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• W2003832016 abstract "The decapeptide gonadotropin-releasing hormone controls reproductive function via interaction with a heptahelical G protein-coupled receptor. Because a molecular model of the receptor predicts that Lys121 in the third transmembrane helix contributes to the binding pocket, the function of this side chain was studied by site-directed mutagenesis. Substitution of Arg at this position preserved high affinity agonist binding, whereas Gln at this position reduced binding below the limits of detection. Leu and Asp at this locus abolished both binding and detectable signal transduction. The EC50 of concentration-response curves for coupling to phosphatidyl inositol hydrolysis obtained with the Gln121 receptor was more than 3 orders of magnitude higher than that obtained for the wild-type receptor. In order to determine whether the increased EC50 obtained with this mutant reflects an altered receptor affinity, the effect of decreases in wild-type receptor density on concentration-response curves was determined by irreversible antagonism. Progressively decreasing the concentration of the wild-type receptor increased the EC50 values obtained to a maximal level of 2.4 ± 0.2 nM. Comparison of this value with the EC50 of 282 ± 52 nM observed with the Gln121 receptor mutant indicates that the agonist affinity for this mutant is reduced more than 100-fold. In contrast, antagonist had comparable high affinities for the wild-type, Arg121, and Gln121 mutants. The results indicate that a charge-strengthened hydrogen bond donor is required at this locus for high affinity agonist binding but not for high affinity antagonist binding. The decapeptide gonadotropin-releasing hormone controls reproductive function via interaction with a heptahelical G protein-coupled receptor. Because a molecular model of the receptor predicts that Lys121 in the third transmembrane helix contributes to the binding pocket, the function of this side chain was studied by site-directed mutagenesis. Substitution of Arg at this position preserved high affinity agonist binding, whereas Gln at this position reduced binding below the limits of detection. Leu and Asp at this locus abolished both binding and detectable signal transduction. The EC50 of concentration-response curves for coupling to phosphatidyl inositol hydrolysis obtained with the Gln121 receptor was more than 3 orders of magnitude higher than that obtained for the wild-type receptor. In order to determine whether the increased EC50 obtained with this mutant reflects an altered receptor affinity, the effect of decreases in wild-type receptor density on concentration-response curves was determined by irreversible antagonism. Progressively decreasing the concentration of the wild-type receptor increased the EC50 values obtained to a maximal level of 2.4 ± 0.2 nM. Comparison of this value with the EC50 of 282 ± 52 nM observed with the Gln121 receptor mutant indicates that the agonist affinity for this mutant is reduced more than 100-fold. In contrast, antagonist had comparable high affinities for the wild-type, Arg121, and Gln121 mutants. The results indicate that a charge-strengthened hydrogen bond donor is required at this locus for high affinity agonist binding but not for high affinity antagonist binding. INTRODUCTIONGnRH, 1The abbreviations used are: GnRHgonadotropin-releasing hormoneGnRHRgonadotropin-releasing hormone receptorTMHtransmembrane helixPIphosphatidylinositolTNBS2,4,6-trinitrobenzenesulfonic acid. a decapeptide secreted from neurons in the medial-basal hypothalamus, has a central role in regulating the mammalian reproductive system. GnRH induces its biological effect by interacting with high affinity pituitary receptors. cDNA cloning of the GnRH receptor (GnRHR) from five mammalian species(1Tsutsumi M. Zhou W. Millar R.P. Mellon P.L. Roberts J.L. Flanagan C.A. Dong K. Gillo B. Sealfon S.C. Mol. Endocrinol. 1992; 6: 1163-1169Crossref PubMed Scopus (218) Google Scholar, 2Reinhart J. Mertz L.M. Catt K.J. J. Biol. Chem. 1992; 267: 21281-21284Abstract Full Text PDF PubMed Google Scholar, 3Kakar S.S. Musgrove L.C. Devor D.C. Sellers J.C. Neill J.D. Biochem. Biophys. Res. Commun. 1992; 189: 289-295Crossref PubMed Scopus (351) Google Scholar, 4Chi L. Zhou W. Prikhozhan A. Flanagan C. Davidson J.S. Golembo M. Illing N. Millar R.P. Sealfon S.C. Mol. Cell. Endocrinol. 