# Constitutive Activation of the Opioid Receptor by Mutations in Transmembrane Domains III and VII

Katia Befort, Christelle Zilliox, Dominique Filliol, Shiyi Yue, Brigitte L Kieffer

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Katia Befort, Christelle Zilliox, Dominique Filliol, Shiyi Yue, Brigitte L Kieffer. Constitutive Activation of the Opioid Receptor by Mutations in Transmembrane Domains III and VII. Journal of Biological Chemistry, 1999, 274 (26), pp.18574-18581. 10.1074/jbc.274.26.18574. hal-03432873

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# Constitutive Activation of the δ Opioid Receptor by Mutations in Transmembrane Domains III and VII*

(Received for publication, February 2, 1999, and in revised form, March 31, 1999)

Katia Befort‡, Christelle Zilliox‡, Dominique Filliol‡, ShiYi Yue§, and Brigitte L. Kieffer‡¶¶

From the ‡Ecole Supérieure de Biotechnologie, Parc d'Innovation, Boulevard Sébastien Brandt, F-67400 Illkirch-Graffenstaden, France and the §Departments of Chemistry and Molecular Biology, Astra Research Center Montreal, Montreal, Quebec H4S 1Z9, Canada

We have investigated whether transmembrane amino acid residues Asp¹²⁸ (domain III), Tyr¹²⁹ (domain II), and Tyr³⁰⁸ (domain VII) in the mouse δ opioid receptor play a role in receptor activation. To do so, we have used a [³⁵S]GTPγS (where GTPγS is guanosine 5'-3'-O-(thio)triphosphate) binding assay to quantify the activation of recombinant receptors transiently expressed in COS cells and compared functional responses of D128N, D128A, Y129F, Y129A, and Y308F point-mutated receptors to that of the wild-type receptor. In the absence of ligand, [³⁵S]GTPγS binding was increased for every mutant receptor under study (1.6-2.6-fold), suggesting that all mutations are able to enhance constitutive activity at the receptor. In support of this finding, the inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (where Aib represents α-aminobutyric acid) efficiently reduced basal [³⁵S]GTPγS binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [³⁵S]GTPγS binding above basal levels with similar (D128N, Y129F, and Y129A) or markedly increased (Y308F) efficacy compared with wild-type receptor. BW373U86 potency was maintained or increased. In conclusion, our results demonstrate that the mutations under study increase functional activity of the receptor. Three-dimensional modeling suggests that Asp¹²⁸ (III) and Tyr³⁰⁸ (VII) interact with each other and that Tyr¹²⁹ (III) undergoes H bonding with His²⁷⁸ (VI). Thus, Asp¹²⁸, Tyr¹²⁹, and Tyr³⁰⁸ may be involved in a network of interhelical bonds, which contributes to maintain the δ receptor under an inactive conformation. We suggest that the mutations weaken helix-helix interactions and generate a receptor state that favors the active conformation and/or interacts with heterotrimeric G proteins more effectively.

Opiates elicit their potent biological actions through three classes of opioid receptors, μ, δ, and κ. Three homologous opioid receptor genes have been identified that encode each of the previously characterized receptor sites (for a review see Ref. 1). The cloned receptors belong to the G protein-coupled receptor superfamily, endowed with a seven transmembrane (Tm)<fnref n="1" /> topology.

All three opioid receptors are coupled to heterotrimeric G proteins of the G₁/G₀ type, and receptor stimulation by an opioid agonist modulates a number of intracellular pathways (2–4) leading to inhibition of neuronal activity. Little is known about the molecular mechanisms of transmembrane signaling at the receptor level.

Activation mechanisms of G protein-coupled receptors (GPRs) are being extensively investigated both by computer modeling and mutagenesis or spectroscopy (for reviews see Refs. 5–8). Rhodopsin, muscarinic, and adrenergic receptors have been most studied, and a number of hypotheses have been proposed. In particular it has been suggested that transmembrane motions are involved in processes that would make intracellular loops of the receptor accessible to G proteins. Tm-Tm interactions seem to be critical in maintaining the receptor in an inactive conformation, and modifications of H bonding by proton transfer have been suggested as part of the activation phenomenon. The question of how binding interactions do propagate through Tm domains down to intracellular loops and trigger G protein activation has not been solved. It is nevertheless predictable that some residues that belong to the binding pocket may also participate in signaling.

According to low structure resolution of bovine rhodopsin (9, 10), Tm III and VII are the most buried regions within the helical bundle, with limited access to the lipidic membrane environment, and they most probably interact with each other. We have previously mutated single amino acid residues of Tm III (Asp¹²⁸ and Tyr¹²⁹) and VII (Tyr³⁰⁸) of the mouse δ opioid receptor (mDOR) (Fig. 1) and proposed that these residues belong to the opioid binding site and participate in ligand recognition (11, 12). To investigate whether these residues also participate in the transduction process itself, we have examined the effect of mutations on receptor signaling.

The determination of ligand potency and efficacy at mutant recombinant G₁/G₀-coupled receptors usually requires the establishment of stable cell lines for each receptor under study. We have recently developed a method to measure opioid receptor activation using transient expression in COS cells and [³⁵S]GTPγS binding (13). We now have used this approach to evaluate the effect of D128N and D128A (Tm III), Y129F and Y129A (Tm III), and Y308F (Tm VII) mutations on functional activation of the δ receptor. An important result from this study is the finding that structural modifications in Tm III and Tm VII enhance ligand-independent [³⁵S]GTPγS binding. This is the first report of constitutively active mutant (CAM) opioid receptors.

