the transmembrane pocket by first interacting with the surface binding site and then may be guided into the transmembrane binding pocket by fluctuations in the extracellular loops.

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.

Previous mutational and computational studies of the thyrotropin-releasing hormone (TRH) receptor identified several residues in its binding pocket [see accompanying paper, Perlman et al. (1996) Biochemistry 35, 7643-7650]. On the basis of the initial model constructed with standard energy minimization techniques, we have conducted 15 mixed mode Monte Carlo/stochastic dynamics (MC-SD) simulations to allow for extended sampling of the conformational states of the ligand and the receptor in the complex. A simulated annealing protocol was adopted in which the complex was cooled from 600 to 310 K in segments of 30 ps of the MC-SD simulations for each change of 100 K. Analysis of the simulation results demonstrated that the mixed mode MC-SD protocol maintained the desired temperature in the constant temperature simulation segments. The elevated temperature and the repeating simulations allowed for adequate sampling of the torsional space of the complex with successful conservation of the general structure and good helicity of the receptor. For the analysis of the interaction between TRH and the binding pocket, TRH was divided into four groups consisting of pyroGlu, His, ProNH2, and the backbone. The pairwise interaction energies of the four separate portions of TRH with the corresponding residues in the receptor provide a physicochemical basis for the understanding of ligand-receptor complexes. The interaction of pyroGlu with Tyr106 shows a bimodal distribution that represents two populations: one with a H-bond and another without it. Asp195 was shown to compete with pyroGlu for the H-bond to Tyr106. Simulations in which Asp195 was interacting with Arg283, thus removing it from the vicinity of Tyr106, resulted in a stable H-bond to pyroGlu. In all simulations His showed a van der Waals attraction to Tyr282 and a weak electrostatic repulsion from Arg 306. The ProNH2 had a strong and frequent H-bonding interaction with Arg306. The backbone carbonyls show a frequent H-bonding interaction with the OH group of Tyr282 and strong, often multiple, interactions with Arg306. Three structures, which maintained these interactions simultaneously, were selected as candidates for ligand-receptor complexes. These show persistent interactions of TRH with Ile 109 and Ile 116 in HX 3 and with Tyr310 and Ser313 in HX 7, which will be tested to refine the structure of the ligand-receptor complex. The superposition of the three structures shows the extent of structural flexibility of the receptor and the ligand in the complex. The backbone of TRH inside the receptor is in an alpha-helical conformation, suggesting that the receptor, through its interaction with the ligand, provides the energy required for the conformational change in the ligand from an extended to the folded form.

Seven transmembrane (TM) spanning, G protein-coupled receptors (GPCRs) appear to bind large glycoprotein hormones predominantly within their extracellular domains, small nonpeptidic ligands within the TM helical bundle, and peptide ligands within the extracellular domains and TM bundle. The tripeptide thyrotropin-releasing hormone (TRH, pyroGlu-His-ProNH2) may bind entirely within the TM bundle of the TRH receptor (TRH-R). We have previously demonstrated direct binding contacts between the pyroGlu of TRH and two residues in TM helix 3 (TM-3) of TRH-R and proposed a model of the binding pocket of TRH-R [Perlman, J. H., Laakkonen, L., Osman, R., & Gershengorn, M. C. (1994) J. Biol. Chem. 269, 23383-23386]. Here, we provide evidence for two additional direct interactions between TRH and TRH-R. One interaction is between the aromatic ring of Tyr 282 of TM-6 and His of TRH. This is based on a large increase in the half-maximally effective concentration (EC50) of TRH for stimulation of inositol phosphate formation by Y282A TRH-R and a loss of selectivity of this mutant receptor for TRH analogs substituted at His. We provide evidence for another interaction between Arg 306 of TM-7 and the terminal carboxamide of TRH. Using four direct interactions as anchors, a refined model of the TRH-R binding pocket was constructed using geometry optimization through energy minimization. A novel method for modeling GPCRs based on Monte Carlo and stochastic dynamics simulations is presented in the accompanying paper [Laakkonen, L. J., Guarnieri, F., Perlman, J. H., Gershengorn, M. C., & Osman, R. (1996) Biochemistry 35, 7651-7663].

