Mutagenesis and Modeling of the Neurotensin Receptor NTR1
1998; Elsevier BV; Volume: 273; Issue: 26 Linguagem: Inglês
10.1074/jbc.273.26.16351
ISSN1083-351X
AutoresCatherine Labbé‐Jullié, Séverine Barroso, Delphine Nicolas-Etève, Jean-Louis Reversat, J. Botto, Jean Mazella, Jean‐Marie Bernassau, Patrick Kitabgi,
Tópico(s)Neuroendocrine regulation and behavior
ResumoThe two neurotensin receptor subtypes known to date, NTR1 and NTR2, belong to the family of G-protein-coupled receptors with seven putative transmembrane domains (TM). SR 48692, a nonpeptide neurotensin antagonist, is selective for the NTR1. In the present study we attempted, through mutagenesis and computer-assisted modeling, to identify residues in the rat NTR1 that are involved in antagonist binding and to provide a tentative molecular model of the SR 48692 binding site. The seven putative TMs of the NTR1 were defined by sequence comparison and alignment of bovine rhodopsin and G-protein-coupled receptors. Thirty-five amino acid residues within or flanking the TMs were mutated to alanine. Additional mutations were performed for basic residues. The wild type and mutant receptors were expressed in COS M6 cells and tested for their ability to bind125I-NT and [3H]SR 48692. A tridimensional model of the SR 48692 binding site was constructed using frog rhodopsin as a template. SR 48692 was docked into the receptor, taking into account the mutagenesis data for orienting the antagonist. The model shows that the antagonist binding pocket lies near the extracellular side of the transmembrane helices within the first two helical turns. The data identify one residue in TM 4, three in TM 6, and four in TM 7 that are involved in SR 48692 binding. Two of these residues, Arg327 in TM 6 and Tyr351 in TM 7, play a key role in antagonist/receptor interactions. The former appears to form an ionic link with the carboxylic group of SR 48692, as further supported by structure-activity studies using SR 48692 analogs. The data also show that the agonist and antagonist binding sites in the rNTR1 are different and help formulate hypotheses as to the structural basis for the selectivity of SR 48692 toward the NTR1 and NTR2. The two neurotensin receptor subtypes known to date, NTR1 and NTR2, belong to the family of G-protein-coupled receptors with seven putative transmembrane domains (TM). SR 48692, a nonpeptide neurotensin antagonist, is selective for the NTR1. In the present study we attempted, through mutagenesis and computer-assisted modeling, to identify residues in the rat NTR1 that are involved in antagonist binding and to provide a tentative molecular model of the SR 48692 binding site. The seven putative TMs of the NTR1 were defined by sequence comparison and alignment of bovine rhodopsin and G-protein-coupled receptors. Thirty-five amino acid residues within or flanking the TMs were mutated to alanine. Additional mutations were performed for basic residues. The wild type and mutant receptors were expressed in COS M6 cells and tested for their ability to bind125I-NT and [3H]SR 48692. A tridimensional model of the SR 48692 binding site was constructed using frog rhodopsin as a template. SR 48692 was docked into the receptor, taking into account the mutagenesis data for orienting the antagonist. The model shows that the antagonist binding pocket lies near the extracellular side of the transmembrane helices within the first two helical turns. The data identify one residue in TM 4, three in TM 6, and four in TM 7 that are involved in SR 48692 binding. Two of these residues, Arg327 in TM 6 and Tyr351 in TM 7, play a key role in antagonist/receptor interactions. The former appears to form an ionic link with the carboxylic group of SR 48692, as further supported by structure-activity studies using SR 48692 analogs. The data also show that the agonist and antagonist binding sites in the rNTR1 are different and help formulate hypotheses as to the structural basis for the selectivity of SR 48692 toward the NTR1 and NTR2. Many neuropeptide receptors belong to the family of GTP-binding protein-coupled receptors (GPCR) 1The abbreviations used are: GPCR, GTP-binding protein-coupled receptor; TM, transmembrane domain; NT, neurotensin; NTR1, neurotensin receptor 1; NTR2, neurotensin receptor 2; r, rat; m, mouse; h, human; IP, inositol phosphate. 