<footnote>* This work was supported by the Association de la Recherche pour le Cancer, the Institut Universitaire de France, and the Center National de la Recherche Scientifique. 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.</footnote>

<footnote>¶ To whom correspondence should be addressed: CNRS URR 9050, ESBS, Parc d'innovation, Blvd. S. Brandt 67400 Illkirch, France.</footnote>

<footnote n="1">¹ The abbreviations used are: Tm, transmembrane domain; BW-373U86, (±)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di-methyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide; CAM, constitutively active mutant; DADLE, [D-Ala², D-Leu⁵]enkephalin; mDOR, cloned mouse δ-opioid receptor; GPRs, G protein-coupled receptors; ICI174864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (where Aib represents α-aminobu-</footnote>

<footnote>tyric acid); SNC80, ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide); WT, wild type; GTPγS, guanosine 5'-3'-O-(thio)triphosphate.</footnote>
<figure><img src="image_2.png" /><figcaption>**FIG. 1.** Site-directed mutagenesis of mDOR. This schematic representation of the seven-transmembrane topology of the δ receptor highlights the positions of Tm III and Tm VII amino acid residues that have been mutated in this study. The single-letter amino acid codes are used, and the numbers refer to the positions of the mutated residues within the protein sequence.</figcaption></figure>

## EXPERIMENTAL PROCEDURES

**Materials**—DADLE, D-Ala² delforphin II, naloxone, GDP, and GTPγS were purchased from Sigma. Bremazocine was from Research Biochemicals International (Natick, MA). SNC80 and ICI174864 were from Tocris Cookson (Bristol, United Kingdom). BW373U86 was kindly provided by Dr. K. J. Chang (Burroughs Wellcome Co., Research Triangle Park, NC). [³H]NTI (specific activity, 44.5 Ci/mmol) was provided by A. Borsodi (Hungarian Academy of Sciences, Szeged, Hungary), and [³⁵S]GTPγS (specific activity, 1250 Ci/mmol) was from NEN Life Science Products. mDOR cDNA (14) was subcloned into pcDNAI/Amp (Invitrogen) for site-directed mutagenesis and transient expression in COS cells. The carrier plasmid used in the electroporation procedure (pBluescript) was from Stratagene. $K_d$, $K_i$, and $B_{max}$ values were calculated using the EBDA/Ligand program (G. A. McPherson, Biosoft, UK), and $EC_{50}$ values were determined using the Prism software (GraphPad, San Diego, CA).

**Mutagenesis of mDOR and Expression of Wild-type and Mutant Receptors in COS Cells**—mDOR cDNA was modified by site-directed mutagenesis, as described in previous studies (11, 12), to obtain point mutations in Tm III (D128N, D128A, Y129F, and Y129A) and in Tm VII (Y308F). COS cells were transfected using an electroporation procedure as described previously (13). Briefly, $2 \times 10^7$ COS cells were seeded the night before transfection at a density of $10^7$ cells/140-mm dish. Cells were washed two times with phosphate-buffered saline, detached by applying 5 ml of trypsin/EDTA (Eurobio) for 5 min at 37 °C, and diluted with 5 ml of Dulbecco's modified Eagle's medium, and an aliquot was used to determine the total cell number. Cells were collected by centrifugation for 10 min at 1000 rpm and resuspended at a $10^8$ cell/ml density in EP 1× buffer (50 mM $K_2$HPO₄, 20 mM $CH_3$CO₂K, 20 mM KOH, pH 7.4). Plasmidic DNA, prepared using Nucleobond columns (Macherey Nagel) and consisting of 4 µg (standard condition) or a variable amount of receptor-encoding plasmid and a carrier plasmid (pBluescript) up to a final $20-µg$ DNA quantity was diluted into EP 1× buffer to a total volume of 300 µl. The DNA mix was then supplemented with 13 µl of $MgSO_4$ 1 M and incubated with 200-µl cell suspension for 20 min at room temperature. The cell/DNA mixture was then transferred to a 0.4-cm cuvette (Bio-Rad) and electroporated using a Gene Pulser apparatus (Bio-Rad) at a capacitance setting of 2000 microfarads and a voltage setting of 240 V. Cells were then immediately transferred into 50 ml of Dulbecco's modified Eagle's medium with 10% fetal calf serum and seeded into two 140-mm dishes. After 72 h growth the cells were harvested, and membranes were then prepared as described previously (13).

**Membrane Preparation**—The same procedure was used for either [³H]naltrindole or [³⁵S]GTPγS binding. Transfected cells (four 140-mm dishes at a 50–100% confluency) were washed two times with phosphate-buffered saline, scrapped off the plates in phosphate-buffered saline, pelleted by spinning at 1500 rpm for 10 min at 4 °C, frozen at –80 °C for at least 30 min, and thawed in 30 ml of cold 50 mM Tris-HCl, pH 7, 2.5 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride added extemporaneously. The cell lysate was put on ice for 15 min, processed in a Dounce homogenizer, and spun at 2500 rpm for 10 min at 4 °C. The pellet was resuspended in 15 ml buffer, processed in a Dounce homogenizer, and spun again at 2500 rpm for 10 min at 4 °C. Both supernatants were pooled and centrifuged at 40 000 rpm for 30 min at 4 °C. The supernatant was removed, and the pellet was resuspended in 25 ml of

50 mM Tris-HCl, pH 7, processed in a Dounce homogenizer, and spun again at 40000 rpm for 30 min at 4 °C. The pellet was then resuspended in 4 ml of 50 mM Tris-HCl, pH 7, sucrose 0.32 M, and the protein concentration was measured using the Bradford assay. Membranes were aliquoted and stored at –80 °C.

**Saturation and Competition Analysis**—Opioid binding experiments were done as described previously (12). For saturation experiments, 0.8–5 µg of membrane proteins were diluted in 50 mM Tris-HCl, pH 7.4, in a final volume of 0.5 ml and incubated with variable concentrations of [³H]naltrindole (0.03–3 nM) for 1 h at 25 °C. Nonspecific binding was determined in the presence of 2 µM naloxone. For competition studies membrane preparations were incubated for 1 h at 25 °C with [³H]naltrindole (0.5 nM) in the presence of various concentrations ($10^{-5}$ to $10^{-13}$ M) of the opioid competing ligand (SNC80). In both cases, incubation mixtures were rapidly washed using a Brandell cell harvester with cold 50 mM Tris buffer on 0.1% polyethylenimine presoaked GF/B filters (Whatman).

