# A 5-HT4 Receptor Transmembrane Network Implicated in the Activity of Inverse Agonists but Not Agonists*

Lara Joubert, Sylvie Claeysen, Michèle Sebben, Anne-Sophie Bessis, Robin D

Clark, Renee H Martin, Joël Bockaert, Aline Dumuis

► To cite this version:

Lara Joubert, Sylvie Claeysen, Michèle Sebben, Anne-Sophie Bessis, Robin D Clark, et al.. A 5-HT4 Receptor Transmembrane Network Implicated in the Activity of Inverse Agonists but Not Agonists*. Journal of Biological Chemistry, 2002, 277 (28), pp.25502-25511. 10.1074/jbc.M202539200. hal-02486579

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# A 5-HT₄ Receptor Transmembrane Network Implicated in the Activity of Inverse Agonists but Not Agonists*

Received for publication, March 15, 2002, and in revised form, April 19, 2002
Published, JBC Papers in Press, April 25, 2002, DOI 10.1074/jbc.M202539200

Lara Joubert‡, Sylvie Claeysen‡, Michèle Sebben‡, Anne-Sophie Bessis‡, Robin D. Clark§, Renee S. Martin§, Joël Bockaert‡¶||, and Aline Dumuis‡||

*From the ‡Centre National de la Recherche Scientifique, UPR 9023, Montpellier, France and
the §Neurobiology Unit, Roche Bioscience, Palo Alto, California 94304*

Activation of G protein-coupled receptors is thought to involve disruption of intramolecular interactions that stabilize their inactive conformation. Such disruptions are induced by agonists or by constitutively active mutations. In the present study, novel potent inverse agonists are described to inhibit the constitutive activity of 5-HT₄ receptors. Using these compounds and specific receptor mutations, we investigated the mechanisms by which inverse agonists may reverse the disruption of intramolecular interactions that causes constitutive activation. Two mutations (D100³.³²A in transmembrane domain (TMD)-III and F275⁶.⁵¹A in TMD-VI) were found to completely block inverse agonist effects without impairing their binding properties nor the molecular activation switches induced by agonists. Based on the rhodopsin model, we propose that these mutated receptors are in equilibrium between two states R and R* but are unable to reach a third “silent” state stabilized by inverse agonists. We also found another mutation in TMD-VI (W272⁶.⁴⁸A) that stabilized this silent state. This mutant remained fully activated by agonists. Molecular modeling indicated that Asp-100, Phe-275, and Trp-272 might constitute a network required for stabilization of the silent state by the described inverse agonists. However, this network is not necessary for agonist activity.

G protein-coupled receptors (GPCRs)¹ are the most numerous and versatile family of proteins that control cell-cell com-

munications (1-3). Their endogenous ligands include hormones, neurotransmitters, growth and survival factors, smells, tastes, and photons.

Both wild type (WT) and mutant GPCRs have been discovered to be spontaneously active and to adopt an active (R*) conformation in absence of agonists (4-6). In the simplest model, GPCRs exist in a dynamic equilibrium between active (R*) and inactive (R) states governed by an allosteric constant (J = [R]/[R*]). Different models have been proposed to simulate the transition of receptor between ligand or G protein-bound states. In the extended ternary complex, R* interacts with the G protein either spontaneously (R*G) or through agonist binding (AR*G). In the cubic ternary complex model, both the R and R* states interact with the G protein (for a review, see Ref. 7). The efficacy of a ligand is thought to be a function of its relative affinity for R and R*. Therefore, neutral antagonists have equal affinity for both receptor states, agonists preferentially bind to R*, thereby shifting the position of equilibrium toward active conformation, and inverse agonists preferentially bind to R, shifting the position of equilibrium toward the inactive state. A growing body of evidence suggests the existence of multiple active and inactive conformations. A model of these multiple conformations has been proposed (2, 7), which accurately simulates the biochemical results of rhodopsin studies (8, 9).

Understanding the function of GPCRs at a molecular level requires better understanding of the mechanisms by which the binding of agonists and inverse agonists preferentially stabilizes the R* and R conformations, respectively. An important contribution to this understanding was the identification of key residues, which, when altered by mutation, led to increased constitutive activity of GPCRs (i.e. accumulation of R* in the absence of agonists). These residues have been identified not only in the intracellular domains known to be involved in G protein activation, but also in transmembrane domains (TMDs) and even in some cases in extracellular domains.

Most of these residues have been proposed to participate in networks of intramolecular interactions, which stabilize the receptors in inactive conformations R. Disruption of these networks results in a conformational shift from R to R* (2, 10-15).

In the case of rhodopsin, a salt bridge constraint between Glu-113³.²⁸² in TMD-III and Lys-296⁷.⁴³ in TMD-VII stabilizes the receptor in the R state (10). Similar intramolecular interactions have been demonstrated for α₁A-adrenoceptors between Asp-125³.³² of TMD-III and Lys-331⁷.⁴³ in TMD-VII. Activation of this receptor is believed to result from the disruption of the Asp-125-Lys-331 salt bridge by the neurotransmitter biogenic

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¶ To whom correspondence should be addressed: CNRS, UPR 9023, 141 rue de la Cardonille, 34094 Montpellier, Cedex 05, France. Tel.: 33-4-67-14-29-30; Fax: 33-4-67-54-24-32; E-mail: bockaert@ccipe.montp.inserm.fr.

|| These authors contributed equally to this work.