1The abbreviations used are: GPCR, GTP-binding protein-coupled receptor; TM, transmembrane domain; NT, neurotensin; NTR1, neurotensin receptor 1; NTR2, neurotensin receptor 2; r, rat; m, mouse; h, human; IP, inositol phosphate.with seven putative transmembrane domains (TMs). The past decade has witnessed the discovery of a number of nonpeptide antagonist ligands of GPCRs for neuropeptides such as cholecystokinin, tachykinins, or angiotensin (1Woodruff G.N. Hughes J. Annu. Rev. Pharmacol. Toxicol. 1991; 31: 469-501Crossref PubMed Scopus (209) Google Scholar, 2Watling K.J. Krause J.E. Trends Pharmacol. Sci. 1993; 14: 81-84Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 3Smith R.D. Chiu A.T. Wong P.C. Herblin W.F. Timmermans M.W.M. Annu. Rev. Pharmacol. Toxicol. 1992; 32: 135-165Crossref PubMed Scopus (249) Google Scholar). Most often, these compounds are selective for a given neuropeptide receptor subtype and, unlike peptides, cross the blood-brain barrier. As such, they have proven extremely useful for exploring the central and peripheral physiopathological functions associated with the receptor they antagonize. It seems to be a general feature that peptide agonist and nonpeptide antagonist bind to distinct epitopes of GPCRs. Site-directed mutagenesis of GPCRs, like vasopressin, tachykinins, or angiotensin receptors, has allowed for the determination of several conserved or specific residues that are involved in agonist and/or antagonist binding (4Mouillac B. Chini B. Balestre M.-N. Elands J. Trumpp-Kallmeyer S. Hoflack J. Hibert M. Jard S. Barberis C. J. Biol. Chem. 1995; 270: 25771-25777Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 5Gether U. Johansen T.E. Snider R.M. Lowe III, J.A. Nakanishi S. Schwartz T.W. Nature. 1993; 362: 345-348Crossref PubMed Scopus (203) Google Scholar, 6Perlman S. Costa-Neto C.M. Miyakawa A.A. Schambye H.T. Hjorth S.A. Paiva A.C.M. Rivero R.A. Greenlee W.J. Schwartz T.W. Mol. Pharmacol. 1997; 51: 301-311Crossref PubMed Scopus (71) Google Scholar). In some studies, mutagenesis data were complemented by computer-assisted modeling to construct tridimensional models of ligand-receptor complexes (7Hibert M.F. Trumpp-Kallmeyer S. Hoflack J. Bruinvels A. Trends Pharmacol. Sci. 1993; 14: 7-12Abstract Full Text PDF PubMed Scopus (95) Google Scholar). This approach has led to the proposal of spatial models for the binding sites of a number of GPCRs that interact with small nonpeptide ligands (4Mouillac B. Chini B. Balestre M.-N. Elands J. Trumpp-Kallmeyer S. Hoflack J. Hibert M. Jard S. Barberis C. J. Biol. Chem. 1995; 270: 25771-25777Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 8Trumpp-Kallmeyer S. Hoflack J. Bruinvels A. Hibert M. J. Med. Chem. 1992; 35: 3448-3462Crossref PubMed Scopus (424) Google Scholar, 9Befort K. Tabbara L. Kling D. Maigret B. Kieffer B.L. J. Biol. Chem. 1996; 271: 10161-10168Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Multiplication of such tridimensional models of GPCRs, depicting the binding site for nonpeptide antagonists, would define a general binding epitope for these small nonpeptide ligands. This approach will be of great interest for the design of new selective high affinity molecules. The neuropeptide neurotensin (NT) exerts central actions that include hypothermia (10Bissette G. Nemeroff C.B. Loosen P.T. Prange Jr., A.J. Lipton M.A. Nature. 