**[³⁵S]GTPγS Binding Assays**—Ligand-stimulated [³⁵S]GTPγS binding on COS cell membrane preparation expressing mDOR and mDOR mutants was examined as described previously (13). 5 µg of membrane proteins were incubated for 2 h at 4 °C both with and without the agonist ($10^{-5}$ to $10^{-13}$ M) or inverse agonist ICI174864 ($10^{-5}$ M) in assay buffer containing 50 mM Hepes, pH 7.6, 5 mM $MgCl_2$, 100 mM NaCl, 10 mM EDTA, 1 mM dithiothreitol, 0.1% bovine serum albumin, 10 µM GDP, and 0.2 nM [³⁵S]GTPγS. Nonspecific binding was determined in the presence of 10 µM GTPγS, and basal binding was assessed in the absence of agonist. As for saturation experiments, the incubation was terminated by rapid filtration through H₂O presoaked GF/B filters, followed by three washes with ice-cold 50 mM Tris-HCl, pH 7, 5 mM $MgCl_2$, 50 mM NaCl. Bound radioactivity was determined by scintillation counting. Mock transfected control cells showed no ligand-induced [³⁵S]GTPγS binding.

## RESULTS

**[³⁵S]GTPγS Binding in COS Cells Transiently Expressing the WT Receptor: Efficacies and Potencies of Opioid Agonists**—To better evaluate the sensitivity of the [³⁵S]GTPγS assay in our expression system (13), we have measured the potency and efficacy of a number of opioid agonists. COS cells were electroporated with transfection efficiencies of 50% at least, as assessed by control β-galactosidase activity measurements (13) (not shown). Receptor expression was assessed 72 h after transfection. Scatchard analysis of [³H]naltrindole binding performed on membrane preparations from several transfection experiments (WT I; Table I) showed high affinity sites for the ligand ($K_d$ = 0.33 ± 0.08 nM) and a $B_{max}$ value of $51.2 \pm 4.2$ pmol/mg protein, indicating high receptor expression level under our standard transfection conditions. These membrane preparations were then subjected to stimulation by agonists, and specific [³⁵S]GTPγS binding was measured using a $10^{-13}$ to $10^{-5}$ M concentration range of the following agonists: BW373U86 and SNC80 (two δ selective alkaloids), delforphin II and DADLE (two δ selective peptides), and bremazocine (a nonselective alkaloid). Results are shown in Fig. 2 and Table II [³⁵S]GTPγS binding increased 1.7–3-fold above basal level upon agonist stimulation. BW373U86, SNC80, and delforphin II were most efficacious with 275%, 273%, and 264% activation at saturating agonist concentrations. Bremazocine was the least effective compound (167% of basal level) and behaved as a partial agonist. The order of potencies was BW373U86 > delforphin II > DADLE > bremazocine > SNC80, and $EC_{50}$ values correlate with previously determined affinity values (11). BW373U86, which displays highest affinity for the receptor, is also most potent at stimulating [³⁵S]GTPγS binding ($EC_{50}$ 0.62 ± 0.18 nM), in accordance with previous data (15). Together these results show that our assay conditions are able to discriminate ligands with distinct activation properties. Recently, [³⁵S]GTPγS binding has been successfully used to evaluate agonists potencies and efficacies at the cloned µ (16) and κ (17) receptors using Chinese hamster ovary cell lines stably expressing the receptors. Here we demonstrate that this assay
<caption>**TABLE I**
Receptor densities ($B_{max}$) at mutants and their corresponding WT receptor membrane preparations</caption>

This table shows receptor densities in WT and mutant receptor preparations that have been compared in this study. WT I and the five mutant receptors were expressed in COS cells under standard conditions (4000 ng/2 × 10⁷ cells; see “Experimental Procedures”). For WT II to WT V, a lower amount of transfected cDNA (2000–4 ng/2 × 10⁷ cells) was allowed to obtain expression levels comparable with that of each mutant receptor (see “Experimental Procedures” and Fig. 3). Membranes were prepared from several independent transfection experiments, and $B_{max}$ values are measured by Scatchard analysis of [<sup>3</sup>H]naltrindole binding. Values are the means ± S.E. from *n* experiments. Membrane preparations were paired as follows: WT II/D 128N, WT III/Y 129F, WT IV/Y129A, WT IV/Y308F, and WT V/D128A.

<table><thead><tr><th rowspan="3"></th><th colspan="3">Wild-type receptors</th><th colspan="3">Mutant receptors</th></tr><tr><th>DNA</th><th>$B_{max}$</th><th>$n$</th><th>DNA</th><th>$B_{max}$</th><th>$n$</th></tr><tr><th>ng/2 × 10<sup>7</sup> cells</th><th>pmol/mg protein</th><th></th><th>ng/2 × 10<sup>7</sup> cells</th><th>pmol/mg protein</th><th></th></tr></thead><tbody><tr><td>WT I</td><td>4000</td><td>51.2 ± 4.2</td><td>10</td><td></td><td></td><td></td></tr><tr><td>WT II</td><td>2000</td><td>31.1 ± 2.3</td><td>12</td><td>D128N</td><td>4000</td><td>34.6 ± 6.7</td></tr><tr><td>WT III</td><td>400</td><td>20.8 ± 2.7</td><td>5</td><td>Y129F</td><td>4000</td><td>20.9 ± 3.4</td></tr><tr><td>WT IV</td><td>80</td><td>12.2 ± 1.9</td><td>5</td><td>Y308F</td><td>4000</td><td>11.1 ± 1.3</td></tr><tr><td></td><td></td><td></td><td></td><td>Y129A</td><td>4000</td><td>9.65 ± 1.56</td></tr><tr><td>WT V</td><td>4</td><td>1.99 ± 0.14</td><td>5</td><td>D128A</td><td>4000</td><td>2.22 ± 0.47</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**FIG. 2.** Agonist-promoted stimulation of [<sup>35</sup>S]GTPγS binding to membranes of COS cells transiently expressing the WT receptor. COS cells were transfected with the WT cDNA under standard conditions (4000 ng of DNA/2 × 10⁷ cells) leading to a receptor density of 51.2 pmol/mg protein (Table I, WT1). Membranes were incubated with 0.2 nM [<sup>35</sup>S]GTPγS, 10 µM GDP, and increasing concentrations of BW373U86, SNC80, DADLE, delforphin II, and bremazocine. Data are the means ± S.E. from three (BW373U86, SNC80, and DADLE) to four (delforphin II and bremazocine) independent experiments performed in triplicate. 100% is defined as specific [<sup>35</sup>S]GTPγS binding in the absence of agonist. Maximal activation and EC₅₀ values from curve fitting of these data are shown in Table II.</figcaption></figure>

<caption>**TABLE II**
Efficacy and potency of opioid agonists for the stimulation of [<sup>35</sup>S]GTPγS binding in COS cells transiently expressing the WT mDOR receptor</caption>

This table displays maximal activation levels and EC₅₀ values determined from experiments shown in Fig. 2. 100% is defined as specific [<sup>35</sup>S]GTPγS binding in the absence of agonist. Values from these experiments are the means ± S.E. from *n* separate experiments performed in triplicate. Ligand binding affinity values ($K_i$) are from previously reported competition experiments (11), except for SNC80 (see “Experimental Procedures.”)