1993; 91: R1-R6Crossref PubMed Scopus (210) Google Scholar, 5Kaiser U.B. Zhao D. Cardona G.R. Chin W.W. Biochem. Biophys. Res. Commun. 1992; 189: 1645-1652Crossref PubMed Scopus (175) Google Scholar, 6Eidne K.A. Sellar R.E. Couper G. Anderson L. Taylor P.L. Mol. Cell. Endocrinol. 1992; 90: R5-R9Crossref PubMed Scopus (138) Google Scholar, 7Perrin M.H. Bilezikjian L.M. Hoeger C. Donaldson C.J. Rivier J. Haas Y. Vale W.W. Biochem. Biophys. Res. Commun. 1993; 191: 1139-1144Crossref PubMed Scopus (97) Google Scholar, 8Illing N. Jacobs G. Becker I.I. Flanagan C.A. Davidson J.S. Eales A. Zhou W. Sealfon S.C. Millar R.P. Biochem. Biophys. Res. Commun. 1993; 196: 745-751Crossref PubMed Scopus (85) Google Scholar, 9Kakar S.S. Rahe C.H. Neill J.D. Domest. Anim. Endocrinol. 1993; 10: 335-342Crossref PubMed Scopus (97) Google Scholar) has revealed that the receptor is a member of the large family of homologous seven-transmembrane helix (TMH) G protein-coupled receptors, which includes receptors for neurotransmitters and peptides(10Kobilka B. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (312) Google Scholar, 11Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (677) Google Scholar, 12Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar).Modulation of the pituitary-gonadal axis via the GnRHR has proven to be therapeutically important, and extensive research has led to the development of several thousand peptide analogs(13Karten M. Rivier J.E. Endocr. Rev. 1986; 7: 44-66Crossref PubMed Scopus (474) Google Scholar). In contrast to some other peptides such as the tachykinins and cholecystokinin, for which small nonpeptide analogs have been identified, all GnRHR ligands reported thus far are peptides. Delineation of the precise contact sites between GnRH and its receptor is critical for developing an understanding of the relationship of the GnRHR binding pocket to that of other neurotransmitter and peptide receptors and for determining the molecular mechanisms underlying receptor activation. Ultimately this insight may lead to the design of novel GnRHR ligands.We have previously reported that a mutation in TMH 7 of the GnRHR restored binding, which had been eliminated by a mutation in TMH 2 (14Zhou W. Flanagan C.A. Ballesteros J. Konvicka K. Davidson J.S. Weinstein H. Millar R.P. Sealfon S.C. Mol. Pharmacol. 1994; 45: 165-170PubMed Google Scholar). The revertant character of mutations at these loci suggested that these two sites are in spatial proximity, a hypothesis that facilitated the refinement of a preliminary three-dimensional model of the receptor helix bundle constructed according to a set of integrated methods (15Ballesteros J.A. Weinstein H. Methods Neurosci. 1995; 25: 366-428Crossref Scopus (2413) Google Scholar). This receptor model predicts that Lys121, located in the third TMH, is positioned in the ligand binding pocket of the receptor and would be accessible to GnRH. Lys121 is found in all six cloned mammalian GnRH receptors at a locus that corresponds to the position of the conserved Asp of the cationic amine receptors (Asp113 in the β-adrenergic receptor), a residue required for high affinity neurotransmitter binding(12Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar, 16Strader C.D. Sigal I.S. Candelore M.R. Rands E. Hill W. Dixon R.A.F. J. Biol. Chem. 1988; 263: 10267-10271Abstract Full Text PDF PubMed Google Scholar, 17Fraser C.M. J. Biol. Chem. 1989; 264: 9266-9270Abstract Full Text PDF PubMed Google Scholar). In order to investigate the role of Lys121 in ligand binding and activation of the GnRHR, a series of mutations was introduced at this position, and the resulting receptors were expressed and characterized in COS-1 cells. The results identify the role of charge-augmented hydrogen bonding at this position for the affinity of agonists but not antagonists.EXPERIMENTAL PROCEDURESReagents and PeptidesAll chemicals, unless otherwise specified, were obtained from Sigma. GnRH (pyro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and GnRH-A ([des-Gly10, D-Ala6, Pro-ethylamide9]-GnRH), a GnRH agonist, were purchased from Bachem (Torrance, CA). Antagonist 27 ([Ac-D-Nal(2)1,D-a-Me-pCl-Phe2,D-Trp3,N-ε-Ipr-Lys5,D-Tyr6,D-Ala10]-GnRH (18Van der Spuy Z.M. Pillay L. Hardie F. van der Watt M. de Chalain T. Kaplan H. Roeske R. Millar R.P. Early Clinical Studies with LHRH Antagonists. NTP Press, Lancaster, UK1987: 293-301Google Scholar) was a gift of R. Roeske.Receptor Constructs and Site-directed MutagenesisThe coding region of the human GnRHR cDNA (4Chi L. Zhou W. Prikhozhan A. Flanagan C. Davidson J.S. Golembo M. Illing N. Millar R.P. Sealfon S.C. Mol. Cell. Endocrinol. 