1976; 262: 607-609Crossref PubMed Scopus (262) Google Scholar), analgesia (11Clineschmidt B.V. Martin G.E. Veber D.F. Ann. N. Y. Acad. Sci. 1982; 400: 283-304Crossref PubMed Scopus (48) Google Scholar), and a number of effects that involve the modulation of nigrostriatal and meso-cortico-limbic dopaminergic pathways (12Nemeroff C.B. Biol. Psychiatry. 1980; 15: 283-302PubMed Google Scholar, 13Kitabgi P. Neurochem. Int. 1989; 14: 111-119Crossref PubMed Scopus (108) Google Scholar). Two NT receptors termed NTR1 and NTR2 (or NTRH and NTRL) have been cloned so far (14Tanaka K. Masu M. Nakanishi S. Neuron. 1990; 4: 847-854Abstract Full Text PDF PubMed Scopus (480) Google Scholar, 15Vita N. Laurent P. Lefort S. Chalon P. Dumont X. Kaghad M. Gully D. Le Fur G. Ferrara P. Caput D. FEBS Lett. 1993; 317: 139-142Crossref PubMed Scopus (227) Google Scholar, 16Chalon P. Vita N. Kaghad M. Guillemot M. Bonnin J. Delpech B. Le Fur G. Ferrara P. Caput D. FEBS Lett. 1996; 386: 91-94Crossref PubMed Scopus (241) Google Scholar, 17Mazella J. Botto J.M. Guillemare E. Coppola T. Sarret P. Vincent J.P. J. Neurosci. 1996; 15: 5613-5620Crossref Google Scholar). They share 60% homology and both belong to the family of GPCRs. The NTR1 has high affinity for NT, whereas the NTR2 has lower affinity for the peptide and is selectively recognized by levocabastine, an anti-histamine H1 receptor antagonist (14Tanaka K. Masu M. Nakanishi S. Neuron. 1990; 4: 847-854Abstract Full Text PDF PubMed Scopus (480) Google Scholar, 15Vita N. Laurent P. Lefort S. Chalon P. Dumont X. Kaghad M. Gully D. Le Fur G. Ferrara P. Caput D. FEBS Lett. 1993; 317: 139-142Crossref PubMed Scopus (227) Google Scholar, 16Chalon P. Vita N. Kaghad M. Guillemot M. Bonnin J. Delpech B. Le Fur G. Ferrara P. Caput D. FEBS Lett. 1996; 386: 91-94Crossref PubMed Scopus (241) Google Scholar, 17Mazella J. Botto J.M. Guillemare E. Coppola T. Sarret P. Vincent J.P. J. Neurosci. 1996; 15: 5613-5620Crossref Google Scholar). These receptors have widespread, though not identical, central and peripheral distributions and exhibit distinct ontogenic profiles (15Vita N. Laurent P. Lefort S. Chalon P. Dumont X. Kaghad M. Gully D. Le Fur G. Ferrara P. Caput D. FEBS Lett. 1993; 317: 139-142Crossref PubMed Scopus (227) Google Scholar, 16Chalon P. Vita N. Kaghad M. Guillemot M. Bonnin J. Delpech B. Le Fur G. Ferrara P. Caput D. FEBS Lett. 1996; 386: 91-94Crossref PubMed Scopus (241) Google Scholar). SR 48692, a nonpeptide NT antagonist (18Gully D. Canton M. Boigegrain R. Jeanjean F. Molimard J.C. Poncelet M. Gueudet C. Heaulme M. Leyris R. Brouard A. Pelaprat D. Labbé-Jullié C. Mazella J. Soubrié P. Maffrand J.P. Rostène W. Kitabgi P. Le Fur G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 65-69Crossref PubMed Scopus (400) Google Scholar), preferentially binds to the NTR1 (16Chalon P. Vita N. Kaghad M. Guillemot M. Bonnin J. Delpech B. Le Fur G. Ferrara P. Caput D. FEBS Lett. 1996; 386: 91-94Crossref PubMed Scopus (241) Google Scholar, 17Mazella J. Botto J.M. Guillemare E. Coppola T. Sarret P. Vincent J.P. J. Neurosci. 1996; 15: 5613-5620Crossref Google Scholar) and has provided a useful tool to define the functions associated with this receptor (19Gully D. Jeanjean F. Poncelet M. Steinberg R. Soubrié P. Le Fur G. Maffrand J.P. Fundam. Clin. Pharmacol. 1995; 9: 513-521Crossref PubMed Scopus (23) Google Scholar). In particular, SR 48692 blocks many of the effects attributed to NT interaction with mesencephalic dopaminergic neurons (20Rostène W. Azzi M. Boudin H. Lepee I. Souaze F. Mendez-Ubach M. Betancur C. Gully D. Ann. N. Y. Acad. Sci. 1997; 814: 125-141Crossref PubMed Scopus (48) Google Scholar). In contrast, it does not antagonize the hypothermic and analgesic responses to NT, suggesting that these effects are not initiated through the NTR1 (21Dubuc I. Costentin J.P. Terranova J.P. Barnouin M.C. Soubrié P. Le Fur G. Rostène W. Kitabgi P. Br. J. Pharmacol. 