¹ The abbreviations used are: GPCR, G protein-coupled receptor; 5-HT, 5-hydroxytryptamine; 5-HT₄ receptor, 5-hydroxytryptamine receptor of class 4; Gs, G protein that stimulates adenylate cyclase; TMD, transmembrane domain; WT, wild type; R, inactive receptor conformation; R*, active receptor conformation; Rg, ground state receptor conformation; GR113808, [1-[2(methylsulfonyl-amino)ethyl]4-piperidinyl]-methyl-1-methylindole-3 carboxylate, maleate; GR125487, [1-[2-(methylsulfonylamino)ethyl]4-piperidinyl]methyl-5-fluoro-2-methoxy-1H-indole-3-carboxylate, hydrochloride; BIMU8, [endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl]-2,3-dihydro-3-isopropyl-2-oxo-1H-benzimidazole-1-carboxamide hydrochloride; RS100235, 1-(8-amino-7-chloro-2,3-dihydrobenzo-(1,4)-dioxin-5-yl)-3-[1-[3-(3,4-dimethoxyphenyl)propyl]-piperidin-4-yl]propan-1-one hydrochloride salt; RO116-0086, 2,3-dihydrobenzo-(1,4)-dioxine-5-carboxylic acid 1-butylpiperidin-4-ylmethyl ester hydrochloride salt; RO116-1148, 2,3-dihydrobenzo-(1,4)-dioxine-5-carboxylic acid 1-butylpiperidin-4-ylmethylamide hydrochloride salt; DMEM, Dulbecco's modified Eagle's medium; dFBS, decomplemented fetal bovine serum.

<footnote n="2">² For numbering of residues, we have followed either the convenient method proposed by Van Rhee and Jacobson, which includes both numbering in the sequence and position in a particular helix referred to its most conserved residue, or only the position in this helix.</footnote>
amine that acts as counter-ion of the Asp-125 (11). However, this mechanism of receptor activation is likely to be specific for α₁B-adrenergic receptor, since the Lys in TMD-VII is not conserved within related GPCRs sequences. β₂-Adrenergic receptors are kept under an inactive state by an ionic lock between the adjacent Arg and Asp of the DRY (D3.49R3.50Y3.51) sequence (cytoplasmic end of TMD-III) and a conserved Glu in the cytoplasmic end of TMD-VI (Glu-2686.30 in β2-adrenergic receptor) (13). For several GPCRs, charge-neutralizing mutations of these residues increase constitutive activity by disrupting this lock (15–17). In rhodopsin, similar disruption is observed following protonation of the Glu in the ERY sequence (equivalent to the DRY sequence) and another acidic residue that could be the conserved Glu of TMD-VI (18). Taken together, these findings support the idea that the disruption of intramolecular interactions constitutes the receptor activation switch, although the precise residues involved in these interactions may vary between different receptors. Such a hypothesis is also supported by the observation that constitutively active receptors are characterized by structural instability (16, 19, 20).

In contrast to the rapid progress in understanding molecular mechanisms underlying the transition from R to R*, mechanisms by which inverse agonists favor the conversion from R* to R (inactive conformation) remain unclear. An important observation was that mutations of residues that constrain the receptor in the inactive state lead to constitutively activated receptors that can be switched back to an inactive state by inverse agonists. This suggests to us that inverse agonist action may implicate different constraining networks than those disrupted by agonists.

The present study, combining mutagenesis, pharmacology, and molecular modeling of 5-HT₄ receptor, sheds new light on this issue. We previously reported that 5-HT₄ receptors display high constitutive activity under wild type and mutated forms (20, 21). In this study, three mutations are reported (D1003.32A in TMD-III and F2756.51A and W2726.48A in TMD-VI) that completely blocked the effects of inverse agonists while keeping their specific binding on the receptor. Besides, when combined with other potent constitutively activating mutations, these three mutations were discovered to be “dominant” as inverse agonist effects were still blocked. Surprisingly, they also left unchanged molecular activation switches induced by agonists. Molecular modeling revealed a network of interactions between these three residues.

## EXPERIMENTAL PROCEDURES

**Construction of Mutated m5-HT₄(a) Receptor cDNA**—The mutants were generated by exchanging the endogenous residues to alanine in the m5-HT₄(a) R cDNA sequence with the QuikChange site-directed mutagenesis kit from Stratagene. The sense primers used were: F2756.51A, 5'-CTG TTT CTG CTG GGC CCC CGC CTT TG-3'; D1003.32A, 5'-ACC TCT CTG GCT GTC CTA CTC ACC-3'; W2726.48A, 5'-CTG TTT CTG CGC GGC CCC CTT CTT-3'; S1975.45A, 5'-TAT GCT ATC ACC TGC GCT GTG GT-3'; R1183.50A, 5'-TTC CCT GGA CGC TTA TTA CGC CAT-3'; N3087.49D, 5'-GGT TGG ACC CTT TTC TCT ATG CCT-3'; T1344.38A, 5'-TGC GTA GAG GGG CCA TCT TGT TCC-3'.

**Cell Culture and Transfection**—The cDNAs, subcloned into pRK5, were introduced into COS-7 cells by electroporation. Briefly, cells were trypsinized, centrifuged, and resuspended in EP buffer (50 mM K₂HPO₄, 20 mM CH₃CO₂K, 20 mM KOH, 26.7 mM MgSO₄, pH 7.4) with 25–2000 ng of receptor cDNA. The total amount of cDNA was kept constant at 15 µg per transfection with wild type pRK5 vector. After 15 min at room temperature, 300 ml of cell suspension (10⁷ cells) were transferred to a 0.4-cm electroporation cuvette (Bio-Rad, Ivry sur Seine, France) and pulsed with a gene pulser apparatus (setting 1000 millifarads, 270 V). Cells were diluted in Dulbecco's modified Eagle's medium (DMEM; 10⁶ cells/ml) containing 10% dialyzed and decomplemented fetal bovine serum (dFBS) and plated on 10-cm Falcon Petri dishes or into 12-well clusters at the desired density.

**Determination of Cyclic AMP (cAMP) Production in Intact Cells**—Six hours after transfection, the surrounding cell medium was exchanged for DMEM without dFBS with 2 mM of [³H]adenine/ml to label the ATP pool and incubated overnight. cAMP accumulation was measured as described previously (22).

**Membrane Preparation and Radioligand Binding Assay**—Membranes were prepared from transiently transfected cells plated on 15-cm dishes and grown in DMEM with 10% dFBS for 6 h, followed by incubation for 20 h in DMEM without dFBS. The cells were washed twice in phosphate-buffered saline, scraped with a rubber policeman, harvested in phosphate-buffered saline, and centrifuged at 4 °C (200 × g for 4 min). The pellet was resuspended in buffer containing 10 mM HEPES, pH 7.4, 5 mM EGTA, 1 mM EDTA, and 0.32 M sucrose and homogenized 10 times with a glass-Teflon potter at 4 °C. The homogenate was centrifuged at 20,000 × g for 20 min, and the membrane pellet was resuspended in 50 mM HEPES, pH 7.4, (5 mg of protein in 1 ml of solution) and stored at -80 °C until use. 5-HT₄ receptor densities were estimated with the specific radioligand [³H]GR113808 at saturating concentration (0.4–0.6 nM, K_d = 0.12 nM) as described previously (23). 5-HT (50 µM) or RS100325 (10 µM) was used to determine nonspecific binding. Protein concentration in the samples was determined with the Bio-Rad protein assay (24).