1993; 91: R1-R6Crossref PubMed Scopus (210) Google Scholar) was digested with EcoRI/XhoI and subsequently subcloned into the EcoRI/SalI sites of the pAlter vector (Promega, Madison, WI). Mutations were introduced as previously reported(14Zhou W. Flanagan C.A. Ballesteros J. Konvicka K. Davidson J.S. Weinstein H. Millar R.P. Sealfon S.C. Mol. Pharmacol. 1994; 45: 165-170PubMed Google Scholar). Mutated receptor cDNA inserts, confirmed by automated DNA sequencing (Bio-Rad) were isolated after EcoRI/SphI digestion and ligated to pcDNAI/Amp (Invitrogen, San Diego, CA) previously digested by the same restriction enzymes. One of the constructs studied, the Asp121 GnRHR, was generated from the mouse GnRHR using pAlter digested with EcoRI and XbaI(14Zhou W. Flanagan C.A. Ballesteros J. Konvicka K. Davidson J.S. Weinstein H. Millar R.P. Sealfon S.C. Mol. Pharmacol. 1994; 45: 165-170PubMed Google Scholar).Tissue Culture and DNA TransfectionCOS-1 cells (American Type Culture Collection, Rockville, MD) were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and seeded into 100 mM plates at 3 million cells/plate the day before the transfection. A mixture of 10 μg of plasmid DNA and 100 μl of lipofectamine (Life Technologies, Inc.) was used to transfect each plate of cells in serum-free Dulbecco's modified Eagle's medium, and an equal volume of media containing 20% fetal bovine serum was added 5 h after transfection.Phosphoinositol Hydrolysis AssayCells were harvested with trypsin 24 h after transfection and plated into 12-well dishes. 56 h after transfection, cells were labeled in Dulbecco's modified Eagle's medium containing 0.5 μCi/ml of [myo-3H]inositol (DuPont NEN). 12-16 h later, cells were washed and exposed to GnRH agonist (plus antagonist for Schild analysis) and 20 mM of LiCl for 45 min at 37°C. Cell extracts in 10 mM formic acid at 4°C were loaded on the Dowex ion-exchange column and eluted with 1 M ammonium formate and 0.1 M formic acid.Irreversible Receptor Antagonism with 2,4,6-Trinitrobenzenesulfonic AcidCells were transfected, replated in 12-well dishes, and labeled with [myo-3H]inositol as described above. After rinsing at room temperature with phosphate-buffered saline (pH 7.4), cells were exposed to varying concentrations of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in phosphate-buffered saline at pH 8.0 for 30 min at 37°C. After rinsing, GnRH-stimulated phosphoinositol hydrolysis was assayed as above.Radiolabeled Ligand Binding AssayIodination of GnRH-A was catalyzed in the presence of Iodogen (Pierce) following published protocols(19Laws S.C. Beggs M.J. Webster J.C. Miller W.L. Endocrinology. 1990; 127: 373-380Crossref PubMed Scopus (81) Google Scholar). 72 h after transfection, cells were scraped from the culture plates and centrifuged. Cell pellets were stored at −80°C for up to 2 months. 20-50 μg of crude membrane protein adjusted to 0.4 ml with assay buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 0.1% bovine serum albumin) was incubated with 60,000 cpm of [125I]GnRH-A (~2.8 × 10−11M) at 4°C for 90 min and processed through a Brandel harvester (Life Technologies, Inc.). Nonspecific binding was determined in the presence of 10−7M cold ligand.Data and Statistical AnalysisThe counts obtained from PI hydrolysis assays were plotted using Kaleidagraph software (Synergy Software, Reading, PA) on a Macintosh computer and fitted against the formula, E = Emax/(1 + EC50/D), where Emax is the maximal response, EC50 is the concentration of the agonists that gives half of the maximal response, and D is the concentration of the agonist. To determine antagonist affinity, a Schild analysis (20Arunlakshana O. Schild H.O. Br. J. Pharmacol. 1959; 14: 48-58Crossref PubMed Scopus (4270) Google Scholar) was performed. The ratio of EC50 in the presence and absence of antagonist was plotted against the corresponding antagonist concentration and fitted to the equation, log(EC50′/EC50− 1) = log[antagonist] − log(Kd antagonist). The slope of the best fit line obtained was found not to be significantly different from the theoretical slope of 1 (two-tailed t test). A paired t test was used to evaluate the significance of differential PI stimulation by Arg121 and wild-type receptors. Ki and Bmax values were determined using the LIGAND program(21Munson P.J. Robard D. Anal. Biochem. 