<table><thead><tr><th rowspan="3">Ligand</th><th colspan="3">[<sup>35</sup>S]GTPγS binding</th><th colspan="2">Ligand binding</th></tr><tr><th>Maximal activation</th><th>EC<sub>50</sub></th><th>$n$</th><th colspan="2">$K_i$</th></tr><tr><th>% above basal</th><th>nM</th><th></th><th colspan="2">nM</th></tr></thead><tbody><tr><td>Bremazocine</td><td>167 ± 9</td><td>7.4 ± 2.1</td><td>4</td><td>2.79 ± 0.14</td><td></td></tr><tr><td>DADLE</td><td>210 ± 28</td><td>5.8 ± 2.4</td><td>3</td><td>10 ± 1.4</td><td></td></tr><tr><td>Delforphin II</td><td>264 ± 22</td><td>3.9 ± 0.9</td><td>4</td><td>9.23 ± 0.94</td><td></td></tr><tr><td>SNC80</td><td>273 ± 55</td><td>12.5 ± 2.1</td><td>3</td><td>8.23 ± 2.27</td><td></td></tr><tr><td>BW373U86</td><td>275 ± 51</td><td>0.62 ± 0.18</td><td>3</td><td>1.63 ± 0.58</td><td></td></tr></tbody></table>

is applicable to transient expression conditions and should allow for the detection of subtle alterations of agonist-induced stimulation in mutant receptors.

**[<sup>35</sup>S]GTPγS Binding in COS Cells Transiently Expressing the WT Receptor: Effect of Receptor Density**—The amplitude of cellular responses are depending on the number of functional receptor molecules expressed by the host cell. We therefore

expected levels of [<sup>35</sup>S]GTPγS binding to vary as a function of receptor density. To correlate the functional activation of mDOR with different expression conditions, we transfected variable amounts of WT cDNA (2 ng to 20 µg) into COS cells. We measured expression levels of the receptor using [<sup>3</sup>H]naltrindole binding, and Fig. 3 shows the increase of receptor density as a function of transfected DNA amounts. $B_{max}$ values ranged from 1.8 ± 0.4 (2 ng) to 62 ± 4 pmol/mg protein (20 µg). We then subjected membrane preparations to stimulation by BW373U86 at a saturating concentration (10⁻⁵ M) and measured [<sup>35</sup>S]GTPγS binding (Fig. 3). Our results confirm that the extent of agonist-stimulated activation is indeed dependent on receptor expression level because [<sup>35</sup>S]GTPγS binding increased (122–327% of basal activity) with mDOR density. It is noteworthy that [<sup>35</sup>S]GTPγS binding plateaus at high receptor densities only (WT I), suggesting that G proteins are not limiting under the expression conditions further considered in this study (WT II to WT V, <31.1 pmol/mg protein; see “Expression of Mutant Receptors”).

**Expression of Mutant Receptors**—We have used site-directed mutagenesis to create the following point mutations in mDOR: D128A, D128N, Y129A, Y129F, and Y308F (Refs. 11 and 12 and Fig. 1). Plasmid DNAs encoding the mutant receptors were electroporated in COS cells under our standard conditions (4 µg DNA/2.10⁷ cells; see “Experimental Procedures”), and expression levels were measured by [<sup>3</sup>H]naltrindole binding (Table I, right columns). Receptor densities appeared lower for all mutant receptors than for the WT receptor expressed under identical experimental conditions (WT I, 51.2 pmol/mg protein), and the $B_{max}$ values varied from 2.22 to 34.6 pmol/mg protein depending on the construct. Because [<sup>35</sup>S]GTPγS binding changes with receptor density, a direct comparison of [<sup>35</sup>S]GTPγS binding in mutant preparations with the WT I preparation would be misleading. To compare [<sup>35</sup>S]GTPγS binding activity of WT and mutant receptors, we therefore matched WT and mutant receptor membrane preparations having comparable receptor expression levels: WT II for D128N mutant, WT III for Y129F mutant, WT IV for Y129A and Y308F mutants, and WT V for D128A mutant receptor (Table I).

**Basal [<sup>35</sup>S]GTPγS Binding at WT and Mutant Receptors**—We measured specific [<sup>35</sup>S]GTPγS binding to membrane preparations of COS cells expressing either mutant receptors or their WT counterparts in the absence of ligand. First, the analysis of [<sup>35</sup>S]GTPγS binding to WT preparations expressing variable receptor densities indicated no significant difference between the basal activity of high expressing preparations or low expressing preparations (not shown), suggesting that a ligand-independent activity of the WT mDOR receptor is not detectable in our expression system. This was further corroborated by
<figure><img src="image_4.png" /><figcaption>FIG. 3. Increased receptor density ($B_{max}$) and receptor activation ([³⁵S]GTPγS) as a function of transfected DNA amounts. 2–20,000 ng of WT mDOR-encoding vector were transfected in COS cells (see "Experimental Procedures"), and expression levels in each membrane preparation were measured by [³H]naltrindole binding. $B_{max}$ values are the means ± S.E. from two to twelve independent experiments performed in triplicate. In parallel experiments, membrane preparations were subjected to maximal BW373U86 stimulation (10⁻⁵ M), and specific [³⁵S]GTPγS binding was measured. Data are the means ± S.E. from four to sixteen independent experiments performed in triplicate. Results are shown as the percentage of activation/basal, and 100% is defined as specific [³⁵S]GTPγS binding in the absence of agonist. WT II to WT V refer to membrane preparations that were further used for the study of mutant receptors (Table I).</figcaption></figure>