**Data Analysis**—Using Kaleidagraph software, the dose-response curves were fitted according to the equation,

$$Y = \frac{(y_{\max} - y_{\min})}{(1 + x/EC_{50})^{n_{\max}}} + y_{\min} \quad (\text{Eq. 1})$$

where EC₅₀ (or EC₅₀inv) is the concentration of agonist (or inverse agonist) that evokes a half-maximal response; y_max and y_min correspond to the maximal and minimal responses, respectively; and n_H is the Hill coefficient. Statistical differences were examined with the Stat-View Student program (Abacus Concepts, Berkeley, CA) with *t* tests.

**Drugs**—GR113808 ([1-[2-(methylsulfonyl-amino)ethyl]4-piperidinyl]methyl-1-methyl-indole-3 carboxylate, maleate) and GR125487 ([1-[2-(methylsulfonyl-amino)ethyl]-4-piperidinyl]methyl-5-fluoro-2-methoxy-1H-indole-3-carboxylate, hydrochloride) were generously donated by Glaxo (Ware, Herts, UK); [³H]GR 113808 was purchased from Amersham Biosciences; 5-HT (serotonin) was purchased from Sigma. BIMUS ([end-o-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl]-2,3-dihydro-3-isopropyl-2-oxo-1H-benzimidazole-1-carboxamide hydrochloride) was obtained from Boehringer Ingelheim (Milan, Italy).

RS100235 (1-(8-amino-7-chloro-2,3-dihydrobenzo(1,4)dioxin-5-yl)-3-[1-[3-(3,4-dimethoxyphenyl)propyl]-piperidin-4-yl]-propan-1-one hydrochloride salt), RO116-0086 (2,3-dihydrobenzo-(1,4)-dioxine-5-carboxylic acid 1-butyl-piperidin-4-ylmethyl ester hydrochloride salt), RO116-1148 (2,3-dihydrobenzo-(1,4)-dioxine-5-carboxylic acid 1-butyl-piperidin-4-ylmethylamide hydrochloride salt) were generously donated by Roche Bioscience (Palo Alto, CA).

**Molecular Modeling of the 5-HT₄ Receptor**—A model of the transmembrane domain in the mouse 5-HT₄ receptor was constructed using the bovine rhodopsin structure (Protein Data Bank code: 1F88:A) as a template. Receptor sequence alignment was determined using GRAP Mutant data base (tGRAP.uit.no/fam1asel.html). All 5-HT₄ receptor modeling was conducted following virtual truncation of certain amino acid residues: 1–10 of the N-terminal region, 163–183 of the e2 loop, 219–250 of the i3 loop, and 332–387 of the C-terminal region. Five different models were developed using the program Modeler in the Insight-II environment (Molecular Simulation Inc., San Diego, CA) on a Silicon Graphics R10000 O₂ work station. Although these models generated very similar molecular structures, the findings reported here are based on the model that returned the best three-dimensional likeness of rhodopsin. This model was submitted to energy minimization using the program Discover 3 and the consistent force field. Cα-trace was thresholded by applying a force constant value of the quadratic potential of 100. A steepest descent followed by a conjugate gradient method then was applied until convergence with 0.5 kcal mol⁻¹ Å⁻¹ root mean square energy gradient difference between successive minimization steps.

## RESULTS

**Novel and Highly Potent 5-HT₄ Receptor-specific Inverse Agonists**—GR125487 and SB207266 have previously been described to be inverse agonists at the 5-HT₄ receptor (21). To find new 5-HT4 receptor inverse agonists we synthesized numerous compounds having common pharmacophoric features with the SB204070, a specific benzoate dioxane 5-HT₄ receptor antagonist (25). Among them, two highly potent inverse agonists were identified, RO116-0086, a benzoate dioxane that is
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /><figcaption>FIG. 1. New highly potent 5-HT₄ receptor inverse agonists. A, chemical structure of 5-HT₄ receptor inverse agonists RO116-0086 and RO116-1148. B, concentration effect curves of the new potent 5-HT₄ receptor inverse agonists on the agonist-independent cAMP production in COS-7 cells transiently expressing m5-HT₄(a) receptors at a level of 1500 ± 130 fmol/mg of protein. cAMP production was measured for 15 min and expressed as a percentage of basal cAMP formation. Basal cAMP production, defined as 100%, was equal to 720 ± 50% of the mock-transfected controls. In mock-transfected cells, the percentage conversions of [³H]ATP to [³H]cAMP was equal to 0.12 ± 0.04%. The results are representative of three independent experiments performed in triplicate.</figcaption></figure>

the des-amino, des-chloro version of SB204070, and RO116-1148, which is the corresponding amide.

By measurement of cAMP accumulation in cell lines that transiently express wild type 5-HT₄(a) receptor, both RO116-0086 and RO116-1148 were found to have high potencies (EC₅₀ᵢₙv = 0.3 ± 0.1 nM) and high efficacies. Indeed, they inhibited 80% of basal constitutive activity, i.e. cAMP accumulation above mock cAMP accumulation, in the absence of agonist (Fig. 1B). Both are much more potent and efficacious than previously described 5-HT₄ inverse agonists (21).