1980; 58: 220-239Crossref Scopus (7760) Google Scholar).RESULTS AND DISCUSSIONAffinity and Relative Efficacy of Agonists for the Wild-type and Mutant ReceptorsIn order to test the hypothesis that Lys121 interacts with GnRH, the effects on receptor function of substituting Arg, Gln, Leu, and Asp at this position were investigated. These mutations were designed to probe the characteristics of this proposed interaction. Arg preserves a charge-strengthened hydrogen bond donor at this locus. Gln retains a hydrogen bond donor but not an ionic charge. Leu eliminates all polar and ionic side chain interactions. Asp was introduced because it reverses the charge of Lys and is found in this position in neurotransmitter receptors. The progressive divergence from Lys of these substitutions allows the side chain properties required at this locus to be evaluated.Expression of the wild-type receptor in COS-1 cells generated high affinity binding of the radiolabeled agonist [125I]GnRH-A (Ki of GnRH-A = 1.8 ± 0.3 nM). When Arg was substituted for Lys121, affinity was comparable with that of the wild-type receptor (Ki = 3.3 ± 1.0 nM, Table1, Fig. 1). However, with the substitution of Gln, Leu, and Asp at this position, agonist binding was reduced below detectable limits. In order to provide further insight into the function of the mutant receptors, agonist-stimulated PI hydrolysis was also evaluated. The wild-type, Arg121, and Gln121 receptors were all able to mediate phosphatidyl inositol hydrolysis. No stimulation was detected in cells transfected with the Leu121 or Asp121 receptor mutants (Fig. 2). The EC50 obtained with the Arg121 construct was comparable with that found with the expression of the wild-type receptor. The EC50 obtained with the Gln121 receptor, however, was more than 3 orders of magnitude higher (Fig. 2 and Table 1). The EC50 values obtained for stimulation with GnRH-A of the wild-type, Arg121, and Gln121 mutants showed the same trend, including the large relative increase for the Gln121 mutant (Table1).Tabled 1 Open table in a new tab Figure 2:GnRH-stimulated PI hydrolysis in COS-1 cells expressing mutant GnRHRs. The cells were transfected with the expression vector pcDNAI/Amp (▪), Leu121 (□), Gln121 (▴), Arg121 (•), or wild-type (○) GnRHRs, and the resulting increase in intracellular inositol phosphates was measured. The response was normalized to that obtained in the wild-type receptor. Data shown are the mean ± S.E. Each curve is representative of four to five replicate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To evaluate the cause of the increased EC50 values obtained with the Gln121 receptor, the relationship between receptor expression and EC50 was examined. In several receptor expression systems, the EC50 has been found to depend on the number of receptors expressed(22Mei L. Lai J. Yamamura H.I. Roeske W.R. J. Pharmacol. Exp. Ther. 1989; 251: 90-97PubMed Google Scholar, 23Adham N. Ellerbrock B. Hartig P. Weinshank R.L. Branchek T. Mol. Pharmacol. 1993; 43: 427-433PubMed Google Scholar, 24Whaley B.S. Yuan N. Birnbaumer L. Clark R. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar). Such findings are compatible with the presence of “spare receptors” in these systems (25Kenakin T. Pharmacologic Analysis of Drug-Receptor Interaction. Raven Press, New York1993: 39-68Google Scholar). If the wild-type GnRHR were expressed in COS-1 cells with a high proportion of spare receptors, the increased EC50 observed with the Gln121 receptor could, in principle, be due to a marked decline in the level of mutant receptor expression. To evaluate this possibility, the effect of decreasing levels of receptor expression was studied using partial chemical modification to irreversibly antagonize an increasing proportion of receptors(26Furchgott R.F. Harper N.J. Simmonds A.B. Advances in Drug Research. Academic Press, New York1966: 21-55Google Scholar). Cells previously transfected with the wild-type GnRHR were exposed to varying concentrations of TNBS, which eliminates binding sites by reacting with free amino groups(27Haynes R. Osuga D.T. Feeney R.E. Biochemistry. 