1994; 112: 352-354Crossref PubMed Scopus (112) Google Scholar). Preliminary data have indicated that NT and SR 48692 bind to different regions of the NTR1 (22Labbé-Jullié C. Botto J.M. Mas M.V. Chabry J. Mazella J. Vincent J.P. Gully D. Maffrand J.P. Kitabgi P. Mol. Pharmacol. 1995; 47: 1050-1056PubMed Google Scholar). In the present study we attempted, through mutagenesis and computer-assisted modeling, to identify residues in the NTR1 that are involved in antagonist binding and to provide a tentative molecular model of the SR 48692 binding site. Because of the hydrophobic and aromatic nature of SR 48692 (Fig. 1), we initially chose to mutate hydrophobic and aromatic residues present in or close to the putative TMs of the NTR1 (Fig. 2). The wild type and mutant receptors were expressed in COS M6 cells and tested for their ability to bind125I-NT and [3H]SR 48692. A first tridimensional model of the SR 48692 binding site in the NTR1 was constructed using bacteriorhodopsin as a template (23Henderson R. Baldwin J.M. Ceska T.A. Zemlin F. Beckmann E. Downing K.H. J. Mol. Biol. 1990; 213: 899-929Crossref PubMed Scopus (2525) Google Scholar). SR 48692 was docked into the receptor, taking into account the mutagenesis data for orienting the antagonist in the transmembrane region. This initial model pointed to a number of hydrophilic residues (Met, Thr, His, Gln, Lys, and Arg) that might also interact with SR 48692 (Fig. 2), thus leading us to construct and test a second series of mutant receptors.Figure 2Alignments of the sequences in the seven transmembrane domains of rNTR1, hNTR1, rNTR2, mNTR2, and bovine rhodopsin (bR). The residues conserved between the rNTR1 and the other proteins are represented by periods. The TMs are numbered in circles on theright. They are oriented so that their extracellular side face the left of the figure. Shaded residues are those that were mutated in the present study.View Large Image Figure ViewerDownload (PPT) Bacteriorhodopsin, though not a GPCR, was at the time we initiated these studies the only protein with seven transmembrane α-helices whose tridimensional structure was known with some resolution (23Henderson R. Baldwin J.M. Ceska T.A. Zemlin F. Beckmann E. Downing K.H. J. Mol. Biol. 1990; 213: 899-929Crossref PubMed Scopus (2525) Google Scholar). Recently, structural data on the arrangement of the transmembrane helices of rhodopsin, the prototype GPCR, has become available (24Unger V.M.U. Hargrave P.A. Baldwin J.M. Schertler G.F.X. Nature. 1997; 389: 203-206Crossref PubMed Scopus (481) Google Scholar,25Baldwin J.M. Schertler G.F. Unger V.M. J. Mol. Biol. 1997; 213: 899-929Google Scholar). Therefore, the representation of the SR 48692-NTR1 complex reported here was achieved by using rhodopsin instead of bacteriorhodopsin as a template for modeling the NTR1 and by positioning SR 48692 into the core of the seven helices so as to fit the mutagenesis data. Our model identifies a number of residues in TMs 6 and 7 that are involved in SR 48692 binding. Two of these residues, Arg327 in TM 6 and Tyr351 in TM 7, play a key role in antagonist/receptor interactions. The data also show that the agonist and antagonist binding sites in the rNTR1 are different. Finally, they help formulate hypotheses as to the structural basis for the selectivity of SR 48692 toward the NTR1 and NTR2. Neurotensin was from Neosystem and SR 48692, SR 48527, and SR 49711 (Fig. 1) from Sanofi Recherche. Monoiodo-[125I-Tyr3]neurotensin (125I-NT) was prepared as described in Ref. 26Bidard J.N. de Nadai F. Rovere C. Moinier D. Laur J. Martinez J. Cuber J.C. Kitabgi P. Biochem. J. 1993; 291: 225-233Crossref PubMed Scopus (34) Google Scholar. [3H]SR 48692 was from Amersham Pharmacia Biotech. The rNTR1 cDNA is a generous gift of Dr. Nakanishi (14Tanaka K. Masu M. Nakanishi S. Neuron. 