*Effects of Asp-100 (D100)<sup>3,32</sup>A Mutation on Responsivity to Agonists and Inverse Agonists*—Screening for mutations that would impair the efficacy of inverse agonists without modifying the agonist-induced activation, we analyzed the mutation of Asp-100<sup>3,32</sup>. This highly conserved residue within the biogenic amine receptor family (26) is believed to make an ionic interaction with neurotransmitters protonated amine. As expected, 5-HT was completely unable to stimulate the D100A mutated receptor (Fig. 2A), due to its loss of binding (Fig. 2B). Interestingly, the binding of the labeled antagonist [³H]GR113808 was only slightly affected by the mutation (*K*<sub>D</sub> = 0.12 ± 0.05 nM and 0.26 ± 0.04 nM at WT and D100A receptors, respectively). In contrast to 5-HT, the benzimidazolone derivative BIMU8 re-

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>FIG. 2. Mutation of the conserved Asp-100<sup>3,32</sup> in TMD-III impedes effect and binding of 5-HT but not of the synthetic agonist, BIMU8. A, the effects of a range on concentrations of 5-HT or BIMU8 on intracellular cAMP levels were measured in COS-7 cells in which 5-HT₄(a) or D100A was expressed at equivalent levels (1350 ± 60 and 1580 ± 110 fmol/mg of protein, respectively). Results are expressed as a percentage of the cAMP production in mock-transfected cells. In mock-transfected cells, 0.10 ± 0.08% of [³H]ATP was converted to [³H]cAMP. B, competition of agonists for [³H]GR113808 binding (0.38 nM) at membranes derived from the same batches of transfected COS-7 cells as in A. The results are expressed as a percent of the mean specific binding in the absence of competing ligand. The results are the mean of four independent determinations.</figcaption></figure>

mained fully and even showed a higher potency on the D100A mutant than on the WT 5-HT₄ receptor. The EC₅₀ values for cAMP stimulation were equal to 4 ± 1.5 and 1 ± 0.5 nM for WT and D100A, respectively (Fig. 2A). Affinity of BIMU8 for 5-HT₄ receptor was also slightly better on the D100A mutant than on the WT. K<sub>D</sub> values for BIMU8 measured by competition with the [³H]GR113808 radioligand were 30 ± 11 nM and 6.5 ± 3 nM for WT and D100A, respectively (Fig. 2B).

These data suggest that the D100<sup>3,32</sup>A mutant has not lost the ability to be activated as long as the agonist is able to bind. When expressed at densities ranging from 200 up to 10,000 fmol/mg of protein, the basal level of cAMP synthesis of the D100A mutant was 1.5–2-fold higher than that of WT. Receptor expression levels of WT or D100A were also not significantly different following transfection of 300 ng of cDNA (1350 ± 60 and 1580 ± 60 pmol/mg of protein for WT and D100A, respectively).

Whereas, as already discussed, highly potent inverse agonists (RO116-0086, RO116-1148) blocked the WT constitutive activity, they were absolutely unable to inhibit basal constitutive activity of the D100A mutant (Fig. 3A). This effect is not
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

FIG. 3. Lack of effect of inverse agonists following mutation of the conserved Asp-100<sup>3,32</sup> in TMD-III. A, COS-7 cells transiently expressing 2500 ± 220 fmol/mg of D100A mutant 5-HT<sub>4</sub>(a) receptor were incubated with the potent and selective 5-HT<sub>4</sub> inverse agonists (RO116-0086 and RO116-1148). B and C, dose-response curves of the same inverse agonists on BIMU8-mediated cAMP accumulation in COS-7 cells expressing 1900 ± 150 fmol/mg of protein WT 5-HT<sub>4</sub> receptors (B) or 2500 ± 220 fmol/mg of D100A (C). cAMP production was measured for 15 min and expressed as a percentage of cAMP formation in mock-transfected control cells. In mock-transfected cells, the percentage conversion of [<sup>3</sup>H]ATP to [<sup>3</sup>H]cAMP was equal to 0.11 ± 0.02%. BIMU8 concentration was 3 × 10<sup>-8</sup> M, and cAMP production was measured as a percent of mock. The results are representative of three independent experiments performed in triplicate.

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

FIG. 4. The binding of the 5-HT<sub>4</sub> receptor inverse agonists was conserved in the mutated D100<sup>3,32</sup> A receptor. Competition binding was performed for the inverse agonists (RO116-0086 (A) and RO116-1148 (B)), in the presence of 0.24 nM [<sup>3</sup>H]GR113808. The assays were carried out on membranes derived from COS-7 cells and expressing similar levels of WT or D100A receptor (2500 ± 180 and 2650 ± 210 fmol/mg of protein, respectively). Results are expressed as a percent of the specific binding in the absence of a competing ligand. Representative data are presented from one of six replicate experiments.

unique to these two newly highly potent inverse agonists; similar results were obtained with the previously described 5-HT<sub>4</sub> receptor inverse agonists, GR125487 and SB207266 (21) (data not shown). This effect can therefore not be attributed to a loss of binding to the D100A mutant receptor, as shown in Fig. 4, A and B. RO116-0086 competition binding experiments showed a very small decrease in affinity for D100A (K<sub>d</sub> = 3 ± 1.4 × 10<sup>-10</sup> M) relative to WT (K<sub>d</sub> = 8.4 ± 2.6 × 10<sup>-11</sup> M). RO116-1148 showed a 10-fold decrease in affinity for D100A (K<sub>d</sub> = 5.1 ± 1.8 × 10<sup>-9</sup> M) as compared with the WT (K<sub>d</sub> = 4.7 ± 1.5 × 10<sup>-10</sup> M).

The binding of these inverse agonists to the D100A mutant was confirmed by the capacity of these compounds to behave as antagonist of the BIMU8-stimulated cAMP accumulation (Fig. 3, B and C).

The influence of receptor expression on inverse agonist activity was then analyzed. While the receptor constitutive activity increased as expected as a function of the receptor concentration, RO116-1148 remained totally inactive as inverse agonist on the D100A mutant (Fig. 5B). In comparison, its effect on WT receptor was constant whatever the receptor concentration (Fig. 5A).
<figure><img src="image_11.png" /><figcaption>FIG. 5. Effects of receptor density on the effects of agonists and inverse agonists on constitutively active WT and D100A receptors. Levels of cAMP accumulation in COS-7 cells expressing WT and D100A over a range of receptor densities (from 200 to 9500 fmol/mg of protein) were determined in the absence (basal) or presence of inverse agonist or agonists. A and B, cAMP accumulation in WT or D100A transfected cells in the absence or presence of the inverse agonist (RO116-1148 at 10⁻⁷ M) is presented as a function of receptor expression levels. C and D, cAMP accumulation was also determined in the absence or presence of agonists (5-HT or BIMU8 at 10⁻⁶ M). cAMP accumulation was measured for 15 min and expressed as a percentage of cAMP production in mock-transfected cells. In mock-transfected cells, the percentage conversions of [³H]ATP to [³H]cAMP was equal to 0.12 ± 0.04%. Representative data are presented from one of three replicate experiments.</figcaption></figure>

Thus, a mutant was generated that was fully activated by but insensitive to inverse agonists despite the ability of these later to bind to the receptor.