1967; 6: 541-547Crossref PubMed Scopus (218) Google Scholar, 28Fields R. Methods Enzymol. 1972; 25: 464-468Crossref PubMed Scopus (517) Google Scholar). Concentration-response curves were then obtained in cells containing varying GnRHR concentrations (Fig. 3). TNBS did not interfere with the capacity for the cells to mediate signal transduction, as demonstrated by the lack of effect of this treatment on the capacity of aluminum fluoride to stimulate PI hydrolysis (data not shown). Thus the increasing EC50 values and decreasing Emax observed with TNBS treatment reflects a decrease in GnRHR receptors/cell and allows a precise assessment of the effect of decreasing receptor expression on EC50. Prior exposure to TNBS at concentrations that decreased the maximal level of PI hydrolysis led to EC50 values of 2.4 ± 0.2 nM. Since this value is more than 100-fold lower than the EC50 obtained for the Gln121 receptor, the difference in the concentration-response curves observed cannot be attributed to altered receptor expression. These results demonstrate, therefore, that the affinity of agonist for the Gln121 mutant receptor is significantly reduced.Figure 3:Effect of varying receptor expression on PI hydrolysis dose-response relationships. COS-1 cells were transfected with the wild-type receptor DNA and exposed to TNBS at the concentrations indicated prior to obtaining the concentration-response curves for GnRH.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Antagonist Binding Properties Suggest Differences from Agonist RecognitionThe effects of the Lys121 substitutions on antagonist binding were also investigated. The lack of [125I]GnRH-A binding to the Gln121 receptor prevented a direct measurement of its affinity for antagonist in competition binding. We have previously studied the expressed mouse GnRHR using radiolabeled antagonist(14Zhou W. Flanagan C.A. Ballesteros J. Konvicka K. Davidson J.S. Weinstein H. Millar R.P. Sealfon S.C. Mol. Pharmacol. 1994; 45: 165-170PubMed Google Scholar). Direct antagonist binding has not proven possible for the human receptor, presumably because antagonist binding is associated with high levels of nonspecific binding, and the relatively low levels of expression (and thus specific binding) obtained with expression of the human receptor hinder direct assay with radiolabeled antagonist. Therefore, a Schild analysis was performed, which allows the determination of antagonist affinity from the functional response(20Arunlakshana O. Schild H.O. Br. J. Pharmacol. 1959; 14: 48-58Crossref PubMed Scopus (4270) Google Scholar). Whereas agonists have a low affinity for the Gln121 receptor, the affinity of antagonist is comparably high for the Gln121, Arg121, and wild-type receptors (Fig. 4 and Table 1). Thus, in contrast to high affinity agonist binding, which appears to require a positive charge at this position, antagonist binding is not sensitive to this locus of the receptor. Such a distinction of GnRHR binding sites for agonists and antagonists is consistent with a recent photoaffinity-labeling study of the GnRH receptor, which suggests that agonists and antagonists are oriented differently in the receptor(29Janovick J.A. Haviv F. Fitzpatrick T.D. Conn P.M. Endocrinology. 1993; 133: 942-945Crossref PubMed Scopus (20) Google Scholar).Figure 4:Blockade by antagonist of GnRH stimulation of PI hydrolysis by the wild-type GnRHR (panels A and C) and Gln121 mutant receptor (panels B and D). A and B, GnRH stimulation of PI hydrolysis was performed in the absence of antagonist 27 (•) or in the presence of 3 × 10−10M (○), 10−9M (×), 3 × 10−9M (□), 10−8M (Δ), or 3 × 10−8M (∇) of antagonist 27. C, Schild regression from one representative experiment with the wild-type receptor. D, Schild regression representing data obtained in four experiments with the Gln121 receptor. Antagonist 27 alone did not cause any PI stimulation at concentrations up to 10−6M (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mammalian GnRH contains an Arg in position 8, whereas chicken I GnRH, which has low affinity for the mammalian receptor, has a Gln in this position(30Millar R.P. Flanagan C.A. del Milton R.C. King J.A. J. Biol. Chem. 1989; 264: 21007-21013Abstract Full Text PDF PubMed Google Scholar). We have previously found that an acidic residue in the third extracellular loop of the GnRH receptor is required for this selectivity for