1990; 4: 847-854Abstract Full Text PDF PubMed Scopus (480) Google Scholar). The rNTR1HindIII-NotI fragment, 1.45 kilobase pairs corresponding to the total reading frame plus the 5′-noncoding end, was used as a template for oligonucleotide site-directed mutagenesis as described previously (27Botto J.M. Chabry J. Nouel D. Paquet M. Séguéla P. Vincent J.P. Beaudet A. Mazella J. Mol. Brain Res. 1997; 46: 311-317Crossref PubMed Scopus (10) Google Scholar). Oligonucleotides were synthesized by Eurogentec. Correct sequences of the mutant receptors cDNA were verified by ABI Prism dye terminator cycle sequencing ready reaction kit following the manufacturer's protocol. TheHindIII-NotI fragments were then subcloned into pcDNA3 eucaryotic vector (Invitrogen). Restriction and modification enzymes were from Eurogentec. COS M6 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Dutcher) and 50 μg/ml gentamicin (Sigma). For transient transfection, 100-mm cell culture dishes seeded with 106 cells the day before were washed twice with Tris-buffered saline (200 mm Tris, 137 mm NaCl, 2.3 mm CaCl2, 0.5 mmMgCl2, 0.4 mm Na2HPO4, pH 7.4) and incubated for 30 min with 1 μg of recombinant pcDNA3 plasmid in the presence of DEAE-dextran (0.5 mg/ml) at room temperature. After 3 h in culture medium supplemented with 100 μm chloroquine, cells were washed twice with Tris-buffered saline and cultured for 48–72 h. Transfected cells were washed twice with phosphate-buffered saline, collected in ice-cold 5 mm Tris/HCl, pH 8. After homogenization by repeated passages through a syringe needle and centrifugation at 4 °C for 30 min at 100,000 × g, cell membranes were resuspended in 500 μl/dish of 5 mm Tris/HCl, pH 7.5, and stored at −70 °C. Membrane protein concentration was determined by the Bio-Rad protein assay. 24 h after transfection with the wild type rNTR1 or the R327M receptor, cells were trypsinized and grown for 18 h in 12-well plates in culture medium in the presence of 0.5 μCi of [3H]myo-inositol (ICN). After two washes with Earle buffer (25 mm Hepes, 25 mm Tris, 140 mm NaCl, 5 mm KCl, 1.8 mm CaCl2, 0.9 mm MgCl2, 5 mm glucose) containing 0.1% bovine serum albumin, cells were incubated for 15 min at 37 °C in 1 ml of 20 mm LiCl in Earle buffer, with or without increasing concentrations of antagonists. Then, NT was added at the indicated concentrations in 10 μl of Earle buffer for 15 min. The reaction was stopped by 750 μl of ice-cold 10 mm HCOOH. After 1 h at 4 °C, the supernatant was collected and neutralized by 3 ml of 5 mmNH4OH. Total [3H]inositol phosphates (IP) were separated from free [3H]inositol on Dowex AG1-X8 (Bio-Rad) chromatography by eluting successively with 5 ml of water and 4 ml of 40 mm and 1 m ammonium formate buffer, pH 5.5. The radioactivity contained in the 1 m fraction was counted after addition of 5 ml of EcoLume (ICN). Binding experiments for the two radioligands were carried out with 1–100 μg of cell membrane proteins in a final volume of 250 μl of 50 mm Tris/HCl, pH 7.5, containing 0.1% bovine serum albumin and 0.8 mm1,10-phenantroline, for 20 min at room temperature. The reaction was stopped by addition of 2 ml of ice-cold buffer and filtration on cellulose acetate filter (0.2 μm, Sartorius) followed by two washes of the tube and filter with 2 ml of the same buffer. Nonspecific binding was determined in the presence of 1 μm unlabeled ligand. For saturation experiments, concentrations of radioligand ranging from 0.01 to 2 nm for 125I-NT or from 0.1 to 10 nm for [3H]SR 48692 were tested. For competitive inhibition experiments, increasing concentrations of unlabeled ligands were incubated with 0.05 nm125I-NT. Saturation and competition data were analyzed by the LIGAND program (28Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7771) Google Scholar). B max values derived from saturation binding data with either radioligand did not vary by more than 50% for a