D100<sup>3.32</sup>A Mutation Is a Dominant Mutation, Which Prevents the Effect of Inverse Agonists Whatever the Level or Nature of the Constitutive Activation of the 5-HT<sub>4</sub> Receptor—In a previous study we showed that either truncation of the C-terminal domain of 5-HT<sub>4</sub> receptor (Δ327<sup>7.70</sup>) or the A258<sup>6.34</sup>L mutation in the third intracellular loop elevated the constitutive activity relative to the WT 5-HT<sub>4</sub> receptor (21) (Fig. 6A). At both of these mutations, 5-HT and BIMU8 increased cAMP synthesis over basal (Fig. 6B), and RO116-1148 decreased cAMP synthesis relative to basal (Fig. 6, C and D). Following dual mutation of D100A+Δ327 or D100A+A258L, the inverse agonist property of RO116-1148 was lost (Fig. 6, C and D). Furthermore, as expected 5-HT was inactive on these double mutants, whereas as on D100A, BIMU8 remained fully efficient (Fig. 6B). Taken together, these results showed that the D100A+Δ327 and D100A+A258L dual mutants were qualitatively similar in their pharmacological behavior to the D100A mutant on its own. Thus, the D100A mutant is a dominant mutant. It blocked the effects of inverse agonists on constitutively activated 5-HT<sub>4</sub> receptors, whatever the nature or inten-

sity of the constitutive activation.

Among Other Mutations Leading to Different Constitutively Active 5-HT4 Receptors, Mutations of Two Aromatic Residues, F275A and W272A, Block RO116-1148 Inverse Agonist Activity—We investigated the effects of mutation of other residues known to be involved in the binding of 5-HT or the constitutive activation of the receptor (Fig. 7A). The T134<sup>4.38</sup>A mutation is located in the second intracellular loop (Thr-134<sup>4.38</sup> is a conserved threonine in many 5-HT receptors (27)). The R118<sup>3.50</sup>A mutation is located at the putative interface between TMD-III and the i₂ loop (Arg-118 residue belongs to the DRY sequence). In addition to the D100A mutant, four mutants were generated in the membrane-spanning regions. The Ser-197<sup>5.43</sup> of TMD-V, which likely interacts via a hydrogen bond with the OH of 5-HT, was mutated into Ala (S197A). Two highly conserved aromatic residues of TMD-VI (F275<sup>6.51</sup>A and W272<sup>6.48</sup>A) (28) were mutated into Ala. The Asn residue in of TMD-VII (Asn-308<sup>7.49</sup>), which is conserved across the family 1 GPCRs, was mutated to Asp.

The S197A, T134A, and F275A mutants were similar to the WT receptor in their levels of constitutive activity. The N308D and R118A mutants had levels of constitutive activity, similar to that of D100A (Fig. 7B). Of all the mutants investigated, we
<figure><img src="image_12.png" /><figcaption>FIG. 6. Lack of effect of inverse agonists in D100<sup>3,32</sup>A mutation is dominant over constitutively activating mutations. A, diagram of the double mutant 5-HT<sub>4</sub>(a) receptors used in this study: D100A+Δ327 and D100A+A258L. B, cAMP accumulation in the absence or presence of 5-HT and BIMU8 in COS-7 cells expressing WT or mutant 5-HT<sub>4</sub>(a) receptors D100A, Δ327, D100A+Δ327 (truncation of C-terminal domain), or A258L and D100A+A258L (mutation in the I3 loop). The expression levels, expressed in fmol/mg of protein, were for WT (3500 ± 420), D100A (3850 ± 350), Δ327 (3470 ± 250), D100A+Δ327 (3620 ± 530), A258L (3750 ± 450), and D100A+A258L (3250 ± 410). C and D, efficacy of the inverse agonist, RO116-1148, on basal constitutive activities of the WT and of these three mutated 5-HT<sub>4</sub> receptors expressed as the same level as in B. Levels of cAMP accumulation were measured after 15-min incubation and expressed as a percentage of basal cAMP production measured in mock-transfected COS-7 cells. The conversion percentage of [<sup>3</sup>H]ATP to [<sup>3</sup>H]cAMP in mock-transfected cells was 0.150 ± 0.05. Each value represents the mean ± S.E. determined from three independent determinations.</figcaption></figure>

found only F275A to be similar to D100A in its insensitivity to the potent inverse agonist, RO116-1148 (Fig. 7, B and C). As with the D100A mutation, the absence of effect of RO116-1148 cannot be attributed to an absence of binding. Competition binding experiments with RO116-1148 demonstrated a very small decrease in the affinity for F275A (9 ± 2.3 × 10<sup>-10</sup> M) relative to WT (4.7 ± 1.5 × 10<sup>-10</sup> M) (Fig. 8A). In addition, the 5-HT-mediated cAMP accumulation at this F275A mutant was antagonized by RO116-1148 (Fig. 8B).

The W272A mutant expressed at a standard level of 450 fmol/mg, was totally silent, with a level of basal cAMP accumulation similar to that of mock-transfected controls (Fig. 7B). However, as shown in Fig. 8B, the 5-HT efficiency to stimulate cAMP accumulation was the same on the W272A mutant as on WT 5-HT<sub>4</sub> receptor. Since it displayed no constitutive activity at all, this silent mutant was, by definition, insensitive to the inverse agonist activity of RO116-1148. To check if this mutation was also a dominant mutation that prevents the effect of inverse agonists, we performed dual mutation studies with the

constitutively active mutant, A258L. The W272A+A258L mutant was found to have a modest level of constitutive activity (Fig. 7B). Note that this modest constitutive activity could not be further increased, due to a low expression level of W272A mutant. The same phenomenon was already observed with a mutation of the 5-HT<sub>2A</sub> receptor when the analogous tryptophan was mutated into alanine at W336<sup>6.48</sup>A (29).

