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• W2095683872 abstract "A prototypic study of the molecular mechanisms of activation or inactivation of peptide hormone G protein-coupled receptors was carried out on the human B2 bradykinin receptor. A detailed pharmacological analysis of receptor mutants possessing either increased constitutive activity or impaired activation or ligand recognition allowed us to propose key residues participating in intramolecular interaction networks stabilizing receptor inactive or active conformations: Asn113 and Tyr115 (TM III), Trp256and Phe259 (TM VI), Tyr295 (TM VII) which are homologous of the rhodopsin residues Gly120, Glu122, Trp265, Tyr268, and Lys296, respectively. An essential experimental finding was the spatial proximity between Asn113, which is the cornerstone of inactive conformations, and Trp256 which plays a subtle role in controlling the balance between active and inactive conformations. Molecular modeling and mutagenesis data showed that Trp256 and Tyr295 constitute, together with Gln288, receptor contact points with original nonpeptidic ligands. It provided an explanation for the ligand inverse agonist behavior on the WT receptor, with underlying restricted motions of TMs III, VI, and VII, and its agonist behavior on the Ala113 and Phe256 constitutively activated mutants. These data on the B2 receptor emphasize that conformational equilibria are controlled in a coordinated fashion by key residues which are located at strategic positions for several G protein-coupled receptors. They are discussed in comparison with the recently determined rhodopsin crystallographic structure. A prototypic study of the molecular mechanisms of activation or inactivation of peptide hormone G protein-coupled receptors was carried out on the human B2 bradykinin receptor. A detailed pharmacological analysis of receptor mutants possessing either increased constitutive activity or impaired activation or ligand recognition allowed us to propose key residues participating in intramolecular interaction networks stabilizing receptor inactive or active conformations: Asn113 and Tyr115 (TM III), Trp256and Phe259 (TM VI), Tyr295 (TM VII) which are homologous of the rhodopsin residues Gly120, Glu122, Trp265, Tyr268, and Lys296, respectively. An essential experimental finding was the spatial proximity between Asn113, which is the cornerstone of inactive conformations, and Trp256 which plays a subtle role in controlling the balance between active and inactive conformations. Molecular modeling and mutagenesis data showed that Trp256 and Tyr295 constitute, together with Gln288, receptor contact points with original nonpeptidic ligands. It provided an explanation for the ligand inverse agonist behavior on the WT receptor, with underlying restricted motions of TMs III, VI, and VII, and its agonist behavior on the Ala113 and Phe256 constitutively activated mutants. These data on the B2 receptor emphasize that conformational equilibria are controlled in a coordinated fashion by key residues which are located at strategic positions for several G protein-coupled receptors. They are discussed in comparison with the recently determined rhodopsin crystallographic structure. G-protein coupled receptor wild-type transmembrane domain constitutively activated mutant BK, bradykinin inositol phosphate The understanding of the mechanisms of G-protein coupled receptor (GPCR)1 activation has been improved by numerous experimental supports to the existence of multiple conformations, active or inactive (1Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar). Mutation-induced constitutive activation phenomena have been widely exploited to dissect the intramolecular interaction networks which stabilize inactive receptor conformations (1Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar), including random mutagenesis approaches to get deeper insight into the obvious complexity of these networks (2Lu Z.L. Hulme E.C. J. Biol. Chem. 1999; 274: 7309-7315Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar,3Spalding