HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 44, 41100-41111, November 2, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/44/41100    most recent M104875200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marie, J. Articles by Bonnafous, J.-C. Search for Related Content PubMed PubMed Citation Articles by Marie, J. Articles by Bonnafous, J.-C. Social Bookmarking                      What's this?

Control of Conformational Equilibria in the Human B₂ Bradykinin Receptor

MODELING OF NONPEPTIDIC LIGAND ACTION AND COMPARISON TO THE RHODOPSIN STRUCTURE^*

Jacky Marie, Eric Richard, Didier Pruneau^§, Jean-Luc Paquet^§, Christian Siatka, Renée Larguier, Cecilia Poncé, Philippe Vassault, Thierry Groblewski^¶, Bernard Maigret, and Jean-Claude Bonnafous^**

From  INSERM U439, 70 rue de Navacelles 34090 Montpellier, the ^§ Laboratoires Fournier, 50 rue de Dijon, and  Laboratoire de Chimie théorique, Université de Nancy 1, 54506 Vandoeuvre-les-Nancy Cédex, 21121 Daix

Received for publication, May 29, 2001, and in revised form, July 17, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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), Trp²⁵⁶ and 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1). Mutation-induced constitutive activation phenomena have been widely exploited to dissect the intramolecular interaction networks which stabilize inactive receptor conformations (1), including random mutagenesis approaches to get deeper insight into the obvious complexity of these networks (2, 3).

In a preceding work (4) 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 (5) and the constitutive activation of the Ala¹¹³ mutant is reminiscent of previous observations for the AT₁ receptor mutated at the homologous Asn (6). The exceptionally high constitutive activation of the Ala¹¹³ B₂ 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 (4). 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 (7, 8), 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 (9, 10), the recent crystallographic data for rhodopsin (11) 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).

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Location and conservation in some GPCR of residues playing key or switch roles in the B2 receptor. Sequence comparisons of TMs II, III, VI, and VII of the B2 receptor with those of some other GPCR reveal the residues, located at privileged positions, which have been shown to be involved in ligand binding or activation properties, or both. The residues located in boxes have been experimentally shown to be important for the function of a given receptor. The roles of the mentioned B2 receptor residues have been assessed in previous works (4, 19) or constitute the matter of the present paper.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents and Ligands-- 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).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Structure of nonpeptidic ligands specific of the human B2 bradykinin receptor. The compounds LF 16-0335 and LF 16-0687 are quite similar: they possess the same substituted quinoline at one extremity and a benzamidine moiety at the other, the latter compound displaying a significantly higher affinity for the receptor. LF 18-1300 differs from LF 16-0687 by the imidazole substitution on the quinoline moiety, resulting in improved affinity. LF 18-1833 possesses the same imidazole-substituted quinoline, while being shortened at its other extremity which no longer possesses the benzamidine function.

Site-directed Mutagenesis and Receptor Expression-- The human B₂ receptor sequence has been determined by Hess et al. (12). 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 (4). Receptors were transiently expressed in COS-7 cells using the electroporation transfection method (4). 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.

Binding Assays-- The binding of [³H]BK or ¹²⁵I-HPP-HOE 140 to crude membranes from transfected COS-7 cells was carried out as previously described (4).

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. 4. 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 mM NaCl, 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 mM CaCl₂, 4.7 mM KCl, 140 µg/ml bacitracin, 10 µM captopril, pH 7.4.

Molecular Modeling-- 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 AT₁ receptor model (13) in which the position of TM VI has been refined (about 30° rotation) so as to fit pharmacological data relative to losartan (5). 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 Tyr²⁹⁵ residues.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strategy: Rationale of AT₁ and B₂ Receptor Comparison

Our previous modeling studies of the AT₁ receptor (5, 13) 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), His²⁵⁶ located 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) (5) to make it consistent with the pharmacological properties of the nonpeptidic ligand losartan (5, 14). 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 (13) and an intrahelical interaction between Trp²⁵³ and His²⁵⁶. The experimental supports can be summarized as follows: receptor inactivation upon Y292F (15), W253F (5), and H256A mutations (5, 16); strong constitutive activation upon Asn¹¹¹ mutations (6, 17); detailed analysis of the induction of intermediary or activated conformations (17, 18). Interestingly these residues are conserved in the B₂ receptor, with the exception of His²⁵⁶ which is replaced by Phe²⁵⁹. That this latter residue is a BK interaction site (proposed for the rat receptor (19), and extended to the human receptor in the present work) and previous results demonstrating a role for Asn¹¹³ and Trp²⁵⁶ (4) reinforces the interest of a mechanistic comparison of the AT₁ and B₂ receptors which display a sequence identity similar to that of the AT₁/AT₂ or B₁/B₂ pairs (about 30%).

Role of Trp²⁵⁶ in the Activation Process

Our previous study (4) showed that Trp²⁵⁶ mutation to Phe or Gln induced marked increases in the human B₂ receptor 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. 3 shows 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-Trp²⁵⁶ interaction.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Basal IP production activities in WT or mutant B2 receptors. The WT and mutant receptor were expressed at several mean levels, in the range 3 × 105-6 × 105 sites/cell, as described under "Experimental Procedures" and basal activities have been compared for similar expression levels within each experiment. The indicated values represent the mean of three to 10 experiments.

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.

Biochemical Verification of the Asn¹¹³-Trp²⁵⁶ Proximity

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. 4A show 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 (20-22), 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.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Histidine engineering and Zn2+ effects on BK binding and BK-induced IP production. Asn113 or Trp256 or both residues were mutated to His and the WT and mutant B2 receptors were transiently expressed in COS-7 cells as described under "Experimental Procedures." Zn2+ ion affinities for the various receptors were indirectly evaluated by their ability to inhibit [3H]BK binding (1 nM) to intact cells (A) or 0.5 nM BK-induced IP production (B).