However, if modest, this basal level of cAMP accumulation was sufficient to reveal that RO116-1148 did not induce detectable decreases in basal cAMP accumulation (Fig. 7C).

Competition binding studies with [<sup>3</sup>H]GR113808 demonstrated that RO116-1148 binds to the W272A mutant (1.14 ± 0.3 × 10<sup>-9</sup> M) with a very small decrease in affinity relative to WT (Fig. 8A). In addition, the 5-HT-mediated cAMP accumulation at this W272A mutant was antagonized by RO116-1148 (Fig. 8B).

Taken together, these results revealed that two mutations (W272<sup>6.48</sup>A and F275<sup>6.51</sup>A), in addition to the D100<sup>3.32</sup>A mutation, inhibit the effects of inverse agonists on the 5-HT<sub>4</sub> recep-
<figure><img src="image_13.png" /></figure>

FIG. 7. Among the highly conserved residues, two aromatic residues are necessary for the inverse agonist activity. A, diagram of all the mutations performed on the 5-HT<sub>4</sub>(a) receptor used in this study. B, native and mutant receptors were transiently expressed in COS-7 cells at 2500–3000 fmol/mg of protein, WT (2560 ± 120), D100A (2720 ± 250), F275A (2520 ± 240), S197A (2900 ± 340), N308D (2570 ± 280), R118A (2950 ± 380), and T134A (2800 ± 430). W272A and W272A+A258L mutants were expressed only at 500 ± 44 and 570 ± 65 fmol/mg of protein, respectively. Basal cAMP production was determined by measuring the accumulation of cAMP over 15 min and expressed as a percent of cAMP accumulation in mock-transfected cells. The basal cAMP levels calculated for all mutated 5-HT<sub>4</sub> receptors ranged from 100 to 1250% of the cAMP levels measured in mock-transfected cells. In mock-transfected cells, the percentage conversion of [<sup>3</sup>H]ATP to [<sup>3</sup>H]cAMP was equal to 0.13 ± 0.05%. C, effects of 10<sup>-7</sup> M RO116-1148 in cells transfected as described in B. cAMP accumulation over 15 min was determined in the absence or presence RO116-1148. Data are expressed as a percent of basal cAMP accumulation. The percentage conversion of [<sup>3</sup>H]ATP to [<sup>3</sup>H]cAMP in mock-transfected cells was 0.12 ± 0.04.

for constitutive activity, but not the ability of these compounds to bind to the receptor.

*Molecular Modeling of the 5-HT<sub>4</sub> Receptor*—A model of the molecular structure of the 5-HT<sub>4</sub> receptor was developed, using a template based on the corresponding region of high structural resolution recently determined for rhodopsin (Fig. 9A). With

<figure><img src="image_14.png" /></figure>

FIG. 8. Responses to 5-HT<sub>4</sub> receptor agonist and antagonist were conserved in F275A and W272A mutant receptors. A, competition of the inverse agonist RO116-1148 for [<sup>3</sup>H]GR113808 binding (0.28 nM) in membranes derived from COS-7 cells expressing mutated F275A and W272A receptors. B, cAMP accumulation was measured in COS-7 cells transiently expressing WT, F275A, or W272A receptors expressed at similar densities (620 ± 40, 750 ± 50, and 650 ± 35 fmol/mg of protein, respectively) in the absence and presence of 10<sup>-5</sup> M 5-HT. The efficacy of RO116-1148 to inhibit 5-HT-mediated cAMP accumulation was tested in the same COS-7 cells. Levels of cAMP accumulation were measured after 15-min incubation and expressed as a percentage of basal cAMP production measured in mock-transfected COS-7 cells. The conversion percentage of [<sup>3</sup>H]ATP to [<sup>3</sup>H]cAMP in mock-transfected cells was 0.150 ± 0.05. Each value represents the mean ± S.E. determined from three independent determinations.

this model we aimed to investigate the relationships between the three residues (Asp-100<sup>3.32</sup>, Phe-275<sup>6.51</sup>, Trp-272<sup>6.48</sup>) that were experimentally determined to contribute to the effects of inverse agonists. With this model we hoped to elucidate the relative positions of these three residues in three-dimensional space.

Using this model, the distances between Asp-100<sup>3.32</sup>, Trp-272<sup>6.48</sup>, and Phe-275<sup>6.51</sup> side chains were determined to range from 3 to 4 Angstroms (Fig. 9B). In addition the side chains of Phe-275 and Trp-272 contribute to an aromatic cluster of π-interactions, similar to those described previously for catecholamine receptors (30). The acidic side chain of Asp-100 was
<figure><img src="image_15.png" /><figcaption>FIG. 9. **Molecular modeling of the 5-HT₄ receptor.** A, a model of the 5-HT₄ receptor, based on the high resolution structure elucidated for bovine rhodopsin. Three-dimensional side view of the Cα-trace with transmembrane helix (represented as ribbons) colored as follows: TMD-I in dark blue, TMD-II in light blue, TMD-III in violet, TMD-IV in pink, TMD-V in orange, TMD-VI in red, and TMD-VII in green. The side chains of Asp-100 (TMD-III), Trp-272, and Phe-275 (TMD-VI), located within the binding pocket of the 5-HT₄ receptor, are shown in black. B, side view of the binding pocket of the m5-HT₄ receptor. Cα-traces are represented as ribbons; residues shown are known to be involved in 5-HT binding or surrounding Asp-100, Trp-272, and Phe-275. Molecular modeling reveals the tight network of these three side chains distant from 3.5 to 4.6 Angstroms. Color coding of Asp-100, Trp-272, and Phe-275 atoms: oxygen, red; nitrogen, cyan; carbon, green; hydrogen, white.</figcaption></figure>

found to be oriented toward the amine of Trp-272 indole group and might participate in a polar interaction that would be stabilized by the aromatic ring of the Phe-275 residue that is predicted to reside immediately above it. The intramolecular interactions predicted by this model would be expected to be disrupted by the D100A, W272A, and F275A mutations, which were found to inhibit the activity (but not significantly affinity) of inverse agonists.