mammalian Arg8-GnRH(31Flanagan C.A. Becker I.L. Davidson J.S. Wakefield I.K. Zhou W. Sealfon S.C. Millar R.P. J. Biol. Chem. 1994; 269: 22636-22641Abstract Full Text PDF PubMed Google Scholar). The effect of having an acidic residue at this position, however, is smaller for constrained GnRH agonist analogs like GnRH-A. Thus, substitution of Gln for Glu301 in the mouse GnRH receptor induces a 50-fold decrease in the affinity for GnRH but only a 4-5 fold change in the affinity for GnRH-A. These results are consistent with the presence of different, although presumably overlapping, binding sites for different classes of agonists. The binding site of GnRH requires an acidic residue in the third extracellular loop, whereas the binding site of GnRH-A has minimal involvement of this locus. Agonist interaction sites that play a fundamental role in positioning the pharmacophore for receptor activation or in transmitting the activation itself are likely to be conserved among all agonist classes. Because both GnRH activity and GnRH-A activity require a positive charge at Lys121, interaction with this site is likely to play a crucial role in receptor activation. Taken together, these considerations identify at least three types of determinants for ligand-receptor interaction: residues required for antagonist binding, residues required for binding of certain agonists, and residues involved in binding of all agonists. Clarification of the role of various receptor loci in ligand interaction should facilitate the understanding of the mechanism of agonist activation of the receptor.The Role of the 121 Locus in GnRHR in Determining the Nature and Consequences of Interactions with LigandsWith mutation of the ligand docking site, the effects of each mutation must correlate with the altered energy of interaction arising from the side chain properties of the amino acids introduced. Analysis of this correlation in the present study provides insight into the nature of the interaction between GnRH and the residue at position 121. A basic amino acid, either Lys or Arg, is required for high affinity agonist binding, yet a direct ionic interaction can be excluded by the lack of a suitable counterion on the ligand. The results, however, are consistent with a hydrogen bond interaction. Both Lys and Arg are strong hydrogen bond donors(32Jeffrey G.A. Saenger W. Hydrogen Bonding in Biological Structures. Springer-Verlag, Berlin1991Crossref Google Scholar). The presence of the weaker hydrogen bond donor Gln at this position would reduce the strength of this interaction and lead to the decreased agonist affinity observed. The Leu and Asp mutant receptors lack hydrogen bond donors at this position, and both demonstrate no detectable agonist binding or coupling. The results are therefore consistent with a charge-strengthened hydrogen bond as the underlying mechanism for Lys121 to interact with GnRH. Because Lys121 contributes to the high affinity of agonists but not antagonists, a limited number of candidate hydrogen bond acceptors in GnRH can be proposed. Structure-activity studies of GnRH have indicated the importance of His2 and Trp3 for agonist activity(13Karten M. Rivier J.E. Endocr. Rev. 1986; 7: 44-66Crossref PubMed Scopus (474) Google Scholar), and these two residues, therefore, represent the best candidates for interacting with Lys121. In forming the hydrogen bond, Lys121 could interact with the electron-dense aromatic rings of His2 or Trp3, the type of interaction postulated to be responsible for the binding of the nonpeptide antagonist CP96345 to the substance P receptor(33Fong T.M. Cascieri M.A. Yu H. Bansal A. Swain C. Strader C.D. Nature. 1993; 362: 350-353Crossref PubMed Scopus (178) Google Scholar). Alternatively, Lys121 may hydrogen bond to the polar imino group of His2. Further molecular modeling and experimental studies should allow the precise site of interaction on GnRH to be determined.The two potential contact sites between GnRH and the receptor identified to date, Lys121 and Glu301, have similarities in their tolerance for variability at each position. In both cases little alteration in affinity is induced by substitution with a similarly charged residue. The Glu in extracellular loop 3 of the mouse GnRHR, which is involved in high affinity GnRH binding, can be replaced by an Asp with little effect on receptor affinity(31Flanagan C.A. Becker