T.A. Burstein E.S. Henderson S.C. Ducote K.R. Brann M.R. J. Biol. Chem. 1998; 273: 21563-21568Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In a preceding work (4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar) we evidenced constitutive activation of the human B2 bradykinin receptor induced by mutations of Asn113 in TM III and Trp256 in TM VI. The role of this tryptophan residue, which is fairly conserved in the GPCR family, was suggested by our previous modeling studies of the AT1 receptor (5Groblewski, T. (1997) Health Biology, Ph. D. Thesis, Montpellier.Google Scholar) and the constitutive activation of the Ala113 mutant is reminiscent of previous observations for the AT1 receptor mutated at the homologous Asn (6Groblewski T. Maigret B. Larguier R. Lombard C. Bonnafous J.C. Marie J. J. Biol. Chem. 1997; 272: 1822-1866Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The exceptionally high constitutive activation of the Ala113B2 receptor demonstrated the crucial role of Asn113 in stabilizing an inactive B2 receptor conformation and the extreme conformational flexibility of this receptor. This flexibility was further supported by the striking changes in the pharmacological properties of peptidic or nonpeptidic ligands induced by Asn113 or Trp256 mutations (4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar). These data make the B2 receptor an interesting model to study conformational changes associated to activation. In this respect the original nonpeptidic ligands designed by Fournier Research Laboratories, which constitute potential anti-inflammatory drugs (7Pruneau D. Luccarini J.M. Fouchet C. Defrene E. Franck R.M. Loillier B. Duclos H. Robert C. Cremers B. Belichard P. Paquet J.L. Br. J. Pharmacol. 1998; 125: 365-372Crossref PubMed Scopus (29) Google Scholar,8Pruneau D. Paquet J.L. Luccarini J.M. Defrene E. Fouchet C. Franck R.M. Loillier B. Robert C. Belichard P. Duclos H. Cremers B. Dodey P. Immunopharmacology. 1999; 43: 187-194Crossref PubMed Scopus (87) Google Scholar), turned out to be interesting tools to modulate conformational equilibria. The purpose of the present work was to take advantage of constitutive activation phenomena to delineate some features of inactive receptor conformations and to propose a model of nonpeptide antagonist interaction with the B2 receptor. These models are discussed with reference to those built for other GPCR (9Baldwin J.M. Schertler G.F. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (632) Google Scholar, 10Herzyk P. Hubbard R.E. J. Mol. Biol. 1998; 281: 741-754Crossref PubMed Scopus (45) Google Scholar), the recent crystallographic data for rhodopsin (11Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5038) Google Scholar) and current knowledge about their activation processes. This study emphasizes the roles of some key residues that are located at strategic positions for several GPCR, including rhodopsin, and simultaneously control ligand recognition and conformational equilibria (Fig.1). BK was purchased from Sigma,myo-[2-3H] inositol and [3H]BK (specific radioactivity about 100 Ci/mmol) were purchased from Amersham Pharmacia Biotech. Hydroxyphenyl-propionyl-HOE 140 (HPP-HOE 140) was kindly supplied by Professor J. Martinez (CNRS, Montpellier, France); it was radioiodinated using Na125I (2,000 Ci/mmol) and IODO-GEN as oxidizing agent. The nonpeptidic derivatives (Fig.2) were designed and synthesized by Fournier Research Laboratories (Daix, France). COS-7 cells were from the European Cell Type Collection (Salisbury, United Kingdom). The human B2 receptor sequence has been determined by Hess et al. (12Hess J.F. Borkowski J.A. Young G.S. Strader C.D. Ransom R.W. Biochem. Biophys. Res. Commun. 