Concerted Roles of Trp²⁵⁶ and Asn¹¹³ in the Human B₂ Receptor Activation

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 (4), 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 (Table I). 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 K_i values relative to LF 16-0335 and LF 16-0687, determined in competition binding assays using ¹²⁵I-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).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Incidence of the residue at position 256 on the properties of HOE 140 (A) and LF 16-0335 (B). The WT or Phe256, Gln256, or Ala256 mutant B2 receptors were transiently expressed in COS-7 cells at similar levels (6 × 105-106 sites/cell) and the ability of the peptidic and nonpeptidic ligands to modulate IP production evaluated as indicated under "Experimental Procedures." The presented typical experiment is representative of three separate experiments. In these specific experiments designed for mutant receptors and ligand comparisons, the basal WT activity was not high enough to evidence the inverse agonist activity of HOE 140 and LF 16-0335, as previously observed (4). The agonist efficacies of HOE 140 for the Phe256, Gln256, and Ala256 receptors were 86, 79, and 27% of the BK efficacies, respectively. The efficacy of LF 16-0335 for the Phe256 mutant was 42% of that of BK in the reported typical experiments. The Kd values of 125I-HPP-HOE 140 and the Ki values of LF 16-0335 are collected in Table I. Similar results we found in three separate experiments and were also obtained with LF 16-0687 as nonpeptidic ligand.

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 (4), 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.

Characterization of New Mutations Modulating Activation Properties: Roles of Tyr¹¹⁵ and Tyr²⁹⁵

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 (3, 20) 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 (21), 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 findings refer to the role of Tyr¹¹⁵, located two residues below Asn¹¹³ and homologous of the rhodopsin Glu¹²². Important constitutive activations were reproducibly elicited by Tyr¹¹⁵ mutation: while its mutation to Phe (the residue located at the homologous position in the B₁ receptor) had no effect, its mutation to Ala reproducibly induced a 5-fold increase in the basal IP production (Fig. 3). The pharmacological properties of the nonpeptidic compound LF 16-0687 for the Y115A receptor were unchanged as it behaved as in inverse agonist, at variance with the CAM N113A and W256F receptors. Examination of the various models suggested some residues as possible partners of Tyr¹¹⁵; a role for some of them was experimentally ruled out: Ser¹⁶², Asn²⁰², and Phe²⁰⁶ located in the vicinity of Gly²⁰⁵, homologous of rhodopsin His²¹¹ which was shown to interact with Glu¹²² (11).

The mutation of the conserved Asn⁴⁸ in TM I did not induce constitutive activation. These results differ from those reported for the _1B and TRH receptors (22, 23) and does not allow to draw any conclusion about the connections between TMs I, II, and VII (23-25).

We checked for possible interactions between Trp²⁵⁶ and residues of TM V which are expected to face TM VI. Phe²⁰⁶ mutation to Ala neither induced constitutive activation, nor significant modification of BK activation properties, leading to the conclusion that the proposed interaction between the TM VI Trp and TM V Phe in the TRH receptor (26) cannot be extrapolated. No significant perturbation of activation properties could be detected for the D202A mutant.

Tyr²⁹⁵ mutation to Phe (4) or to Ala in TM VII of the B₂ bradykinin receptor changed neither its basal IP production activity nor its BK activation properties (Fig. 3 for basal activities, data not shown for BK activation properties). These results show that Tyr²⁹⁵ does not play, by itself, a role similar to that of the homologous Tyr²⁹² in the AT₁ receptor. Nevertheless, as this residue, homologous of the rhodopsin Lys²⁹⁶, was demonstrated to be an essential interaction site with nonpeptidic compounds, together with Gln²⁸⁸ (see modeling section), we constructed some double mutants to check a possible role of Tyr²⁹⁵ in association with other residues. Interestingly we found that while the Y295A and Q228A mutant displayed moderately decreased recognition of BK and HOE140 (Table I), the Q228A,Y295A double mutant no longer recognized BK, and displayed biphasic binding curves for ¹²⁵I-HPP-HOE 140 with the appearance of low affinity binding sites. Moreover the simultaneous Ala mutation of Tyr²⁹⁵, Trp²⁵⁶, and Phe²⁵⁹ drastically affected the maximal BK stimulated IP production, which was not observed for the W256A,F259A double mutant despite decreased BK potency (not shown). It indicated that Tyr²⁹⁵ plays some subtle role in receptor activation.

Ala mutations of other TM VII residues, Thr²⁸⁷, Ser²⁹¹, Ser²⁹⁶, and Ser²⁹⁸, did not significantly alter the activation properties (Fig. 3), while the properties of the Asn²⁹⁷ mutant could not be adequately evaluated because of its poor expression. In this respect the behavior of helix VII appeared significantly different in AT₁ and B₂ receptors, as, in addition to Tyr²⁹² mutation, mutations of Asn²⁹⁴ and Asn²⁹⁵ caused inhibition of hormone-induced AT₁ receptor activation (27).