I.L. Davidson J.S. Wakefield I.K. Zhou W. Sealfon S.C. Millar R.P. J. Biol. Chem. 1994; 269: 22636-22641Abstract Full Text PDF PubMed Google Scholar). In the present study, we found that the Arg121 substitution, presenting the hydrogen bond donor group at either ε or η positions that can mimic the ζ position of the group in Lys, gives comparable affinity. In either locus of the GnRHR, substitution by Gln significantly decreases GnRH affinity. Thus alterations in the length of the side chain of charged residues in TMH 3 and in the third extracellular loop are both well tolerated, suggesting flexibility in the spatial constraints for the binding interaction.In contrast to affinity, the receptor activation mechanism may be sensitive to the length of the side chain at position 121. Thus, the maximal level of GnRH-stimulated inositol phosphate accumulation obtained with expression of the Arg121 receptor was significantly higher than that generated by the wild-type receptor in all experiments (paired two-tailed t test, p < .05, n = 5 experiments). Expression of the Gln121 receptor led to a lower maximal stimulation. Because the Gln121 receptor was undetectable in radioligand binding, the possibility that the reduction in Emax was due to a lower level of expression of this receptor cannot be definitively excluded. In the case of the Arg121 construct, however, the higher level of stimulation was accompanied by a reduction in the level of receptor expression, suggesting that the activated state of the Arg121 receptor is more efficient at G protein coupling than the activated wild-type receptor. A qualitatively similar effect of side chain length on efficacy was observed in the β-adrenergic receptor. Asp113, which is located at a position homologous to that of Lys121 in the GnRHR, serves as the counterion for binding of the catecholamine head group of the ligand. Substitution of Glu at this position led to the development of partial agonist activity from antagonists for the wild-type β-adrenergic receptor(34Strader C.D. Candelore M.R. Hill W.S. Dixon R.A. Sigal I.S. J. Biol. Chem. 1989; 264: 16470-16477Abstract Full Text PDF PubMed Google Scholar). In the GnRHR, replacing Lys121 with Arg leads to an augmentation of the efficiency of signal transduction by GnRH. In both receptors, altering the length of this charged side chain leads to an alteration of measured drug efficacy. This similarity between a neurotransmitter and a peptide receptor suggests that the interaction with this locus can contribute to activation, possibly by serving to position the component of the ligand that triggers the receptor.We have identified a transmembrane domain site in the GnRHR that is specifically involved in docking GnRH agonists. We have previously provided evidence for the proximity of specific side chains in helix 2 and helix 7 (14Zhou W. Flanagan C.A. Ballesteros J. Konvicka K. Davidson J.S. Weinstein H. Millar R.P. Sealfon S.C. Mol. Pharmacol. 1994; 45: 165-170PubMed Google Scholar) and have proposed another site of GnRH docking in the third extracellular domain(31Flanagan C.A. Becker I.L. Davidson J.S. Wakefield I.K. Zhou W. Sealfon S.C. Millar R.P. J. Biol. Chem. 1994; 269: 22636-22641Abstract Full Text PDF PubMed Google Scholar). This study provides insight into the structure of the receptor and of the GnRH binding site. The results obtained constitute a useful basis for extending and refining an experimentally testable model of the structure of the receptor-hormone complex that will make possible the elucidation of the molecular dynamics of receptor activation. INTRODUCTIONGnRH, 1The abbreviations used are: GnRHgonadotropin-releasing hormoneGnRHRgonadotropin-releasing hormone receptorTMHtransmembrane helixPIphosphatidylinositolTNBS2,4,6-trinitrobenzenesulfonic acid. a decapeptide secreted from neurons in the medial-basal hypothalamus, has a central role in regulating the mammalian reproductive system. GnRH induces its biological effect by interacting with high affinity pituitary receptors. cDNA cloning of the GnRH receptor (GnRHR) from five mammalian species(1Tsutsumi M. Zhou W. Millar R.P. Mellon P.L. Roberts J.L. Flanagan C.A. Dong K. Gillo B. Sealfon S.C. Mol. Endocrinol. 1992; 6: 1163-1169Crossref PubMed Scopus (218) Google Scholar, 2Reinhart J. Mertz L.M. Catt K.J. J. Biol. Chem. 1992; 267: 21281-21284Abstract Full Text PDF PubMed Google Scholar, 3Kakar S.S. Musgrove L.C. Devor D.C. Sellers J.C. Neill J.D. Biochem. Biophys. Res. Commun. 