1992; 184: 260-268Crossref PubMed Scopus (448) Google Scholar). The WT and mutated receptors were systematically tagged through the addition of a 10-amino acid epitope from the c-myc oncogene at the N termini of receptors truncated at the Asn3 residue. The cDNA sequences included a Kozak sequence. The various mutations were carried out as described previously (4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar). Receptors were transiently expressed in COS-7 cells using the electroporation transfection method (4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar). Pharmacological characterizations were performed on cells cultured for 2 days at 37 °C in Dulbecco's modified Eagle's medium, 4.5 g/liter glucose, 10% fetal calf serum, 100 units/ml penicillin, 100 units/ml streptomycin. The binding of [3H]BK or125I-HPP-HOE 140 to crude membranes from transfected COS-7 cells was carried out as previously described (4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar). The binding of [3H]BK to intact cells (24 or 48 well dishes), including evaluation of expression levels, and IP production (12-well dishes) were routinely carried out as described in Ref. 4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar. In experiments devoted to the effects of Zn2+ ions, the media were modified to ensure their perfect solubility: [3H]BK binding was carried out in 25 mm HEPES, 140 mmNaCl, 140 µg/ml bacitracin, 10 µm captopril, pH 7.4; IP accumulation was measured in 20 mm HEPES, 116 mm NaCl, 11 mm glucose, 2.5 mmCaCl2, 4.7 mm KCl, 140 µg/ml bacitracin, 10 µm captopril, pH 7.4. Models were built on an “Octane” Silicon Graphics computer, using software from Molecular Simulations Inc. (Insight, Discover, Homology, Modeler). Molecular dynamics simulations carried out in the absence of restraints of some transmembrane helices were performed using the computing facilities of CINES. The B2 receptor “experimental model” was built using the Modeler software, using as template a first AT1receptor model (13Joseph M.P. Maigret B. Bonnafous J.C. Marie J. Scheraga H.A. J. Protein Chem. 1995; 14: 381-398Crossref PubMed Scopus (67) Google Scholar) in which the position of TM VI has been refined (about 30° rotation) so as to fit pharmacological data relative to losartan (5Groblewski, T. (1997) Health Biology, Ph. D. Thesis, Montpellier.Google Scholar). As compared with the AT1 template a slight rotation of helix VII was introduced to take into account binding properties on nonpeptidic ligands to receptors mutated at some positions of these helices (Thr287 and Gln288). A second B2 receptor model was built using the rhodopsin crystallographic structure as template (“rhodopsin-like” model). For both models the positions of the transmembrane helix peptidic backbones were strictly ascribed to those of their respective templates with the exception of transmembrane domains displaying differences in proline residues: in the “rhodopsin-like model” the 35–48 (N-terminal part of TM I) and 285–290 (N-terminal part of TM VII) sequences were ascribed to helix structures; in the experimental model the 278–288 sequence was forced to an helix structure. Molecular dynamics was initially applied to the conformational analysis of LF 16-0687. The analysis of the corresponding trajectory revealed, as expected, a great flexibility of the molecule. The lowest energy conformations were compact, with an interaction between the quinoline and benzamidine moieties. However, it appeared impossible to perform any preliminary positioning of such conformations into our receptor model. As a consequence we selected extended conformations and performed their manual docking so as to fit the experimental data. The quinoline of LF 16-0687 or LF 18-1300 was positioned so as to interact simultaneously with Tyr295 and Trp256. Then the rest of the molecule was orientated toward the extracellular side of the TMs, applying appropriate torsion angles to the ligand bonds, with the exception of the dichlorophenyl-SO2-prolyl portion which was considered as playing an essential orientation role; therefore the geometry of this portion was kept close to that delineated by the ligand conformational analysis. No remarkable interaction could be found for the dichloro-phenyl group of the ligand which was positioned between TMs II and VII inside the intrahelix space. Thr287 and