On the Role of Phe²⁵⁹ in the B₂ Receptor Activation Mechanism-- Phe²⁵⁹, located about one helix turn above Trp²⁵⁶, is supposed to directly interact with BK, together with Thr²⁶³; indeed we found dramatic losses in BK affinity upon Phe²⁵⁹ or (and) Thr²⁶³ mutations to Ala in the human B₂ receptor (Table I), consistent with previous findings for the rat receptor (19). Therefore Phe²⁵⁹ might be a "switch" residue playing a key role in agonist-induced destabilization of inactive conformations. Previous data on the AT₁ receptor have suggested that the transition from inactive to active conformations might involve an interaction between Trp²⁵³ and His²⁵⁶ (5), the residues homologous of the B₂ receptor Trp²⁵⁶ and Phe²⁵⁹ respectively. It is noteworthy that His²⁵⁶ and Asn¹¹¹ were shown to interact with the Phe⁸ (16, 18) and Tyr⁴ (6, 13) residues of angiotensin II, respectively. There exists no evidence for or against the possible interaction of Asn¹¹³ with BK in the B₂ receptor; the expected increase of agonist affinity for CAM receptors, recently observed for the B₁ receptor mutated at its conserved Asn residue (28), was found neither for the Ala¹¹³ B₂ receptor (4) nor for the N111A AT₁ receptor (6). It might result from compensatory effects through the loss of BK-Asn¹¹³ interaction. Answering this question would imply an exhaustive structure-function analysis, as performed for the AT₁ receptor using both receptor mutants and hormone analogs (17, 18). An alternative explanation is that Asn¹¹³ mutation mimicks a receptor perturbation induced by BK through its interaction with Phe²⁵⁹. In this respect it is tempting to postulate that there is some link between the roles of Phe²⁵⁹ and Asn¹¹³; indeed the lack of increase in BK affinity upon Asn¹¹³ to Ala mutation might reflect some "redundancy" between the molecular events (and associated free energy changes) triggered by BK-Phe²⁵⁹ interaction and Asn¹¹³ mutation-induced facilitation of transition to active conformations. It was corroborated by the fact that the N113A,F259A (and also N113A,T263A) double mutant displayed striking increases in BK affinity or potency for IP production as compared with the F259A single mutant (Table II), indicating that the Asn¹¹³ mutation is also able to favor some other molecular events which are mediated by the Phe²⁵⁹-BK (and Thr²⁶³-BK) interaction.

View this table: [in this window] [in a new window]   Table II Binding and activation properties of B2 receptors mutated at positions 113, 256, 259 and 263  Direct binding of [3H]BK or 125I-HPP-HOE140 and competition binding of BK using this latter ligand as tracer were carried out on membrane preparations from COS-7 cells transiently expressing the WT or mutant receptors. EC50 values refer to BK-induced IP production in intact cells. The maximal stimulation (Emax) were determined in the presence of 107 M BK. The values represent the mean ± S.D. of three separate experiments.

Other experimental findings are in favor of a concerted role of Asn¹¹³ and Phe²⁵⁹: the behavior of B₂ receptors possessing either N113A and F259A mutations or N113A and T263A mutations deserves a comment: the properties of these double mutants revealed a discrepancy between the BK K_d values determined by direct [³H]BK binding (not possible in the single F259A and T263A mutants) and the K_i values evaluated in competition binding assays using ¹²⁵I-HPP-HOE 140 as tracer ligand (Table II). It might result from the existence of various conformational states, displaying either high affinity for HOE 140 and low affinity for BK (preferentially binding the tracer ligand) or a high affinity for BK (preferentially binding BK at low concentrations), together with a very slow interconversion between these two states, as already described for other receptors (29). In the B₂ receptor context, the observed discrepancies confirm that the mutations of residues 113 and 259 profoundly affect the pathways of conformational changes; the additional mutation of Phe²⁵⁹ (and therefore suppression of BK-Phe²⁵⁹ interaction) in the CAM N113A receptor might in some way limit the reversibility of specific steps in these pathways.

A concerted role for Phe²⁵⁹ and Trp²⁵⁶ could also be inferred from the properties of receptors mutated at positions 256 and 259. The CAM W256F and W256Q receptors displayed no significant increases in BK affinity. Phe²⁵⁹ was also mutated to His, its AT₁ counterpart. While the F259H receptor displayed properties similar to those of the F259A mutant, i.e. strongly decreased affinity for BK (Table II) and no constitutive activation, the F259H, W256F and F259H, W256Q receptors were constitutively activated, similarly to the single Phe²⁵⁶ and Gln²⁵⁶ mutants (not shown) and the EC₅₀ for BK stimulation were dramatically decreased as compared with the W256H receptor (these effects were much less pronounced for the F259H, W256A receptor, which was not constitutively activated) (Table II).

As a consequence our hypothesis is that the balance between active and inactive conformations might be controlled by a network of concerted interactions involving Asn¹¹³, Trp²⁵⁶, and Phe²⁵⁹ (Fig. 8) but obviously not restricted to these residues. Besides its interaction with Trp²⁵⁶, Asn¹¹³ might interact with another residue located in another TM than TM VI. By analogy to the AT₁ receptor, Tyr²⁹⁵ appeared as a plausible candidate, thus pointing to coordinated roles of TMs III, VI, and VII. As detailed in Fig. 8, all predictions about receptor interaction networks should take into account the possible existence of a statistical distribution of multiple conformational states each displaying only part of the postulated interactions.

Docking of Nonpeptidic Ligands in a B₂ Receptor Model

Nonpeptidic ligands designed by Fournier Laboratories, inverse agonists of the WT receptor, are expected to stabilize or induce inactive receptor conformations. They might induce inactive conformations characterized by a loss of Asn¹¹³-Trp²⁵⁶ interaction. However, the fact that they become agonists of the Ala¹¹³ CAM receptor (4) which is no longer stabilized by this interaction, together with the lack of experimental evidence for their interaction with Asn¹¹³, suggest that the Asn¹¹³-Trp²⁵⁶ interaction is essentially preserved in the nonpeptide-WT receptor complex. As a consequence, nonpeptide ligands were docked into a receptor model built by homology to the AT₁ receptor (13), characterized by the Asn¹¹³-Trp²⁵⁶ and Asn¹¹³-Tyr²⁹⁵ proximities. Interestingly the Asn¹¹¹-Trp²⁵³ interaction resulted from a refinement of TM VI position in initial models (5), so as to allow the double interaction of the tetrazole moiety of the nonpeptide losartan with Lys¹⁹⁹ and His²⁵⁶ (5) (Fig. 6). In parallel, we also performed a docking of nonpeptidic ligands into a B₂ receptor model homology-derived from the rhodopsin crystallographic structure (11). Three main candidates to nonpeptidic ligand recognition emerged from the results collected in Table I: Tyr²⁹⁵, Gln²⁸⁸, and Trp²⁵⁶.