The receptor for thyrotropin-releasing hormone (TRH) is a member of the seven-transmembrane-spanning, GTP-binding protein-coupled receptor family. We showed that tyrosine at position 106 in transmembrane helix 3 of the TRH receptor directly binds the ring carbonyl of the pyroglutamyl moiety of TRH (Perlman, J. H., Thaw, C. N., Laakkonen L., Bowers, C. Y., Osman, R., and Gershengorn, M. C. (1994) J. Biol. Chem. 269, 1610-1613). We now show that asparagine at position 110 of transmembrane helix 3 directly interacts with the ring N-H of the TRH pyroglutamyl moiety. Based on these findings and evidence that two transmembrane arginines are important in binding, we developed a three-dimensional model of the TRH receptor binding pocket using molecular modeling and simulation programs. The model places the binding pocket for TRH within the transmembrane domains of the receptor and predicts that multiple hydrogen-bonding interactions are involved in binding TRH. To our knowledge, this is the first model, at an atomic level of detail, of the interaction of a peptide ligand with a GTP-binding protein-coupled receptor.

Thyrotropin-releasing hormone (TRH, pyroglutamic acid-histidine-proline-amide) binds to a seven-transmembrane-spanning, G protein-coupled receptor. We tested the hypothesis that Tyr106 of the third transmembrane helix of the TRH receptor (TRH-R) binds pyroglutamyl of TRH by mutating Tyr106 to Phe and replacing the ring carbonyl of the TRH pyroglutamyl moiety with a methylene group ([Pro1]TRH). Compared to the affinity of wild-type TRH-R for TRH, the affinities of [Phe106]TRH-R for TRH and of wild-type TRH-R for [Pro1]TRH were 100,000- and 110,000-fold lower, respectively. The affinity of [Phe106]TRH-R for [Pro1]TRH was only 16-fold lower than that for TRH, demonstrating a lack of additivity of the effects of these changes in the receptor and ligand. These data provide compelling evidence that the hydroxyl group of Tyr106 of the TRH-R binds the TRH pyroglutamyl carbonyl group. To our knowledge, this represents the highest affinity, non-covalent bond yet observed between single functional groups of a GPCR and ligand and is the first delineation of a direct binding interaction between a residue in the transmembrane core of a GPCR and a specific moiety of a peptide agonist.

Thyrotropin-releasing hormone, TRH (< Glu-His-Proamide), and [N tau-Me-His]TRH (MeTRH) are present as neutral and positively charged forms at physiologic pH, and it was possible that they bind to the TRH receptor (TRH-R) as charged (protonated) species. Binding affinities of TRH and MeTRH to endogenous rat TRH-Rs and to transfected wild type mouse TRH-Rs decreased below pH 7.1. Half-maximal decreases in binding occurred at the approximate pK alpha values of these ligands. Asp to Ala mutations in extracellular loop 1, TM-4, and TM-5 did not decrease binding affinity, but an Asp to Ala mutation in TM-2 caused the affinity to decrease 8-fold. The pH dependences of binding of MeTRH, however, were similar in wild type and all mutant receptors and were consistent with the protonated form of MeTRH binding less well. Thus, the binding of TRH to its receptor does not involve ionic interactions and may be a prototype for binding of neutral peptide ligands to G protein-coupled receptors.

Previous reports have provided conflicting evidence as to whether the response to TRH desensitizes. Here we show that TRH stimulation of phosphoinositide (PPI) hydrolysis, measured as inositol phosphate accumulation in the presence of LiCl, desensitizes in rat pituitary GH3 cells and in rat glioma C6 cells stably transfected with mouse pituitary TRH receptor complementary DNA. In GH3 cells, the rate of stimulation by 1000 nM TRH of PPI hydrolysis was maximal initially and then decreased by 44 +/- 13% after 20 min. In an experimental paradigm in which PPI hydrolysis was measured by adding 20 mM LiCl at different times after TRH, desensitizations caused by 3, 10, and 1000 nM TRH were 33 +/- 5%, 41 +/- 6%, and 69 +/- 2%, respectively. In transfected C6 cells, TRH-induced desensitization of 76 +/- 9% was found. In GH3 cells, 1 microM phorbol myristate acetate (PMA), an activator of protein kinase C, inhibited the initial response to TRH by 75 +/- 6% and preexposure to PMA and TRH decreased the rate of PPI hydrolysis by 98 +/- 1% after 60 min. One hundred micromolar H-7 (1-(5-isoquinolinesulfonyl)-2-methyl piperazine), an inhibitor of protein kinases, abolished the effect of PMA but did not inhibit TRH-induced desensitization. Elevation of cytoplasmic free Ca2+ by K+ depolarization increased TRH stimulation of PPI hydrolysis. We conclude that TRH stimulation of PPI hydrolysis acutely desensitizes and that this effect is not specific to pituitary cells. TRH-induced desensitization, moreover, does not appear to be mediated by protein kinase C or by elevation of cytoplasmic free Ca2+.