orated by weak or undetectable activity of the inverse agonist ICI174864 at WT receptors (see below). In contrast, each mutant receptor displayed markedly enhanced basal [³⁵S]GTPγS binding activity compared with WT receptor (Fig. 4). [³⁵S]GTPγS binding at Tm III mutant preparations was 215% (D128N), 261% (D128A), 172% (Y129F), and 205% (Y129A) relative to [³⁵S]GTPγS binding at their corresponding WT preparations (WT II to WT V). Membranes expressing the Y308F mutant receptor exhibited a [³⁵S]GTPγS binding level that was 165% from the WT IV value, an effect that was less pronounced than for D128A, D128N, and Y129A mutants and comparable with Y129F mutant receptor activity. We conclude that all the mutations under study confer constitutive activity at the δ receptor, with a prominent effect of alanine and asparagine mutations at amino acid residue Asp¹²⁸.

*Inhibition of [³⁵S]GTPγS Binding at WT and Mutant Receptors by the Inverse Agonist ICI174864*—To confirm that the TM III and Tm VII mutations induce constitutive activation of the receptor, we tested whether an inverse agonist could dampen the basal [³⁵S]GTPγS binding activity of mutant receptors (see review in Ref. 18). ICI174864 (19) is the only reported inverse agonist for the δ receptor. This compound was shown to inhibit basal high affinity GTPase activity in membranes of NG108-15 cells (20) or rat-1 fibroblast (21) and HEK 293 (22) cells stably expressing the δ opioid receptor. Similarly, an inhibitory effect of ICI174864 on basal [³⁵S]GTPγS binding to transiently transfected COS cells would be indicative of constitutive activity at mutant receptors.

At WT receptor, ICI174864 had a tendency to decrease basal [³⁵S]GTPγS binding levels (Fig. 4), but this action was only significant for membrane preparations WT II and WT IV. This confirms the weak ligand-independent activity of mDOR transiently expressed in COS cells suggested by our measurements of [³⁵S]GTPγS binding at different receptor densities (see

above). Our data contrast with the high efficacy of ICI174864 for inhibiting constitutive activity in other cell lines expressing the endogenous receptor (50% inhibition of GTPase activity in NG108-15 (20)) or stably expressing the WT cloned receptor (40% inhibition of GTPase or basal [³⁵S]GTPγS binding activity in Rat-1-DOR cells (21); 4-fold increase of forskolin-stimulated cAMP accumulation in HEK 293-DOR cells (22)). The difference could arise from different experimental procedures or from distinct G protein content and receptor/G protein stoichiometry in host cells. The type of Gα subunits present in COS cells, as well as the high receptor/G protein ratio under transient expression conditions, may be less appropriate for efficient coupling to G proteins than in the other systems that have been studied so far. Alternatively, the presence of other proteins or the membrane composition of COS cells may be less favorable to conformational modifications within the WT receptor.

In mutant receptors ICI174864 decreased [³⁵S]GTPγS binding below basal levels but to variable extends depending on the mutant (Fig. 4). ICI174864 markedly reduced [³⁵S]GTPγS binding in mutant receptors with high constitutive activity, namely D128N (36% inhibition) and D128A (51% inhibition). This result therefore provides additional evidence for constitutive activity at Asp¹²⁸ mutant receptors.

For mutant receptors exhibiting lower ligand-independent activity (Y129F, Y129A, and Y308F), the effect of ICI174864 was less evident. Basal levels were moderately diminished for mutations at Tyr¹²⁹, with a significant 19% inhibition at the Y129A mutant. No ICI174864 activity was detectable at Y308F mutant receptor. Hence the analysis of ICI174864 activity at Tyr¹²⁹ and Tyr³⁰⁸ receptors is less conclusive, presumably because of the low constitutive activity of the latter mutants compared with Asp¹²⁸ mutant receptors. ICI174864 activity may also be less well detected in these mutant receptors for another reason: we observed that the ICI174864 compound was not able to decrease [³⁵S]GTPγS binding in mutant receptor preparations to levels as low as in their corresponding WT preparations. Thus, in the presence of ICI174864, [³⁵S]GTPγS binding levels remain 1.3–2.1-fold higher in mutant preparations than in WT preparations (Fig. 4). One explanation could be that although ICI174864 binding to mutant receptors is maintained (not shown), the point mutations introduced into the receptor protein have altered some interactions between ICI174864 and the receptor that are critical for the inverse agonist activity. Alternatively, the active states of the mutant receptors may be structurally different from the active state of the WT receptor, and mutations may have produced receptor conformations that cannot be fully inactivated by an inverse agonist.

*Stimulation of [³⁵S]GTPγS Binding at WT and Mutant Receptors by the Agonist BW373U86*—To measure agonist-induced stimulation of mutant receptors we used the BW373U86 compound because its binding affinity was not dramatically impaired by the mutations under study (Table III) and because it is one of the most potent agonist in our assay system (Fig. 2). We first compared BW373U86-evoked stimulation of [³⁵S]GTPγS binding to mutant and WT receptor membrane preparations at a saturating agonist concentration (Fig. 4). A strong increase of [³⁵S]GTPγS binding levels was observed for all high expressing receptor preparations (WT II, WT III, WT IV, D128N, Y129F, Y129A, and Y308F), clearly indicating that BW373U86 remains a potent agonist at D128N, Y129F, Y129A, and Y308F mutant receptors. Extremely weak agonist-induced activation could be measured for the D128A preparation, as well as its corresponding WT preparation (WT V). Because of the low receptor densities in those membrane preparations, it is difficult to conclude whether
<figure><img src="image_5.png" /><figcaption>FIG. 4. [³⁵S]GTPγS binding at wild-type and mutant receptors. Specific [³⁵S]GTPγS binding at mutant receptor membrane preparations and their wild-type counterparts (Table I) are shown side by side. Membranes were incubated with 0.2 nM [³⁵S]GTPγS and 10 µM GDP for 2 h at 4 °C in the absence (basal) or presence of saturating concentrations of the agonist BW373U86 (10⁻⁵ M) or the inverse agonist ICI174864 (10⁻⁵ M). Membranes from one (D128A) and two to five (others) transfection experiments were used, and results are from three to nine [³⁵S]GTPγS binding experiments performed in triplicate. Within each WT/mutant group, raw data (cpm) were normalized to the value obtained for the WT receptor preparation in the absence of ligand (100%).</figcaption></figure>