Gln288 thus appeared as possible candidates for an interaction with the carbonyl function of the prolyl ligand moiety. As the mutation of Thr287 to Ala or Leu, its B1 counterpart, had no incidence on affinity, we favored the existence of an hydrogen bond interaction between Gln288 and the carbonyl group of the prolyl moiety and we slightly rotated helix VII, by ∼20°, so as to fulfil this interaction preference while preserving the “sandwich” interaction of the quinoline with the Trp256 and Tyr295residues. Our previous modeling studies of the AT1 receptor (5Groblewski, T. (1997) Health Biology, Ph. D. Thesis, Montpellier.Google Scholar,13Joseph M.P. Maigret B. Bonnafous J.C. Marie J. Scheraga H.A. J. Protein Chem. 1995; 14: 381-398Crossref PubMed Scopus (67) Google Scholar) and experimental data revealed the essential roles of the conserved Asp in TM II (Asp74), Asn111 (TM III), the fairly conserved Trp in TM VI (Trp253), His256located one helix turn above Trp253 and Tyr292(TM VII) which occupies a position homologous to that of the rhodopsin Lys296, the site of retinal covalent binding. An inactive receptor conformation would be stabilized by a double interaction of Asn111 (TM III) with Tyr292 (TM VII) and Trp253 (TM VI), homologous of the rhodopsin Trp265; the Asn111-Trp253 proximity was postulated upon model refinement (helix VI rotation) (5Groblewski, T. (1997) Health Biology, Ph. D. Thesis, Montpellier.Google Scholar) to make it consistent with the pharmacological properties of the nonpeptidic ligand losartan (5Groblewski, T. (1997) Health Biology, Ph. D. Thesis, Montpellier.Google Scholar, 14Noda K. Saad Y. Kinoshita A. Boyle T.P. Graham R.M. Husain A. Karnik S.S. J. Biol. Chem. 1995; 270: 2284-2289Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). We postulated that the interaction of the Tyr4 residue of AII with Asn111 would destabilize the above mentioned network and induce rearrangements resulting in a Asp74-Tyr292 interaction (13Joseph M.P. Maigret B. Bonnafous J.C. Marie J. Scheraga H.A. J. Protein Chem. 1995; 14: 381-398Crossref PubMed Scopus (67) Google Scholar) and an intrahelical interaction between Trp253 and His256. The experimental supports can be summarized as follows: receptor inactivation upon Y292F (15Marie J. Maigret B. Joseph M.P. Larguier R. Nouet S. Lombard C. Bonnafous J.C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar), W253F (5Groblewski, T. (1997) Health Biology, Ph. D. Thesis, Montpellier.Google Scholar), and H256A mutations (5Groblewski, T. (1997) Health Biology, Ph. D. Thesis, Montpellier.Google Scholar, 16Noda K. Saad Y. Karnik S.S. J. Biol. Chem. 1995; 270: 28511-28514Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar); strong constitutive activation upon Asn111 mutations (6Groblewski T. Maigret B. Larguier R. Lombard C. Bonnafous J.C. Marie J. J. Biol. Chem. 1997; 272: 1822-1866Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 17Noda K. Feng Y.H. Liu X.P. Saad Y. Husain A. Karnik S.S. Biochemistry. 1996; 35: 16435-16442Crossref PubMed Scopus (142) Google Scholar); detailed analysis of the induction of intermediary or activated conformations (17Noda K. Feng Y.H. Liu X.P. Saad Y. Husain A. Karnik S.S. Biochemistry. 1996; 35: 16435-16442Crossref PubMed Scopus (142) Google Scholar, 18Miura S. Feng Y.H. Husain A. Karnik S.S. J. Biol. Chem. 1999; 274: 7103-7110Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Interestingly these residues are conserved in the B2receptor, with the exception of His256 which is replaced by Phe259. That this latter residue is a BK interaction site (proposed for the rat receptor (19Jarnagin K. Bhakta S. Zuppan P. Yee C. Ho T. Phan T. Tahilramani R. Pease J.H. Miller A. Freedman R. J. Biol. Chem. 1996; 271: 28277-28286Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and extended to the human receptor in the present work) and previous results demonstrating a role for Asn113 and Trp256 (4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar) reinforces the interest of a mechanistic comparison of the AT1 and B2receptors