DISCUSSION

One of the key findings of this study was the observation that the potent inverse agonists, RO116-0086 and RO116-1148, reverse basal levels of cAMP accumulation in WT but not the D100A mutant. In addition, these agents were ineffective following dual mutation of D100<sup>3.32</sup>A with the constitutively active mutations, Δ327<sup>7.70</sup>L or A258<sup>6.34</sup>L.

Highly conserved among biogenic amine receptors, the Asp
residue Asp-100<sup>3.32</sup> is known to make an ionic bond with the protonated amine of the ligand. Mutation of this Asp to Ala generally leads to a loss of binding for the neurotransmitter structurally related to amine biogene (28). This was also the case for the D100<sup>3.32</sup>A mutant that proved unable to bind 5-HT. However, in contrast to what has been observed for other biogenic amine receptors, this mutation did not significantly affect the binding of the radioligand antagonist [³H]GR113808 or the binding of the synthetic agonist BIMU8. Indeed, this mutant might be considered to be a RASSL (receptor activated solely by synthetic ligand (31)). Since BIMU8 was at least equi-potent and equi-effective at the D100A and WT receptor, we conclude that Asp-100<sup>3.32</sup> is not involved in the conformational shifts that result in receptor activation, at least for this synthetic ligand. In contrast, Asp-100 is essential for the shifts in receptor conformation that result in receptor inactivation by the studied inverse agonists.

The two-state model is now believed to be a simplification of a more general concept of allosteric equilibrium (7, 32, 33). According to this concept, the receptor undergoes transitions between different allosteric states. Agonists preferentially stabilize different R\* states and consequently can stimulate different signal transduction pathways by “receptor trafficking” (7). Our group previously showed that constitutively active 5-HT<sub>4</sub> receptor mutants (Δ327 and A258L) have different R\* conformations (20).

A recent study on agonist-mediated changes in conformation of the G protein-coupling domain of β₂-adrenoceptors found that conformations stabilized by full or partial agonists were different (14). Therefore, it seems likely that the inactive R state is a heterogeneous population of different inactive conformations. Using this logic, the rhodopsin receptor, proposed to exist in at least three different states, might be expected to exist instead in at least three different populations of states. The “ground” silent state (R<sub>g</sub>) of rhodopsin is stabilized by the inverse agonist, cis-retinal. It is distinct from another low activity state, the ligand-free opsin state, R. The third state, metarhodopsin II, is characterized by being bound to all-trans-retinal and is fully active (R\*) (8, 34). The activity of the R state was determined to be 10<sup>-6</sup> times lower than that of the R\* state (34). Stabilization of the R<sub>g</sub> state by cis-retinal, the inverse agonist, is necessary for the physiological activity of rhodopsin as it enables the rod cell to operate as a single photon detector even in the presence of high levels of activated rhodopsin.

The findings of this study might be explained by applying the rhodopsin three-state model to the 5-HT<sub>4</sub> receptor. In this model, the 5-HT<sub>4</sub> receptor exists in an equilibrium between three states (or populations of state), R<sub>g</sub>, R\*, and R, stabilized by inverse agonist, agonist, ligand-free, respectively. The R<sub>g</sub> state would be preferred in the presence of inverse agonists. Indeed, in the presence of inverse agonists, the levels of cAMP accumulation were close to those observed in the absence of functional receptor (the mock-transfected controls, see Figs. 1 and 5A).

Inversely, the absence of constitutive activity and insensitivity to inverse agonists observed with the W272A mutant might indicate that this receptor exist predominantly in the R<sub>g</sub> state. The observed agonist-mediated increases in cAMP accumulation demonstrate that this mutation does not interfere with agonist-mediated conversion from R<sub>g</sub> to R\* (Fig. 8). The lack of effect of inverse agonists in the W272A mutant is possibly due to the very low levels of constitutive activity. However, from separate studies in which basal cAMP accumulation is increased by the additional A258L mutation, the persistent insensitivity to inverse agonists supports the idea that the Trp-272<sup>6.48</sup> required to mediate their effects. The Trp-272<sup>6.48</sup>

residue (TMD-VI) is highly conserved in Family I GPCRs. In the crystal structure of rhodopsin, an analogous tryptophan residue, Trp-265<sup>6.48</sup>, points downwards to the retinylidene group, near the B-ionone ring of cis-retinal and close to its C-13 methyl group (3.8 Å) (34, 35). Since rhodopsin was crystallized in the presence of the inverse agonist cis-retinal (34), this orientation of Trp-265<sup>6.48</sup> is likely to correspond to the R<sub>g</sub> state. Conversely, rhodopsin adopts a conformation in the absence of activation by light (R), in which the cross-linking group located on retinal ionone ring is directly bound to Trp-265<sup>6.48</sup> (36). Finally, deletion of the C-13 methyl group on the retinal results in an increase constitutive activity that might indicate a stabilization of the R\* state (37).

We can apply the three-state model to our observations with the D100A, D100A+Δ327, and D100A+A258L mutants. Although these mutants had different levels of constitutive activity, they retained sensitivity to the agonist. This indicates that the D100A mutation does not impair the agonist-mediated transition to the R\* conformation. Conversely, in these mutants the transition to the R<sub>g</sub> state is no longer favored by inverse agonists. Similar logic might be applied to the influence of the F275A mutant on intramolecular interactions.

In dual mutation studies, the observed failure of the constitutively activating mutations, Δ327 or A258L, to overcome the insensitivity of D100A to inverse agonists suggests that the intramolecular interactions associated with activation of cAMP synthesis, and hence R\*, are different from those associated with inactivation of cAMP synthesis and R<sub>g</sub>.

It is now well established that TMD-III and TMD-VI play a crucial role in GPCR activation (2). For many GPCRs, activation involves disruption of a network of interactions consisting of the Arg<sup>3.50</sup> and the Asp<sup>3.49</sup>/Glu<sup>3.49</sup> residues of the conserved DRY sequence of the cytoplasmic side of TMD-III, and a conserved Glu<sup>6.30</sup> residue at the cytoplasmic end of TMD-VI. It has been proposed that this disruption, possibly following protonation of the Asp<sup>3.49</sup>/Glu<sup>3.49</sup> or Glu<sup>6.30</sup> residues, is critical to receptor activation (15–17). Moreover, in studies of rhodopsin or β₂-adrenoceptors, activation by site-directed labeling of residues on the cytoplasmic side of the TMDs III and VI strongly suggests that activation involves clockwise rotation and separation of TMD-III from TMD-VI (38, 39).