<caption>TABLE III
Efficacy and potency of BW373U86 to stimulate [³⁵S]GTPγS binding at D128N, Y129F, Y129A, and Y308F mutant receptors and their WT counterparts (from Fig 5)</caption>

Agonist efficacy is calculated as the maximal difference between specific [³⁵S]GTPγS binding in the presence and absence of agonist and is expressed in cpm. Noticeably, BW373U86 efficacy was increased by 2.7-fold at Y308F mutant receptor. EC₅₀ values are obtained from curve fitting of dose-response curves shown in Fig. 5. Kᵢ values of BW373U86 for the WT and mutant receptors were determined previously and are from Refs. 11 and 12. The effect of mutations on BW373U86 potency are expressed as mutant/WT EC₅₀ ratios and compared with the effect of mutations on binding affinity (mutant/WT Kᵢ ratios).

<table><thead><tr><th rowspan="2"></th><th rowspan="2">n</th><th rowspan="2">Efficacy<br>cpm</th><th rowspan="2">EC<sub>50</sub><br>nM</th><th rowspan="2">K<sub>i</sub><br>nM</th><th colspan="2">Ratios</th></tr><tr><th>EC<sub>50</sub></th><th>K<sub>i</sub></th></tr></thead><tbody><tr><td>D128N</td><td>3</td><td>10984 ± 374</td><td>4.48 ± 0.35</td><td>43.7 ± 12.8</td><td>7.6</td><td>27</td></tr><tr><td>WT II</td><td>3</td><td>9745 ± 169</td><td>0.59 ± 0.27</td><td>1.63 ± 0.58</td><td></td><td></td></tr><tr><td>Y129F</td><td>3</td><td>9944 ± 976</td><td>1.40 ± 0.66</td><td>13.8 ± 2.1</td><td>0.98</td><td>8.5</td></tr><tr><td>WT III</td><td>3</td><td>7401 ± 288</td><td>1.43 ± 0.63</td><td>1.63 ± 0.58</td><td></td><td></td></tr><tr><td>Y129A</td><td>3</td><td>6271 ± 307</td><td>2.83 ± 1.27</td><td>8.63 ± 1.95</td><td>5.1</td><td>5.3</td></tr><tr><td>Y308F</td><td>4</td><td>10935 ± 1654</td><td>1.09 ± 0.75</td><td>13.1 ± 0.02</td><td>2</td><td>7.9</td></tr><tr><td>WT IV</td><td>4</td><td>4098 ± 313</td><td>0.55 ± 0.26</td><td>1.63 ± 0.58</td><td></td><td></td></tr></tbody></table>

BW373U86-induced activation is altered in the D128A mutant receptor. Transferring higher amount of cDNA (up to 20 µg) failed to increase expression levels of the D128A receptor (data not shown). Therefore agonist activity at this mutant receptor could not be investigated further.

Dose-response curves for D128N, Y129F, Y129A, and Y308F mutants are shown in Fig. 5. These data from a representative experiment show both the constitutive activity and the agonist-induced activation at mutant receptors. Indeed, their enhanced agonist-independent activity combined with the efficient BW373U86-induced activation leads to maximal [³⁵S]GTPγS binding levels that largely exceed those of WT receptors. The efficacy of BW373U86 was evaluated as the difference of [³⁵S]GTPγS binding in the presence and absence of agonist and is shown for each mutant receptor and its WT counterpart (Table III). Results show that agonist efficacy was not impaired in any mutant receptor. On the contrary, a tendency to increased efficacy (1.1–1.5-fold) could be detected for Tm III mutants, and a marked augmentation was measured for the Tm VII mutant (2.7-fold).

The potency of BW373U86 to activate [³⁵S]GTPγS binding was determined from the dose response-curves (Fig. 5), and EC₅₀ values are shown in Table III together with the previously

determined affinity values of the agonist for each mutant receptor. For the Y129A receptor, the EC₅₀ value was decreased to the same extent than affinity (5-fold). At the D128N receptor BW373U86 potency was reduced to a lesser extent (7.6-fold) than affinity (27-fold). Finally no significant change in potency was observed for mutants Y129F and Y308F, although BW373U86 affinity was reduced approximately 8-fold at both mutants. These results suggest that, similar to efficacy, the potency of BW373U86 was generally maintained or even enhanced at the mutant receptors.

## DISCUSSION

Here we have investigated both agonist-dependent and agonist-independent activity of point-mutated δ opioid receptors. Our results show that all the structural changes that we have introduced enhance receptor activity. The most noticeable feature is the observation that each mutation significantly augments ligand-independent receptor-mediated [³⁵S]GTPγS binding activity.

Point mutations at adrenergic receptors originally demonstrated that structural modifications of a GPR can trigger agonist-independent activation (23–25). Mutations that produce constitutive activity have since been found for other
FIG. 5. Dose responses for BW-373U86-stimulated [³⁵S]GTPγS binding at D128N, Y129F, Y129A, and Y308F mutant receptors. Increasing concentrations of BW373U86 (10⁻¹³ to 10⁻⁵ M) were used to stimulate [³⁵S]GTPγS binding. Parallel experiments were performed for each mutant, and wild-type receptors were expressed at comparable densities. Representative data from one transfection experiment are shown. Data are the means ± S.E. from three to four independent [³⁵S]GTPγS binding experiments performed in triplicate. BW373U86 efficacy and potency from curve fitting of these data are shown in Table III.