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Model of losartan interaction with the AT1 angiotensin II receptor. Mutagenesis data from our group or other groups (5, 16) allowed the refinement of the initial AT1 receptor model (13) so as to make possible the double interaction of the losartan tetrazole moiety with Lys199 (TM V) and His256 (TM VI) which is important for receptor activation (5, 16) and AII binding. Rotation of TM VI fulfilled this requirement, allowing Asn111 to interact with Trp253, in addition to its postulated interaction with Tyr292. An essential interaction involves the hydroxyl group of the ligand and the Asn111 residue (13, 17, 18) which is essential for the stabilization of inactive receptor conformations. Top, side view; bottom, view from the extracellular side.

The mutation of Tyr²⁹⁵ to Phe or Ala induced moderate perturbations of [³H]BK and ¹²⁵I-HPP-HOE 140 binding (Table I). Tyr²⁹⁵ appeared to be essential for nonpeptide affinity; its mutation to Phe and Ala induced a gradual increase in K_i values for LF 16-0687 (8-10-fold and 130-140-fold increases, respectively). Interestingly the loss of affinity upon Tyr to Phe mutation was significantly attenuated, irrespective of the tracer ligand used, for the compound LF 18-1300 which possesses an imidazole moiety at position 4 of the quinoline (Table III). The interpretation is that Tyr²⁹⁵ interacts with the quinoline and that the loss of hydroxyl group upon its mutation to Phe is compensated by the presence of the imidazole in LF 18-1300. The similar affinity losses upon Tyr mutation for LF 18-1300 and LF 18-1833, a related compound lacking the benzamidine moiety at its extremity, precludes a Tyr interaction with the benzamidine, which significantly contributes to the affinity of LF 16-0335, LF 16-0687, and LF 18-1300. The Tyr-quinoline interaction is consistent with the essential role of quinoline emerging from the chemical design of LF 16-0687 and the dramatic loss of affinity induced by Ala mutation of Tyr²⁹⁵.

Significant losses in nonpeptide affinity were observed for the W256A mutant (Fig. 5, Table I, Table II); their relatively moderate intensities can be interpreted as resulting from compensatory effects involving the reinforcement of Tyr²⁹⁵-quinoline interaction in the mutant receptor. The decreases in nonpeptide affinity were lower for the W256F and W256Q mutants which were constitutively activated and therefore displayed modified conformational equilibria with increased representations of active conformations. The changes in the pharmacological properties of the nonpeptide for the various mutants support the hypothesis of nonpeptide-Trp²⁵⁶ interaction: as LF 16-0335, LF 16-0687, LF 18-1300, and LF 18-1833 became agonists of the W256F receptor, but not of the W256Q receptor, we postulated that an aromatic-aromatic interaction between the W256F receptor and the ligand was responsible for the agonist action of this latter; as detailed in the recapitulative scheme (Fig. 8) the ligand conformation interacting with the W256F CAM receptor should be different from that interacting with the inactive conformation of the WT receptor.

As a consequence we postulated the existence of a sandwich packing comprising Tyr²⁹⁵-quinoline-Trp²⁵⁶. The drastic loss of nonpeptide affinity for the W256A,Y295A double mutant (Table III) supports this hypothesis. The alternative hypothesis of Tyr²⁹⁵ and Trp²⁵⁶ separate interactions with the quinoline and dichlorophenyl ligand groups, respectively, appeared impossible to be fulfilled, because of the steric hindrance of the dichlorophenyl group and the importance of its link to the SO₂-prolyl portion in the ligand conformation. The hypothesis of Trp²⁵⁶ interaction with the benzamidine moiety of LF 16-0687 and LF 18-1300 was also excluded because of the similar behavior of LF 18-1833.

All nonpeptidic ligands displayed ~10-fold decreased affinities for the Q288A mutant, as compared with the WT receptor (Table III). Here again the behavior of the "shortened" compound LF 18-1833 could not be distinguished from that of compounds possessing the benzamidine. As expected, the simultaneous mutation of Tyr²⁹⁵ and Gln²⁸⁸ elicited drastic losses in nonpeptide affinity (K_i value difficult to evaluate because of multiple sites for the tracer ligand ¹²⁵I-HPP-HOE 140).

These experimental results constituted the support for the docking of nonpeptide ligands into a model of receptor inactive conformation, derived from previous AT₁ and B₂ receptor studies (4-6, 15). The docking strategy (see details under "Experimental Procedures") positioned the benzamidine moiety of LF 16-0687 at the extracellular space boundary of the transmembrane helix bundle. Further experimentation would be required to detect receptor residues responsible for its interaction with benzamidine which is not an absolutely required pharmacophore but nevertheless improves ligand affinity. The possible candidates to an interaction with the benzamidine appeared to be Asp²⁶⁶, Thr²⁶⁷, Asp²⁷⁹, Glu²⁸⁰, and Asp²⁸⁴; this latter residue was excluded on the basis of D284A mutant properties.

The model of the minimized LF 16-0687-receptor complex, termed as experimental model, is represented in Fig. 7A. This minimization did not significantly change the position of TM VII which was kept free of constraints together with the adjacent loops. An opened question is the participation of Asn¹¹³ to ligand interaction, through hydrogen bonding to the oxygen atom directly linked to the quinoline. The lack of incidence of Asn¹¹³ to Ala mutation on LF 16-0687 affinity is not necessarily inconsistent with this possibility as the mutation drastically shifts conformational equilibria to active states, conferring agonist properties to the nonpeptidic ligand. As above indicated, optimization of the geometry of the microdomain constituted by Asn¹¹³, Trp²⁵⁶, and Tyr²⁹⁵ and the ligand quinoline, which might not be unique (and its relationship with the Asn¹¹³-Trp²⁵⁶ interaction) is not easily feasible through use of semi-empirical quantum mechanics calculations, taking into account the difficulty to assess the relevance of expectedly weak differences in energy levels of conformations corresponding to different possibilities of packing the involved chemical structures.