The data presented in this study suggest that a different network of intramolecular interactions exists in the 5-HT<sub>4</sub> receptor between the Asp-100<sup>3.32</sup> residue of TMD-III and the Trp-272<sup>6.48</sup> and Phe-275<sup>6.50</sup> residues of TMD-VI. This network underlies the actions of inverse agonists but not agonists. The resulting structural model of the 5-HT<sub>4</sub> receptor, presented in Fig. 9A was based on the structure of the corresponding sequence of rhodopsin, which has previously been elucidated (35). In this model, the side chains of residues Asp-100<sup>3.32</sup>, Trp-272<sup>6.48</sup>, and Phe-275<sup>6.51</sup> are 3–4 Angstroms apart. In addition, the aromatic cluster formed between the side chains of Phe-275 and Trp-272 has previously been described for other catecholamine GPCRs (40). Finally, we propose that a polar interaction between the acidic side chain of Asp-100 and the amine of Trp-272 is stabilized if the aromatic ring of Phe-275 is localized immediately above it.

We hypothesize that the electrostatic influence of the aromatic Trp-272 residue on its surroundings must be neutralized for the receptor to adopt the R<sub>g</sub> conformation. This occurs when the Trp-272 residue is engaged in the Trp-272-Asp-100-Phe-275 network. Mutations of Asp-100 and Phe-275 to alanine explain the absence of effect of inverse agonists and hence the failure to stabilize the Trp-272-Asp-100-Phe-275 network that defines the R<sub>g</sub> conformation, because under the alanine substitutions, neutralization of the electrostatic influence of Trp-272
is no longer possible. Using the same logic, we predict that the W272A mutation permanently neutralizes the electrostatic influence of the Trp-272, thereby stabilizing the R<sub>g</sub> state.

We propose that 5-HT<sub>4</sub> receptor inverse agonists described here favor a Asp-100<sup>3.32</sup>-Phe-275<sup>6.51</sup>-Trp-272<sup>6.48</sup> constraining network, which is not required for agonist activation. How this occurs is a matter of speculation. One hypothesis is that the aromatic moiety of inverse agonists interact with hydrophobic residues of the network to favor the receptor constrain. Indeed, a shift in the position of the aromatic moiety of RO116-0086, by adding a chloro and an amino substituent to give the SB204070 compound, suppresses the inverse agonist property of the drug.

Further studies may reveal the existence of analogous networks in other Family I GPCRs, which would help to define the molecular determinants of inverse agonists. Exploration of such networks would provide a better understanding of the influences that govern conversion between different receptor conformations and the effects of agonists, inverse agonists, and antagonists in terms of their impact on the GPCR structure.

*Acknowledgements*—We are grateful to M.-L. Parmentier, J.-P. Pin, and M. Bouvier for helpful comments and valuable discussions and to T. Galvez for his skills in molecular modeling. A.-L. Turner-Madeuf and S. Gavarini are acknowledged for help in language revision and M. Passama for preparation of the figures.

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# A 5-HT₄ Receptor Transmembrane Network Implicated in the Activity of Inverse Agonists but Not Agonists

Lara Joubert, Sylvie Claeysen, Michèle Sebben, Anne-Sophie Bessis, Robin D. Clark, Renee S. Martin, Joël Bockaert and Aline Dumuis

J. Biol. Chem. 2002, 277:25502-25511.

doi: 10.1074/jbc.M202539200 originally published online April 25, 2002

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Additions and Corrections

Vol. 277 (2002) 25502-25511

A 5-HT₄ receptor transmembrane network implicated in the activity of inverse agonists but not agonists.

Lara Joubert, Sylvie Claeysen, Michèle Sebben, Anne-Sophie Bessis, Robin D. Clark, Renee S. Martin, Joël Bockaert, and Aline Dumuis

Page 25504, fifth line from the bottom of the first column and page 25506, right-hand column, line 6: Insert the following reference:

Mialet, J., Dahmoune, Y., Lezoualc'h, F., Berque-Bestel, I., Eftekhari, P., Hoebeke, J., Sicsic, S. S., Langlois, M., and Fischmeister, R. (2000) *Br. J. Pharmacol.* **130**, 527-538

Vol. 277 (2002) 28663-28668

Influences of base excision repair defects on the lethal-
ity and mutagenicity induced by Me-lex, a sequence
selective N3-adenine methylating agent.

Paola Monti, Paola Campomenosi, Yari Ciribilli, Raffaella Iannone, Alberto Inga, Dharini Shah, Gina Scott, Philip A. Burns, Paola Menichini, Angelo Abbondandolo, Barry Gold, and Gilberto Fronza

The affiliation for Paola Campomenosi was partially omitted.
The additional affiliation should read:

*Human Genetics Laboratory, Department of Structural and Functional Biology (DBSF), University of Insubria, Varese, Italy*

Also, Paola Monti was supported by a "Fondazione Italiana per la Ricerca sul Cancro" (FIRC) fellowship.

Vol. 277 (2002) 30153-30158

The insulin receptor catalyzes the tyrosine phos-
phorylation of caveolin-1.

Akiko Kimura, Silvia Mora, Satoshi Shigematsu, Jeffrey E. Pessin, and Alan R. Saltiel

In this manuscript, we unintentionally failed to cite two studies (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K. E., and Strålfors, P. (1999) *FASEB J.* **13**, 1961-1971 and Parpal, S., Karlsson, M., Thorn, H., and Strålfors, P. (2001) *J. Biol. Chem.* **276**, 9670-9678) that also demonstrated the presence of the insulin receptor in caveolin-positive structures in 3T3L1 adipocytes. We regret our oversight.

We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate
places where the article to be corrected originally appeared. Authors are urged to introduce these
corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of
these corrections as prominently as they carried the original abstracts.