<figure><img src="image_6.png" /></figure>

GPRs, both from site-directed mutagenesis studies and among spontaneous mutations associated with human diseases (for reviews see Refs. 6 and 26). For rhodopsin, adrenergic, muscarinic, thyroid-stimulating hormone, luteinizing hormone, and melanocortin receptors, constitutive activity mostly arises from mutations clustered in Tms II, III, VI, and VII, as well as in the cytoplasmic extensions of Tms III and VI. It has been suggested that these specific receptor domains form a key structure that is central for the activation process and would operate among most GPRs (for reviews see Refs. 5–8). CAM were also identified in other receptor areas, including extracellular loops for thrombin (27) and thyroid-stimulating hormone (28) receptors or the C-terminal tail for two prostaglandin E receptor splice variants (29). This suggests the possible existence of receptor-specific activation mechanisms.

In this study we have generated constitutive activity at the δ receptor by modifying amino acid residues located in the upper portion of putative helices III (Asp<sup>128</sup> and Tyr<sup>129</sup>) and VII (Tyr<sup>308</sup>). CAM localized in these particular receptor regions have been described in other GPRs. Some are found at a position homologous to Asp<sup>128</sup> and were characterized in rhodopsin (Glu<sup>113</sup>; see Refs. 30 and 31) and in the α<sub>1B</sub>-adrenergic receptor (Asp<sup>125</sup>; see Ref. 32). Mutations at vicinal positions have also been shown to enhance receptor basal activity both for the α<sub>1B</sub>-adrenergic receptor (C128F; see Refs. 32 and 33) and the AT1A angiotensin II receptor (N111A; see Ref. 34). Thus our data showing constitutive activity at D128N, D128A, Y129F, and Y129A mutant δ receptors further strengthen the notion that the exofacial portion of Tm III is critical in receptor signaling. CAMs located at a position homologous to Tyr<sup>308</sup> in Tm VII have also been reported. These include mutations at Lys<sup>296</sup> in rhodopsin (30, 31, 35, 36) and Lys<sup>331</sup> in α<sub>1B</sub>-adrenergic receptor (32). Again our finding of an enhanced, although modest, basal activity at the Y308F mutant is consistent with findings at other GPRs. Altogether our data suggest that receptor domains that have been shown to be critical for the activation process in previously studied GPRs are also important in opioid receptors.

The initial finding that replacement of Ala<sup>293</sup> in the α₁B-adrenergic receptor by any other amino acid residue increases agonist-independent activity, has led to the notion that a number of critical intramolecular interactions would maintain the receptor under an inactive conformation (23). In this hypothesis, agonists would act by disrupting these conformational constraints and induce a “relaxed” receptor state that in turn would productively interact with G proteins. Compelling evi-

dence has now accumulated, from a wide variety of methodological approaches, to show the importance of interhelical interactions in the allosteric transition between inactive and active receptor states (for a recent review see Ref. 8). On the basis of the current hypothesis, it is likely that the three amino acid residues of the δ receptor that we have studied may be part of the stabilizing interhelical network that keeps the receptor inactive. Our results show that conservative mutations in Tm III (D128N and Y129F) and Tm VII (Y308F) trigger constitutive activation of the receptor. Those structural modifications may alter (D128N) or suppress (D128A, Y129F, and Y308F) one hydrogen bond within the helical bundle, and this may be sufficient to enhance conformational flexibility, allowing the receptor to undergo transition from inactive to active state more easily. Further, it seems that the alanine substitution at Tyr<sup>129</sup> produces a slightly higher constitutive activation than does the conservative phenylalanine substitution (Fig. 4). We therefore cannot exclude the possibility that the benzene ring of Tyr<sup>129</sup> also participates in interhelical stabilization through aromatic-aromatic interaction, as previously suggested (12, 37).

Possible interacting partners of Asp<sup>128</sup>, Tyr<sup>129</sup>, and Tyr<sup>308</sup> can be hypothesized based on computer three-dimensional modeling. Most models that have been proposed for the δ opioid receptor poorly describe the empty receptor and mainly identify amino acid residues that could potentially interact with opioid ligands (38–46). From these models Asp<sup>128</sup>, Tyr<sup>129</sup>, or Tyr<sup>308</sup> could establish direct hydrogen bonding with a number of agonists, including BW373U86 (Asp<sup>128</sup> and Tyr<sup>129</sup>; see Ref. 38; Asp<sup>128</sup>; see Footnote 2). On the assumption that agonist binding induces receptor activation by disrupting pre-existing interhelical interactions, those amino acid residues are good candidates for participating in the structural stabilization of the unliganded receptor. We have further investigated possible Tm-Tm interactions in the absence of ligand using the model of Yue *et al.* (43). The model indicates that Asp<sup>128</sup> in Tm III and Tyr<sup>308</sup> in Tm VII could directly interact with each other and be critical in stabilizing the entire helical core (Fig. 6). A similar interaction may exist in other GPRs. For rhodopsin, it has been suggested that a salt bridge between the Tm III glutamic acid residue (Glu<sup>113</sup>), and the Tm VII lysine bond visual chromophore 11-cis-retinal (Lys<sup>296</sup>) would help maintain the constrained inactive conformation of the native protein (30, 31). In

<footnote n="2">² G. Poda and B. Maigret, personal communication.</footnote>
<figure><img src="image_7.png" /><figcaption>FIG. 6. A molecular model of the unliganded δ opioid receptor. The figure shows a view from the membrane of a three-dimensional model of the empty δ opioid receptor (43). The seven-trans-membrane domain helical backbone is in green. Side chains of Asp<sup>128</sup> (Tm III, red), Tyr<sup>129</sup> (Tm III, yellow), His<sup>278</sup> (Tm VI, blue), and Tyr<sup>308</sup> (Tm VII, yellow) residues are highlighted.</figcaption></figure>

the α₁B-adrenergic receptor, the Tm III residue homologous to Asp¹²⁸ has been proposed to serve as a counterion for the Tm VII residue homologous to Tyr³⁰⁸, and disruption of this bond was postulated to initiate receptor activation (32). There is therefore good indication that Tm III and Tm VII are located in close proximity and also that the existence of a Asp¹²⁸-Tyr³⁰⁸ interaction could play a role in the activation process. In the model by Yue *et al*. (Fig. 6) Tyr¹²⁹, the other Tm III residue that we have studied, forms a hydrogen bond with His²⁷⁸ in Tm VI. The same interhelical hydrogen bond has also been hypothesized in the three-dimensional model proposed by Poda and Maigret (46). This specific Tm III-Tm VI interaction may be opioid-receptor specific because no homologous interhelical interaction has been reported for other GPRs. In summary, we propose that Asp¹²⁸ and Tyr¹²⁹ interact with Tyr³⁰⁸ and His²⁷⁸, respectively, within the unliganded receptor. The mutations would mimic the ligand action by altering those interhelical interactions, thereby facilitating receptor isomerization into an active conformation and/or its interaction with G proteins.