View larger version (56K): [in this window] [in a new window]   Fig. 7.   Models of the human B2 receptor interaction with the nonpeptidic compound LF 16-0687. The model represented in A was homology-derived from the AT1 model as described under "Experimental Procedures," with a slight modification of TM VII position. The represented inactive receptor conformation is stabilized by a Asn113-Trp256 interaction, which received experimental support. The nonpeptidic compound binding to the receptor involves interaction of the ligand quinoline moiety with Tyr295 and Trp256 and interaction of the carbonyl function of the prolyl moiety with Gln288. Interestingly receptor residues involved in nonpeptide binding are located at strategic positions, important for the control of conformational equilibria in the B2 receptor itself or other GPCR, including rhodopsin and the AT1 receptor. The model represented in B was homology-derived from the rhodopsin structure. The nonpeptidic ligand docking can be achieved using the same ligand-receptor contact points as in A; the bent form of the ligand extremity bearing the benzamidine moiety results from the arbitrary transposition to the B2 receptor of the lid role of some rhodopsin extracellular loops (11). Top, side view; bottom, view from the extracellular side.

Preliminary positioning of LF 18-1300 into B₂ receptor models made no interaction of additional receptor residues with its imidazole moiety, suggesting that this latter probably induces a reinforcement of the quinoline interaction with its partners Tyr²⁹⁵ and (or) Trp²⁵⁶. Here again the success of theoretical calculations is not guaranteed. As previously mentioned it is noteworthy that a model of losartan-AT₁ receptor interaction (Fig. 6), consistent with abundant experimental structure-function and activation data, was based on similar hypotheses of inactive receptor conformation (5, 13), including the essential stabilizing Asn¹¹¹-Trp²⁵³ interaction.

A B₂ receptor model, homology-derived from the crystallographic data of rhodopsin (termed as rhodopsin-like model), was also built (Fig. 7B). The flexibility of the ligand LF 16-0687 made possible a docking into this model which satisfied the same interactions as in the preceding model, including a sandwich position of the ligand quinoline with Tyr²⁹⁵ and Trp²⁵⁶ which is postulated to be essential for the stabilization of inactive receptor conformations. In this model, the ligand extremity bearing the benzamidine moiety adopts a more bent shape, so as to keep conserved possible constraints of extracellular loops which, in the rhodopsin structure, function as a lid preventing retinal from going out of the binding pocket. In the absence of any data about residues interacting with the benzamidine, it is difficult to support any hypothesis about the position of this ligand extremity as well as the role of the B₂ receptor extracellular loops in the delimitation of the binding site.

The comparison of the experimental and rhodopsin-like models was focused on residues which display interesting activation and ligand recognition properties, more particularly on Asn¹¹³, Trp²⁵⁶, and Tyr²⁹⁵ which are essential for the relative positioning of TMs III, VI, and VII and therefore the positions of other residues located on them (Tyr¹¹⁵, Phe²⁵⁹, Gln²⁶⁰, and Gln²⁸⁸). It is noteworthy that the proximity between Asn¹¹³ and Trp²⁵⁶, which constitutes an essential hypothesis of the present work, is more consistent with the rhodopsin structure than with previous rhodopsin models (9, 10). Indeed the distance between the  carbon atoms of Gly¹²⁰ and Trp²⁶⁵, the rhodopsin residues homologs of the B₂ Asn¹¹³ and Trp²⁵⁶ is 11.5 Å in the crystallographic structure while it is strikingly higher in the model by Herzyk et al. (10) (15.1 Å); the corresponding distance in our experimental AT₁ and B₂ receptor models is 9.5 Å and the spatial arrangement of helices III and VI allows hydrogen bond or amino-aromatic interactions between the side chains, in a B₂ receptor model built by homology to the rhodopsin structure, the distance between the Asn¹¹³ and Trp²⁵⁶ side chains is not compatible with the geometrical possibility of an hydrogen bond interaction, because of a 40° difference in the orientation of TM III, but the addition of a water molecule between the two side chains would account for their functional interaction. The ability of Zn²⁺ ions to coordinate histidine residues simultaneously introduced at these positions is an essential verification of the proximity between Asn¹¹³ and Trp²⁵⁶. A careful analysis of models possessing the histidine mutations at these positions revealed that Zn²⁺ ion coordination is theoretically possible in our experimental B₂ receptor model as well as in the rhodopsin-like model (minimal distances between the -N histidine atoms 5.3 and 5.3 Å, respectively, corresponding distances between the -N histidine atoms 2.0 and 5.4 Å, respectively). Further comparisons revealed that the Asn¹¹¹-Tyr²⁹² or Asn¹¹³-Tyr²⁹⁵ proximity in the experimental AT₁ or B₂ receptor models is consistent with the Herzyk's rhodopsin models (10) while it does not exactly match the rhodopsin-like model (difference in the distances between side chains less than 2 Å in terms of hydrogen bonding possibility). At the present stage of these studies it is difficult to privilege one of the two proposed models or to exclude that an intermediary model would be more realistic.

Summarized Picture of Interaction Networks Controlling B₂ Receptor Conformational Equilibria and Their Modulation by Mutations or Ligand Action

Fig. 8 represents tentative explanations for a wide set of pharmacological data relative to ligand recognition and activation or inactivation properties of the human B₂ bradykinin receptor, with special attention to the key or switch roles of residues involved in the control of conformational equilibria or the binding of nonpeptidic ligands, or both.