which display a sequence identity similar to that of the AT1/AT2 or B1/B2 pairs (about 30%). Our previous study (4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar) showed that Trp256 mutation to Phe or Gln induced marked increases in the human B2receptor constitutive activity, together with changes in the pharmacological properties of the peptidic compound HOE 140 and the nonpeptidic ligand LF 16-0335. We further investigated the crucial role of Trp256 by evaluating the activation properties of the W256F, W256Q, and W256A mutant receptors, as well as the double mutants displaying the additional Asn113 to Ala mutation. Fig. 3shows that, at similar expression levels, the W256F and W256Q mutants were significantly constitutively activated (CAM receptors) as compared with the wild-type receptor, the activation factor varying from 4 to 10 according to experiments. Combined with the previously described exceptionally high constitutive activation displayed by the Ala113 mutant (Fig. 3), we postulated that inactive receptor conformation(s) might be stabilized by an Asn113-Trp256interaction. The first interesting finding was the lack of constitutive activation of the W256A mutant, with preservation of the BK activation properties (Fig. 3). This result has two implications: taking into account the proximity of Asn113 and Trp256 (validated in experiments described in the next paragraph) the hypothesis that Asn113 and Trp256 might stabilize an inactive conformation through interactions with separate unidentified partners appeared unlikely as Trp to Ala mutation would be expected to be activating in such a situation; the role of Trp256 cannot be restricted to an interaction with Asn113 stabilizing an inactive conformation. Trp256 might also contribute to the stabilization or the generation of an active conformation in which the Asn113-Trp256 interaction would be disrupted. One can reasonably postulate that Asn113 and Trp256 belong to a network of concerted interactions comprising one or several other residues and subtly tuned by the residue at position 256. Constitutive activation observed upon Trp mutation to Phe or Gln would then result from the perturbation of a balance between these interactions. Asn113 and Trp256 were substituted, either separately or simultaneously, by histidine, and the binding of Zn2+ ions to the single or double His mutants was evaluated. Results in Fig. 4Ashow that His mutations by themselves induce no or little perturbation of BK or HOE 140 recognition. Competition binding of Zn2+ions using [3H]BK as tracer ligand revealed a striking leftwards shift of the dose-response curve for the double N113H, W256H mutant as compared with the WT receptor (Fig. 4); it indicated an increased affinity for Zn2+ ions, interpreted as a spatial proximity between the two histidines. This effect was not observed for the W256H single mutant. It was much less pronounced for the N113H single mutant; the slight shift observed in this case probably resulted from the ability of Zn2+ ions, which can display different modes of interactions with amino acid side chains (20Konopka J.B. Margarit S.M. Dube P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6764-6769Crossref PubMed Scopus (106) Google Scholar, 21Fathy D.B. Mathis S.A. Leeb T. Leeb-Lundberg L.M. J. Biol. Chem. 1998; 273: 12210-12218Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. EMBO J. 1996; 15: 3566-3578Crossref PubMed Scopus (361) Google Scholar), to coordinate N113H to another residue. These results were confirmed by functional assays which first indicated that His replacement of Asn113, or Trp256 or both induced neither constitutive activation, nor modification of BK-stimulated IP production. Zn2+ ions, which had no effect on basal activities, were able to counteract BK-induced IP production in COS-7 expressing the N113H, W256H mutant (Fig. 4B), with Zn2+ inactivation characteristics similar to those observed for BK binding. The pivotal role of Trp256 was confirmed by the pharmacological properties of B2 receptors mutated at