The originally proposed ternary agonist-receptor-G protein complex theory (47), as well as the now commonly accepted allosteric ternary complex model (24), predict that agonist binding increases binding affinity of the G protein for the receptor and *vice versa*. As a consequence, CAM receptors presumably imitate the active state of the receptor and are likely to bind agonist ligands with higher affinity, the so-called “high affinity state.” In accordance with this hypothesis, most previously reported CAM GPRs exhibit enhanced affinity for agonists (23, 24, 32, 48, 49). Our data are not concordant with these findings. In a previous report, we have shown that agonist binding is diminished in D128N, Y129F, Y129A, and Y308F mutant δ opioid receptors, and we have suggested that this may be due to the loss of appropriate interactions in the binding pocket (11, 12). Because we have modified amino acid residues that seem to be involved both in ligand recognition and in the activation process, one cannot exclude that a putative gain of affinity associated with constitutive activity may be

masked by the loss of binding interactions between the ligand and the receptor.

Further, this observation is not necessarily in opposition with the data obtained at other GPRs. Receptors undergo several processes that include agonist binding, receptor isomerization between active and inactive conformations, and G protein association or dissociation. The relationship between these different steps is complex, the extent to which they are interconnected most certainly depends on the receptor under study and its cellular environment. Each step could be differently influenced by chemical modifications of the receptor (for a review see Ref. 5), and accumulating data now tend to demonstrate increasing diversity in the properties of CAM receptors. For example, the extent of agonist-induced stimulation appears highly variable, depending on the receptor and the mutation under study. As for the Y308F mutant receptor investigated here, agonist-induced stimulation was augmented in a CAM AT1A angiotensin II mutant receptor (34), but agonist efficacy was shown diminished for CAM α₁B-adrenergic (23), luteinizing hormone 32 (50), and VIP (51) receptors or abolished in CAM thyrotropin (48) and prostaglandin EP3 (29) receptors. This suggests that the active conformations of agonist bond receptors may differ widely from one CAM to another, as well as from that of their wild-type counterparts. Both the localization and nature of the mutation certainly are critical to explain the pharmacological properties of a CAM receptor. Our mutations modify amino acid residues that are involved in at least two steps, namely ligand binding and receptor isomerization. The mutations may have suppressed a number of anchor points for the agonist, as well as Tm-Tm hydrogen bonds, impairing both the ligand recognition process and the stability of the inactive conformation. Full agonist-induced G protein activation, however, is preserved. Therefore, modifications of Asp¹²⁸, Tyr¹²⁹, and Tyr³⁰⁸ side chains may have modified the ligand-receptor complex and the receptor itself as well as the receptor-G protein complex, but none of these mutations prevents productive interactions within the ligand-receptor-G protein ternary complex.
The activity profile of the Y308F mutant receptor (Tm VII) differed to some extent from that of the Tm III mutant receptors. Most remarkably, Y308F responded significantly better to agonist stimulation. Currently the BW373U86-induced hyperactivation remains unexplained. The finding that Asp¹²⁸ mutants behave differently from the Y308F receptor suggests that breakage of the proposed Asp¹²⁸-Tyr³⁰⁸ hydrogen bond is not the only event induced by the site-directed mutagenesis. In addition to the hypothesized direct interaction, Asp¹²⁸ and Tyr³⁰⁸ could also be involved in other interactions that, in turn, would implicate them differently in the activation process. The role of amino acid residues homologous to Tyr³⁰⁸ has been investigated in other GPRs, by examining agonist-induced responses in mutant receptors. In the angiotensin AT1 receptor, substitution of the equivalent tyrosine (Tyr²⁹²) by a phenylalanine did not modify agonist and antagonist peptide binding but impaired angiotensin II-stimulated inositol triphosphate hydrolysis (52). A different result was obtained for the m3 muscarinic receptor, where a tyrosine is also found at this position. Mutation into phenylalanine (Y533F) clearly reduced agonist affinity, but maximal stimulation of inositol triphosphate hydrolysis was comparable with that of the WT receptor (53, 54). Similar results were obtained for the homologous residue of adenosine receptor (His²⁷⁸), in which substitution with several amino acid residues abolished high affinity binding of agonist and antagonist, whereas the cAMP production was comparable with that of the WT receptor in the H278A mutant (55, 56). Therefore our finding that the Y308F mutation reduces opioid binding affinity parallels what was observed for muscarinic and adenosine receptors. We also found constitutive activity at the Y308F receptor, as described previously for homologous mutations in rhodopsine and adrenergic receptors (see above), but the enhanced agonist-induced stimulation in Y308F receptor contrasts with findings at angiotensin AT1A, m3, and adenosine mutant receptors. We conclude that there is no obvious common role for this Tm VII amino acid residue. The distinct effect of homologous substitutions at different GPRs suggests the existence of receptor-specific mechanisms at the level of Tm VII, presumably because of distinct structural characteristics of this specific area of the helical bundle.

In conclusion CAMs have already proven to be useful tools to explore activation mechanisms of GPRs. We anticipate that the further exploration of constitutive activity in opioid receptors will help to clarify the general mechanisms of GPRs activation and also highlight opioid receptor-specific processes that accompany receptor signaling.

*Acknowledgments*—We thank G. Poda and B. Maigret for the three-dimensional modeling work at the δ receptor and fruitful comments. We are also grateful to T. Groblewski for helpful discussion and Astra Pain Control for collaborative support. We thank C. Evans and P. Zaki for critical reading of the manuscript, and finally, we especially thank F. Pattus for continuous encouragement.

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