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Elements of networks stabilizing B2 receptor inactive conformations and mechanisms of their mutation or ligand-induced perturbations. Likely or possible interactions stabilizing inactive receptor conformations are indicated by dashed lines; the sluices drawn on these dashed lines materialize a statistical distribution of various conformational states in which the interactions might not be fulfilled simultaneously. The ability of BK or receptor mutations to increase IP production through interaction networks destabilization is materialized by the number of + symbols. The central core of networks stabilizing inactive conformations is postulated to be constituted by the Asn113-Trp256-Phe259 triad, taking into account the Asn113-Trp256 proximity and the role of Phe259 in BK binding. The other residues which are probably connected in direct or indirect ways to this triad are Asp76, Gln260, Gln288, and Tyr295. The basal activity of the WT receptor would be accounted for by the existence of transition or activated states such as state 2, characterized by an intrahelical interaction inside TM VI and the loss of TM III-TM VI contacts, and ultimate states materialized as 3,4, ... , which might display modifications of TM III, VI, and VII (and probably other TMs) positioning. BK contact with Phe259 would greatly contribute to the destabilization of inactive conformations; therefore Phe259 would act as a switch residue, and for instance, might favor the transition to conformations such as 2. The strikingly high constitutive activation induced by Asn113 to Ala mutation would result from the complete loss of TM III-TM VI and TM III-TM VII interactions. It is conceivable that, because of this strong destabilization, the conformational change pathways and resulting activated states are different from those followed by the WT receptor; it is likely that BK activates the WT receptor through more coordinated and more readily reversible conformational changes leading to the loss of specific helix-helix interactions. The constitutive activation of the Phe256 or Gln256 mutants would result from a perturbation of TM III-TM VI contacts, with under-representation of inactive conformations and over-representation of conformations 2. The lack of constitutive activation of the Ala256 mutant or the Ala259 mutant would result from compensation of lost interactions by over-representation of conformations possessing, for instance, Asn113-Tyr295 and Asn113-Trp256 interactions, respectively. The lack of constitutive activation of the W256A,F259A double mutant might result from the preservation or reinforcement of the Asn113-Tyr295 interaction. An interesting question is the role of Tyr115, homologous of the rhodopsin Glu122, in stabilizing inactive conformations. The precise roles of Tyr115, Phe259, Gln260, Tyr295, and Gln288 in intra- or inter-helical stabilizations require further experimentation. Tyr295, Trp256, and Gln288 are likely candidates for the binding of the nonpeptidic ligand LF 16-0687, the ligand quinoline pharmacophore occupying a sandwich position between Tyr295 and Trp256. The changes in the pharmacological properties of this nonpeptidic ligand which became agonist of the CAM N113A and W256F receptors led us to postulate that the most representative conformations of the ligand receptor complexes might display the following properties: the ligand-WT receptor complex should be stabilized by the Asn113-Trp256 interaction (conformations 5), possibly reinforced by a role of the ligand in maintaining the Trp in the adequate position; these ligand-receptor interactions would contribute to restrain the relative motions of TMs III, VI, and VII. In the N113A and W256F receptors, which are constitutively activated and overactivated by the nonpeptidic ligand, the major conformations representative of the agonist ligand-CAM receptor complexes would no longer possess connections between Asn113 and Trp256; the favored motions of TM III, VI, and VII would result in the preferential stabilization of complexes symbolized by 6 and 7 which are expected to be structurally different of 5; this preferential stabilization (or induction) of active conformations of the CAM receptors would be facilitated by the nonpeptidic ligand flexibility allowing this conformational adaptation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The human B₂ bradykinin receptor possesses an extreme conformational flexibility, and thus constitutes a model of choice to investigate the control of conformational equilibria by ligands. Consistently with modeling studies of the AT₁ receptor which originated these studies we hypothesized that an interaction between Asn¹¹³ and Trp²⁵⁶ could play a major role on the stabilization of an inactive B₂ receptor conformation (4). A first essential finding of the work was the biochemical evidence for the proximity between these two residues, provided by the ability of Zn²⁺ ions to coordinate histidine residues simultaneously introduced at these positions. It is noteworthy that this proximity is in better agreement with the recently determined rhodopsin structure (11) than previously published rhodopsin models (9, 10, 31).

Extensive investigations were devoted to the function of Trp²⁵⁶, fairly conserved in many GPCR. A detailed pharmacological study of the various mutants, including nonpeptide ligand recognition and activation properties, led to the conclusion that the residue at position 256 exerts a subtle control of the balance between inactive and active receptor conformations. The importance of this tryptophan in GPCR function is underlined by initial photolabeling experiments demonstrating that the homologous Trp²⁶⁵ in rhodopsin lies in the retinal binding pocket (32); it was confirmed by crystallographic data (11) and a more detailed study of rhodopsin conformational changes associated to retinal photoisomerization suggested that Trp²⁶⁵ keeps 11-cis-retinal as an inverse agonist (33). The same tryptophan was shown to participate in the stabilization of a inactive TRH receptor conformation (26), through interaction with TM V, a finding that could not be extrapolated to the B₂ receptor.

The search for partners interacting with Asn¹¹³ or Trp²⁵⁶ led us to address the possible role of Phe²⁵⁹, which is a BK interaction site and is located one helix turn above Trp²⁵⁶ in TM VI and Tyr²⁹⁵, which is adequately located for a possible interaction with Asn¹¹³ and might play a role in the transition to activated states, in association with other key residues. The inactivation of the AT₁ receptor (15) and the constitutive activation of the -opioid receptor (34) observed when the homologous Tyr residues were mutated probably reflect some conserved features with rhodopsin which covalently binds retinal by its Lys²⁹⁶ located at the same position.

The search for additional residues which might participate in the stabilization of inactive B₂ receptor conformations led us to evidence strong constitutive activation upon Ala mutation of Tyr¹¹⁵. Tyr¹¹⁵, located about half an helix turn below Asn¹¹³ in TM III, is homologous of Glu¹²²_, which is important for rhodopsin function (35) and interacts with His²¹¹ in the inactive crystal form (11, 36).

We constructed a B₂ receptor model inherited of previous building (13) and refinements (5) of an AT₁ receptor model, and based on the Asn¹¹³ proximity to Trp²⁵⁶ and Tyr²⁹⁵. Interestingly these proximities can be considered as in fairly good agreement with rhodopsin crystallographic structure (11). At this stage the question is raised to which extent it is justified to deduce the various GPCR models from the structure of rhodopsin. A first consideration is the possible participation of water molecules in hydrogen bond networks which is liable to modify distances between residues which are supposed to interact on the basis of experimental results. The bacteriorhodopsin and rhodopsin examples demonstrate the importance of water molecules and their redistribution associated to retinal photoisomerization (37, 38). Another consideration is that the inactive rhodopsin conformation is covalently linked to retinal which behaves as an inverse agonist in the absence of light and should contribute to induce a specific conformation which cannot be transposed to other liganded or unliganded GPCR. Indeed the role of either all-trans-retinal in bacteriorhodopsin or 11-cis-retinal in rhodopsin in exerting constraints on transmembrane helices has been well documented and retinal isomerization has been shown to induce significant changes in the helix bundle organization (33, 39-45). An elegant recent study demonstrates that appropriate rhodopsin illumination drastically changes the site of its covalent labeling by photoactivatable retinal derivatives and that the generation of some photointermediates is accompanied by helix motions (33).