this position. HOE 140, which behaved as an inverse agonist of the WT receptor in experimental situations where the basal IP production is high enough (4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar), became a fairly potent agonist of the three W256F, W256Q, and W256A mutant receptors, with a markedly decreased maximal stimulation for the latter mutant (Fig.5A). The binding of a radioiodinated derivative of HOE 140 (125I-HPP-HOE 140) revealed moderate decreases in affinity for the mutant receptors (TableI). The behavior of the nonpeptidic compound LF 16-0335 was strikingly different: it only activated the W256F mutant (Fig. 5B). These properties of LF 16-0335 were reproduced for LF 16-0687, LF 18-1300, and LF 18-1833 which were agonists of the W256F mutant, and not of the W256Q and W256A mutants (not shown). The Ki values relative to LF 16-0335 and LF 16-0687, determined in competition binding assays using125I-HPP-HOE 140 as tracer ligand, were slightly increased for the three mutants, the most significant increase being observed for the W256A receptor (Table I). Similar data were obtained when [3H]BK was used as tracer ligand (not shown). These results suggest that Trp256 might directly interact with the nonpeptidic ligand through an aromatic-aromatic interaction (which does not exclude additional interactions such as hydrogen bonding or amino-aromatic interactions) and that preservation of this interaction in the W256F CAM receptor participates to the ligand agonist behavior (see paragraph devoted to modeling).Table ILigand recognition properties of human B2 mutant receptorsReceptorsTM[3H]BKKd125I-HPP-HOE 140KdLF 16–0335LF 16–0687KiKiKimut/Ki wtnmnmnmnmWT0.51 ± 0.200.28 ± 0.161.08 ± 0.5310.45 ± 0.29N48AI0.39 ± 0.090.31 ± 0.011.60 ± 0.251.47D76AII0.40 ± 0.120.12 ± 0.080.84 ± 0.280.77D76NII1.00 ± 0.520.18 ± 0.071.05 ± 0.070.97N92AII0.86 ± 0.150.57 ± 0.062.11 ± 0.851.94S111AIII1.51 ± 0.40.36 ± 0.041.4 ± 0.501.29S111KIII2.58 ± 1.400.67 ± 0.072.8 ± 1.222.59N113AIII0.27 ± 0.120.20 ± 0.090.62 ± 0.120.57Y115AIII1.25 ± 0.210.38 ± 0.041.60 ± 0.521.47Y115FIII1.15 ± 0.350.31 ± 0.060.62 ± 0.340.57S116AIII0.30 ± 0.050.20 ± 0.011.17 ± 0.081.08S117AIII0.70 ± 0.120.06 ± 0.011.90 ± 0.921.750.35 ± 0.04F120AIII0.42 ± 0.110.27 ± 0.030.47 ± 0.620.43M165AIV0.52 ± 0.040.45 ± 0.123.99 ± 1.103.72.60 ± 0.52M165TIV0.44 ± 0.110.33 ± 0.051.72 ± 0.951.600.75 ± 0.15N198AV0.30 ± 0.042.20 ± 0.80N202AV0.24 ± 0.120.50 ± 0.121.00 ± 0.150.92F206AV0.53 ± 0.081.00 ± 0.251.91 ± 0.551.75F252AVI0.21 ± 0.070.56 ± 0.122.60 ± 0.852.30C255AVI1.090.53 ± 0.05C255SVI1.400.54 ± 0.04W256AVI1.00 ± 0.210.50 ± 0.167.34 ± 1.336.82.94 ± 0.40W256FVI0.52 ± 0.180.32 ± 0.133.24 ± 1.883.000.70 ± 0.08W256QVI0.41 ± 0.150.32 ± 0.074.32 ± 0.5541.09 ± 0.64P258AVI1.21 ± 0.12F259AVI80 ± 141-aDetermined using [3H]BK as tracer ligand.0.28 ± 0.051.15 ± 0.061.06Q260AVI0.55 ± 0.110.38 ± 0.082.56 ± 0.952.30Q260HVI0.45 ± 0.010.31 ± 0.112.70 ± 0.822.50S262AVI1.92 ± 0.940.32 ± 0.101.81 ± 0.651.67S262FVI1.44 ± 0.250.17 ± 0.052.34 ± 0.772.16T263AVI170 ± 421-aDetermined using [3H]BK as tracer ligand.0.15 ± 0.061.00 ± 0.120.92F264AVI0.41 ± 0.130.35 ± 0.132.47 ± 1.252.30D284AVII4.40 ± 0.611.4 ± 0.701.86 ± 0.861.71T287AVII0.88 ± 0.150.62 ± 0.321.17 ± 0.35T287LVII0.62 ± 0.120.27 ± 0.060.18 ± 0.02Q288AVII3.45 ± 0.501.90 ± 0.4016.2 ± 0.9154.20 ± 1.80S291AVII0.30 ± 0.151.40 ± 0.521.29F292AVII0.66 ± 0.200.52F292YVII0.62 ± 0.220.37 ± 0.120.27 ± 0.07Y295AVII4.70 ± 1.102.7 ± 0.7025.0 ± 5.523.542.9 ± 11.5Y295FVII0.35 ± 0.070.48 ± 0.157.80 ± 4.907.23.78 ± 0.45S296AVII0.38 ± 0.172.31 ± 3.102.1N297AVIIUD1-bUD, undetectable.UDS298AVII0.18 ± 0.050.51 ± 0.251.83 ± 0.721.7N301AVII0.78 ± 0.150.50 ± .281.71 ± 0.751.6N301DVII0.25 ± 0.080.24 ± 0.050.97 ± 0.110.9The affinities of [3H]BK and 125I-HPP-HOE 140 were measured through direct binding experiments on membrane preparations from COS-7 cells transiently expressing the WT or mutant receptors. Alternatively in some experiments [3H]BK binding was carried out on intact cells; intact cells were not suitable for125I-HPP-HOE 140 binding because