given receptor between different transfection experiments. However, they ranged from 0.1 to 10 pmol/mg protein for125I-NT binding and from 1 to 30 pmol/mg protein for [3H]SR 48692 binding among the wild type and mutant receptors. For a given receptor, the B max value for the antagonist was two to five times higher than that for the agonist. This is due to the fact that, in the concentration range used,125I-NT labels only those binding sites that are in a high affinity conformation, as described previously (22Labbé-Jullié C. Botto J.M. Mas M.V. Chabry J. Mazella J. Vincent J.P. Gully D. Maffrand J.P. Kitabgi P. Mol. Pharmacol. 1995; 47: 1050-1056PubMed Google Scholar). The rNTR1 sequence was aligned by means of the “Viseur” software 2F. Campagne, J. M. Bernassau, and B. Maigret; http://www.ictn.u-nancy.fr/viseur/viseur.html. with those of the hNTR1, the rNTR2, and mNTR2, other GPCRs, and bovine rhodopsin (25Baldwin J.M. Schertler G.F. Unger V.M. J. Mol. Biol. 1997; 213: 899-929Google Scholar) to define by sequence comparison the regions in the rNTR1 that would most likely correspond to the seven putative TMs thought to exist in all such receptors. These domains are shown in Fig. 2. A three-dimensional model of the TMs of the rNTR1 was built using the Sybyl program by substituting each rhodopsin residue side chain by the corresponding one in the rNTR1 sequence. Then, a global minimization was effected by the Powel method with a dielectric constant value of 4 and with fixed Kollman charges and essential hydrogen atoms, while freezing the helical backbone. Sixteen aromatic and 6 hydrophobic residues located within TMs 1–7 or near the junction between the TMs and the extracellular domains of the rNTR1 were mutated into Ala (Fig. 2 and TableI). The wild type and mutant receptors were expressed in COS M6 cells, and saturation experiments with either125I-NT or [3H]SR 48692 were performed with membranes prepared from each transfectant. K d values for both ligands are shown in Table I. Mutations of the rNTR1 in TMs 1, 3, 4, and 5 did not affect the affinity of either ligand. In contrast, mutations Y324A and F331A in TM 6, and Y351A and Y359A in TM 7, yielded receptors that showed no measurable [3H]SR 48692 binding. In addition, the F358A mutation in TM 7 produced a 3-fold decrease in antagonist affinity. These mutations either did not affect the affinity of NT (Y324A, F358A, and Y359A) or resulted in a 10-fold decrease in affinity (F331A, Y351A) (Table I). Interestingly, the Y347A mutation in TM 7 led to an apparent loss of agonist binding without change in antagonist affinity (Table I).Table IKd and Ki values for neurotensin and SR 48692 binding to wild type and mutated rNTR1TMMutationK d,K iaK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.NeurotensinSR 48692nmWild type0.12 ± 0.022.60 ± 0.200.18 ± 0.01aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.5.88 ± 0.11aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.TM1V65A0.11 ± 0.022.21 ± 0.13L66A0.12 ± 0.022.17 ± 0.16V67A0.07 ± 0.012.59 ± 0.34TM3Y145A0.16 ± 0.034.42 ± 1.12Y146A0.30 ± 0.032.69 ± 0.33F147A0.14 ± 0.012.25 ± 0.26Y154A0.26 ± 0.062.43 ± 0.61TM4F206A0.08 ± 0.022.36 ± 0.25TM5V236A0.12 ± 0.014.21 ± 0.66V237A0.12 ± 0.013.14 ± 0.55I238A0.13 ± 0.013.04 ± 0.46F243A0.13 ± 0.021.42 ± 0.16TM6Y324A0.20 ± 0.07ND0.28 ± 0.03aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.106 ± 7aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.F331A1.29 ± 0.15ND3.02 ± 0.51aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.124 ± 19aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.Y333A0.20 ± 0.052.86 ± 0.61TM7F346A0.22 ± 0.023.12 ± 0.42Y347AND3.99 ± 0.73Y349A0.45 ± 0.103.02 ± 0.77F350A0.23 ± 0.035.37 ± 1.19Y351A0.90 ± 0.27ND1.18 ± 0.12aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.1625 ± 470aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.F358A0.13 ± 