1992; 189: 289-295Crossref PubMed Scopus (351) Google Scholar, 4Chi L. Zhou W. Prikhozhan A. Flanagan C. Davidson J.S. Golembo M. Illing N. Millar R.P. Sealfon S.C. Mol. Cell. Endocrinol. 1993; 91: R1-R6Crossref PubMed Scopus (210) Google Scholar, 5Kaiser U.B. Zhao D. Cardona G.R. Chin W.W. Biochem. Biophys. Res. Commun. 1992; 189: 1645-1652Crossref PubMed Scopus (175) Google Scholar, 6Eidne K.A. Sellar R.E. Couper G. Anderson L. Taylor P.L. Mol. Cell. Endocrinol. 1992; 90: R5-R9Crossref PubMed Scopus (138) Google Scholar, 7Perrin M.H. Bilezikjian L.M. Hoeger C. Donaldson C.J. Rivier J. Haas Y. Vale W.W. Biochem. Biophys. Res. Commun. 1993; 191: 1139-1144Crossref PubMed Scopus (97) Google Scholar, 8Illing N. Jacobs G. Becker I.I. Flanagan C.A. Davidson J.S. Eales A. Zhou W. Sealfon S.C. Millar R.P. Biochem. Biophys. Res. Commun. 1993; 196: 745-751Crossref PubMed Scopus (85) Google Scholar, 9Kakar S.S. Rahe C.H. Neill J.D. Domest. Anim. Endocrinol. 1993; 10: 335-342Crossref PubMed Scopus (97) Google Scholar) has revealed that the receptor is a member of the large family of homologous seven-transmembrane helix (TMH) G protein-coupled receptors, which includes receptors for neurotransmitters and peptides(10Kobilka B. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (312) Google Scholar, 11Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (677) Google Scholar, 12Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar).Modulation of the pituitary-gonadal axis via the GnRHR has proven to be therapeutically important, and extensive research has led to the development of several thousand peptide analogs(13Karten M. Rivier J.E. Endocr. Rev. 1986; 7: 44-66Crossref PubMed Scopus (474) Google Scholar). In contrast to some other peptides such as the tachykinins and cholecystokinin, for which small nonpeptide analogs have been identified, all GnRHR ligands reported thus far are peptides. Delineation of the precise contact sites between GnRH and its receptor is critical for developing an understanding of the relationship of the GnRHR binding pocket to that of other neurotransmitter and peptide receptors and for determining the molecular mechanisms underlying receptor activation. Ultimately this insight may lead to the design of novel GnRHR ligands.We have previously reported that a mutation in TMH 7 of the GnRHR restored binding, which had been eliminated by a mutation in TMH 2 (14Zhou W. Flanagan C.A. Ballesteros J. Konvicka K. Davidson J.S. Weinstein H. Millar R.P. Sealfon S.C. Mol. Pharmacol. 1994; 45: 165-170PubMed Google Scholar). The revertant character of mutations at these loci suggested that these two sites are in spatial proximity, a hypothesis that facilitated the refinement of a preliminary three-dimensional model of the receptor helix bundle constructed according to a set of integrated methods (15Ballesteros J.A. Weinstein H. Methods Neurosci. 1995; 25: 366-428Crossref Scopus (2413) Google Scholar). This receptor model predicts that Lys121, located in the third TMH, is positioned in the ligand binding pocket of the receptor and would be accessible to GnRH. Lys121 is found in all six cloned mammalian GnRH receptors at a locus that corresponds to the position of the conserved Asp of the cationic amine receptors (Asp113 in the β-adrenergic receptor), a residue required for high affinity neurotransmitter binding(12Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar, 16Strader C.D. Sigal I.S. Candelore M.R. Rands E. Hill W. Dixon R.A.F. J. Biol. Chem. 1988; 263: 10267-10271Abstract Full Text PDF PubMed Google Scholar, 17Fraser C.M. J. Biol. Chem. 1989; 264: 9266-9270Abstract Full Text PDF PubMed Google Scholar). In order to investigate the role of Lys121 in ligand binding and activation of the GnRHR, a series of mutations was introduced at this position, and the resulting receptors were expressed and characterized in COS-1 cells. The results identify the role of charge-augmented hydrogen bonding at this position for the affinity of agonists but not antagonists." @default.
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• W2003832016 title "A Locus of the Gonadotropin-releasing Hormone Receptor That Differentiates Agonist and Antagonist Binding Sites" @default.
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