of passive ligand penetration, which led to overestimated values. The expression levels for a given plasmid amount (25–50 ng) was similar for the WT and all mutant receptors (1–3 × 105 sites/cell), with the exception of N198A and P258A, which displayed 10-fold reduced expression, and N297A, which was not expressed at the plasma membrane but was intracellularly detectable. The Ki values relative to the binding of LF 16–0335 or LF 16–0687 were evaluated on membrane preparations, in competition binding assays using 125I-HPP-HOE 140 as tracer ligand. Each value is the mean ± S.D. of at least three separate determinations.1-a Determined using [3H]BK as tracer ligand.1-b UD, undetectable. Open table in a new tab The affinities of [3H]BK and 125I-HPP-HOE 140 were measured through direct binding experiments on membrane preparations from COS-7 cells transiently expressing the WT or mutant receptors. Alternatively in some experiments [3H]BK binding was carried out on intact cells; intact cells were not suitable for125I-HPP-HOE 140 binding because of passive ligand penetration, which led to overestimated values. The expression levels for a given plasmid amount (25–50 ng) was similar for the WT and all mutant receptors (1–3 × 105 sites/cell), with the exception of N198A and P258A, which displayed 10-fold reduced expression, and N297A, which was not expressed at the plasma membrane but was intracellularly detectable. The Ki values relative to the binding of LF 16–0335 or LF 16–0687 were evaluated on membrane preparations, in competition binding assays using 125I-HPP-HOE 140 as tracer ligand. Each value is the mean ± S.D. of at least three separate determinations. The essential role of Trp256 was further supported by the properties of receptors doubly mutated at positions 113 and 256 which were still constitutively activated: as previously described (4Marie J. Koch C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J.C. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar), LF 16-0335 displayed agonist properties on the N113A receptor which possesses an extremely high constitutive activity. This agonist property was only maintained for the N113A,W256F double mutant (not shown). Therefore the residue at position 256 modulates the activation properties of a receptor which is strongly destabilized by Asn113 mutation to Ala. Taken together the data indicate that Trp256 controls in a subtle way the balance between the stabilization of receptor inactive conformations and the stabilization of intermediary or active conformations and that Asn113 and (or) Trp256 possess other interacting partners at some steps of the activation process. The Pro258 or Phe252 mutations to Ala did not induce any constitutive activation, so that the data obtained upon mutation of these residues in TM VI of other GPCR (3Spalding T.A. Burstein E.S. Henderson S.C. Ducote K.R. Brann M.R. J. Biol. Chem. 1998; 273: 21563-21568Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 20Konopka J.B. Margarit S.M. Dube P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6764-6769Crossref PubMed Scopus (106) Google Scholar) cannot be generalized. The mutation of Gln260 (TM VI) to His (B1 homologous residue) did not change the basal activity; its mutation to Ala induced either a weak (2-fold) or no constitutive activation according to experiments (Fig. 3). As Ser111 was postulated to be involved in peptidic ligand recognition specificity (21Fathy D.B. Mathis S.A. Leeb T. Leeb-Lundberg L.M. J. Biol. Chem. 1998; 273: 12210-12218Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), it was mutated to either Ala or Lys, its B1 counterpart. These mutations induced moderate but significant losses of BK affinity. However, none of these mutations induced any significant modification of basal IP production activities. The most clearcut f" @default.
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• W2095683872 title "Control of Conformational Equilibria in the Human B2 Bradykinin Receptor" @default.
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