The postulated interaction network is consistent with the importance of TMs III, VI, and VII motions for GPCR activation (1, 46); it would provide a plausible explanation to the destabilizing action of BK through direct interaction with Phe²⁵⁹. It is noteworthy that similar considerations can be put forward for AII interaction with Asn¹¹¹ and His²⁵⁶ in the AT₁ receptor (5, 16). It is necessary to emphasize that conformational states simultaneously displaying all the possible interactions inside these networks might have no physical reality but the "inactive conformation" might cover a set of conformational states each displaying only part of these interactions. The consequence of mutations or ligand binding would be a change in the statistical distribution of the conformational states or the induction of new states.

Although the characterization of GPCR activated states is not presently available, with the exception of punctual information (47, 48), the Asn¹¹³-Trp²⁵⁶ and Trp²⁵⁶-Phe²⁵⁹ spatial proximities in the B₂ receptor are consistent with specific local rearrangements which can be predicted to take place during activation. Based on the previous hypothesis of Trp²⁵³-His²⁵⁶ interaction in the AT₁ receptor activation process (5), and the properties of B₂ receptors mutated at position 256, we postulated that transition to activated states of the B₂ receptor involves an increase in the representation of conformational states possessing a Trp²⁵⁶-Phe²⁵⁹ interaction (Fig. 8). This hypothesis of intrahelical interaction is in agreement with previous modeling studies of biogenic amine receptors pointing out the role of TM VI aromatic residues (30, 49) and data relative to the ₁ receptor activation process.²

Concerning possible mechanistic connections between TM VI and TM VII the question is raised whether the different behaviors of TM VII residues in the AT₁ and B₂ receptors, including Tyr²⁹² and Tyr²⁹⁵, are related to differences in the role of their TM VI Trp residue, i.e. inhibition of hormone-induced activation for the AT₁ (5) and constitutive activation for the B₂ receptors. Relationships between the roles of the two helices would be consistent with the presence of hormone interaction sites near the extracellular ends of TM VI and VII of the AT₁ (50, 51) and B₂ (52, 53) receptors and the finding that relative motions of TMs VI and VII participate in the activation of muscarinic receptors (54). The construction of chimeric B₁/B₂ receptors (55) and point mutations in the B₁ receptor TMVII (56) emphasize the role of the 3rd extracellular loop and TM VII, respectively, in the specificity of ligand recognition.

Although it is impossible to deduce from literature data general rules about spatial restrains in inactive GPCR conformations, many data indicate that residues of TMs III and VI, some of which are located at homologous or neighboring positions, have been identified as switch residues, i.e. direct interaction sites for agonists whereby these latter initiate destabilization of inactive conformations. For instance, constitutive activation of the platelet-activating factor receptor (57), the _1B or ₂-adrenergic receptors (58, 59), or the B₁ bradykinin receptor (28) could be induced by the mutation of an asparagine or a cysteine located at the homologous position of the TM III Asn of the AT₁ and B₂ receptors. The role of this Asn is consistent with membrane helix studies which emphasize that hydrogen bond interhelical interactions can be driven by Asn residues (60-62). Asn¹¹¹ and His²⁵⁶ were postulated to constitute AII interaction points (16) and are probably involved in the generation of the AT₁ receptor intermediary conformational states (17); that Phe²⁵⁹ is a BK-binding residue in the B₂ receptor is consistent with the postulated role of this residue inside the intramolecular interaction networks. Interestingly residues located at the same position in TM VI of other receptors control the activation process and are involved in ligand binding: Phe³¹⁰ in the _1B receptor (63), Tyr³⁸¹ in the M₁ muscarinic receptor (64), and Tyr²⁸² in the TRH receptor (26). A systematic analysis of randomly mutated receptors (2, 3, 65) emphasize that other residues located in TMs III and VI are involved: for instance, Phe⁴⁵¹ and Asn⁴⁵⁹ in the M₅ muscarinic receptor, homologous of the Phe²⁵² and Gln²⁶⁰ B₂ residues.

A second aspect of the present work was to establish the molecular basis of nonpeptide ligand recognition and inactivation of the B₂ bradykinin receptor. Structure-function analysis allowed precise essential nonpeptide-receptor contacts: the nonpeptide quinoline moiety interacts simultaneously with Tyr²⁹⁵ and Trp²⁵⁶. Gln²⁸⁸ is also involved in ligand binding. On this basis we could propose a docking of the nonpeptidic ligand LF 16-0687 either in a receptor model homology deduced from a previous AT₁ model (experimental model) or in a model derived from the rhodopsin structure (rhodopsin-like structure). It is noteworthy that B₂ receptor nonpeptide antagonists studied in the present work interact with residues of TM VI and VII, possibly restricting movements of these helices required for activation, together with helix III movements. The nonpeptide interaction with these residues, with a major "locking" role of the sandwich interaction of their quinoline moiety with Trp²⁵⁶ and Tyr²⁹⁵, might restrain the essential Trp²⁵⁶ in a position favorable to its interaction with Asn¹¹³ which constitutes the cornerstone of inactive conformations. This stabilizing effect would provide an explanation for the inverse agonist behavior of this ligand and its agonist behavior on the CAM N113A and W256F receptors which no longer possesses the Asn-Trp interaction; in this latter situation the high conformational flexibility of the nonpeptidic derivatives would allow them to fit with or induce CAM receptor active conformations.