0.048.94 ± 1.90.31 ± 0.13aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.29.1 ± 1.6aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.Y359A0.40 ± 0.07ND0.40 ± 0.09aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.29.2 ± 2.2aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.COS M6 cells were transfected with wild type and mutated rNTR1s and used for binding assays as described under “Experimental Procedures.” K d values in nm were derived from Scatchard analysis of saturation binding experiments employing either 125I-NT or [3H]SR 48692 as labeled ligand.a K i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable. Open table in a new tab COS M6 cells were transfected with wild type and mutated rNTR1s and used for binding assays as described under “Experimental Procedures.” K d values in nm were derived from Scatchard analysis of saturation binding experiments employing either 125I-NT or [3H]SR 48692 as labeled ligand. To determine the decrease in antagonist binding affinity for those mutant receptors that did not measurably bind [3H]SR 48692, competition experiments with unlabeled SR 48692 and NT were performed using 125I-NT as the labeled ligand (Fig. 3). K i values were thus derived for both competitors and are shown in Table I. The Y351A mutant, the Y324A and F331A mutants, and the Y359A mutant exhibited 200-fold, 20-fold, and 5-fold increases in K i values for SR 48692, respectively, as compared with the wild type rNTR1. The data also confirmed the decreased antagonist affinity of the F358A mutant and the decreased agonist affinity of the F331A and Y351A mutants observed in saturation experiments. An initial model of the rNTR1 was constructed using bacteriorhodopsin as a template. SR 48692 was docked into the model, taking into account the above mutagenesis data. Visual inspection of the SR 48692-rNTR1 complex predicted that a number of hydrophilic residues might interact with the antagonist. In particular, it suggested that the carboxylic function of the antagonist might form an ionic link with either one of four basic residues in the rNTR1, i.e. Arg143 in TM 3, Lys235near TM 5, Arg327 and Arg328 in TM 6. The model also predicted that Met204 and/or Met208 in TM 4 might interact with the adamantane moiety of SR 48692 and that one or several threonine, histidine, or glutamine residues (Thr68in TM 1, Thr156 in TM 3, Gln239 and Thr242 in TM 5, His325 in TM6, and His348 and Thr354 in TM 7) might stabilize the receptor-antagonist interaction. To assess the above predictions, additional mutant receptors were constructed and tested in binding and pharmacological experiments. The M204A mutation did not significantly affect the binding affinity of either SR 48692 or NT (Table II). In contrast, the M208A mutant exhibited a 10-fold increase inK i for the antagonist and a 6-fold increase inK i and K d for NT, as compared with the wild type rNTR1 (Table II). The T354A mutant showed a 5–6-fold decrease in SR 48692 affinity and no change in NT affinity. The T68A, T156A, Q236A, T242A, H325A, and H348A mutants bound SR 48692 and NT with the same affinity as the wild type rNTR1.Table IIKd and Ki values for neurotensin and SR 48692 binding to wild type and mutated rNTR1TMMutationK d,K iaK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.NeurotensinSR 48692nmWild type0.12 ± 0.022.60 ± 0.200.18 ± 0.01aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.5.88 ± 0.11aK i values in nm were derived from competition binding experiments with unlabeled NT and SR 48692 as the competitors and 125I-NT as the labeled ligand. The values are the means ± S.E. of three to five experiments. ND, not determinable.TM1T68A0.29 ± 0.051.89 ± 0.21TM3R143K0.11 ± 0.011.55 ± 0.29R143Q0.13 ± 0.031.77 ± 0.32R143M0.09 ± 0.042.75 ± 0.62T156A0.22 ± 0.052.32 ± 0.27TM4M204A0.20 ± 0.064.56 ± 0.45M
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