Interestingly the B₂ receptor residues involved in nonpeptide recognition are somehow involved in receptor activation even if the precise role of some of them, located at key positions in other GPCR, requires further elucidation. It indicates that ligands synthesized through semiempirical strategies have been implicitly selected according to their ability to interact with receptor residues which control conformational equilibria. Similar conclusions can be drawn for the AT₁ receptor, inasmuch Asn¹¹¹ and His²⁵⁶, which are switch residues for AII action, are involved in losartan and other nonpeptide ligand binding (together with Asn²⁹⁵ in TM VII) (5, 14, 66, 67). The NK₂ receptor amino acid Tyr²⁶⁶, homologous to the AT₁ receptor His²⁵⁶, was shown to participate in peptide agonist and nonpeptide antagonist binding (68). The question is raised whether such considerations should be taken into account for future attempts of rational design of nonpeptidic GPCR ligands. Experimental evaluation of the conformational flexibility of GPCR (1) and the ability of ligands to induce specific conformational changes will probably constitute a major difficulty in future structure predictions from the rhodopsin crystallographic data (11). Conflicting results were reported about the extent of structural changes associated to rhodopsin activation (33, 69). A recent reassessment of catecholamine action on the ₂-adrenergic receptor (70) demonstrates the usefulness of thermodynamic studies to dissect ligand action in terms of receptor binding and modulation of conformational equilibria. Taken together our data emphasize the role of B₂ receptor key residues which control conformational equilibria and constitute ligand interaction points and are located at strategic positions for the function of other GPCR, including rhodopsin.

	ACKNOWLEDGEMENTS

We thank J. L. Borgna and J. C. Nicolas for reading the manuscript and helpful advice.

	FOOTNOTES

^* This work was supported by the Institut National de la Santé et de la Recherche Médicale, Center National de la Recherche Scientifique (including "Molécules et Cibles Thérapeutiques" and "Physique et Chimie du Vivant" programs), Laboratoires Fournier (Daix, France), and Fondation pour la Recherche Médicale.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche and Association pour la Recherche contre le Cancer. Present address: AstraZeneca, 7171 Frédérick-Banting, Ville Saint-Laurent (Montréal), Québec H4S 1Z, Canada.

^** To whom correspondence should be addressed.

Published, JBC Papers in Press, August 8, 2001, DOI 10.1074/jbc.M104875200

² S. Cotecchia, unpublished results.

	ABBREVIATIONS

The abbreviations used are: GPCR, G-protein coupled receptor; WT, wild-type; TM, transmembrane domain; CAM, constitutively activated mutant; AII: angiotensin II, BK, bradykinin; IP, inositol phosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S. R. Hawtin, J. Simms, M. Conner, Z. Lawson, R. A. Parslow, J. Trim, A. Sheppard, and M. Wheatley Charged Extracellular Residues, Conserved throughout a G-protein-coupled Receptor Family, Are Required for Ligand Binding, Receptor Activation, and Cell-surface Expression J. Biol. Chem., December 15, 2006; 281(50): 38478 - 38488. [Abstract] [Full Text] [PDF]

				L. M. F. Leeb-Lundberg, F. Marceau, W. Muller-Esterl, D. J. Pettibone, and B. L. Zuraw International Union of Pharmacology. XLV. Classification of the Kinin Receptor Family: from Molecular Mechanisms to Pathophysiological Consequences Pharmacol. Rev., March 1, 2005; 57(1): 27 - 77. [Abstract] [Full Text] [PDF]

				X. Lu, W. Huang, S. Worthington, P. Drabik, R. Osman, and M. C. Gershengorn A Model of Inverse Agonist Action at Thyrotropin-Releasing Hormone Receptor Type 1: Role of a Conserved Tryptophan in Helix 6 Mol. Pharmacol., November 1, 2004; 66(5): 1192 - 1200. [Abstract] [Full Text] [PDF]

				R. P. Millar, Z.-L. Lu, A. J. Pawson, C. A. Flanagan, K. Morgan, and S. R. Maudsley Gonadotropin-Releasing Hormone Receptors Endocr. Rev., April 1, 2004; 25(2): 235 - 275. [Abstract] [Full Text] [PDF]

				V. D. Nair and S. C. Sealfon Agonist-specific Transactivation of Phosphoinositide 3-Kinase Signaling Pathway Mediated by the Dopamine D2 Receptor J. Biol. Chem., November 21, 2003; 278(47): 47053 - 47061. [Abstract] [Full Text] [PDF]

				C. Prioleau, I. Visiers, B. J. Ebersole, H. Weinstein, and S. C. Sealfon Conserved Helix 7 Tyrosine Acts as a Multistate Conformational Switch in the 5HT2C Receptor. IDENTIFICATION OF A NOVEL "LOCKED-ON" PHENOTYPE AND DOUBLE REVERTANT MUTATIONS J. Biol. Chem., September 20, 2002; 277(39): 36577 - 36584. [Abstract] [Full Text] [PDF]

				D. S. Kang and L. M. F. Leeb-Lundberg Negative and Positive Regulatory Epitopes in the C-Terminal Domains of the Human B1 and B2 Bradykinin Receptor Subtypes Determine Receptor Coupling Efficacy to G9/11-Mediated Phospholipase Cbeta Activity Mol. Pharmacol., August 1, 2002; 62(2): 281 - 288. [Abstract] [Full Text] [PDF]

				Z.-G. Gao, A. Chen, D. Barak, S.-K. Kim, C. E. Muller, and K. A. Jacobson Identification by Site-directed Mutagenesis of Residues Involved in Ligand Recognition and Activation of the Human A3 Adenosine Receptor J. Biol. Chem., May 17, 2002; 277(21): 19056 - 19063. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/44/41100    most recent M104875200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marie, J. Articles by Bonnafous, J.-C. Search for Related Content PubMed PubMed Citation Articles by Marie, J. Articles by Bonnafous, J.-C. Social Bookmarking                      What's this?