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CELLULAR AND MOLECULAR

µ- Opioid Receptor Functional Interaction: Insight Using Receptor-G Protein Fusions

Département de Neurobiologie, Unité Mixte de Recherche 7104, Institut de Génétique et Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, Illkirch, France (L.A.S., B.L.K., D.M.); and Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom (G.M.)

Received January 11, 2006; accepted May 2, 2006.

	Abstract

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Fusion proteins between a receptor and a pertussis toxin-insensitive G_i subunit were used to gain insight into the molecular interactions that take place upon µ and opioid receptor heterodimerization. When µ opioid receptor-G_i1 fusions were coexpressed with nonfused opioid receptors in human embryonic kidney 293 cells, or vice versa, receptor heterodimers were detected by coimmunoprecipitation. In pertussis toxin-treated cells, receptor coexpression decreased the amount of guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) incorporated in the fused G protein after the addition of agonists specific for the receptor-G_i1 fusion. In addition, activation of the G protein occurred in heterodimers upon addition of an agonist specific for the nonfused receptor. It remained unaffected by an inverse agonist specific for the receptor-G_i1 fusion. These data suggest that signaling through the receptor-G_i1 fusion protein is impaired in heterodimers and support a mechanism in which activation of the G subunit is promoted by a direct interaction with the nonfused receptor. Alternatively, receptor coexpression did not modify the ligand binding properties for the high-affinity state of the receptor-G_i1 fusion nor the EC₅₀ values for agonist-induced [³⁵S]GTPS incorporation in the G_i1 subunit. In addition, no binding competition was observed between and µ ligands. Together, the data point to µ- opioid receptor heterodimers formed by contact interactions between monomers that retain their structural integrity.

GPCR signaling is a complex process whose modulation likely proceeds from many intricate factors. Recently, a possible involvement of the receptor oligomerization state has received growing attention since existence of homo- and heterodimers has been postulated for an increasing number of GPCRs (for reviews, see Milligan, 2004; Park et al., 2004; Prinster et al., 2005). Arguments in favor of a spatial proximity between receptor molecules mainly arose from coimmunoprecipitation studies and more recently from bioluminescence and fluorescence resonance energy transfer experiments (for review, see Milligan and Bouvier, 2005). However, establishing physical proximity between receptors does not necessarily imply functional interactions between them. Even when modifications of the signaling properties seemed correlated with receptor colocalization, the underlying mechanisms remained largely unknown (Gomes et al., 2004).

Heterodimerization between µ and opioid receptor types has already been reported (George et al., 2000; Gomes et al., 2000) and seems of particular interest because it may modulate opioid analgesia (Gomes et al., 2004). In this context, receptor-G protein fusions may help to elucidate the molecular interactions taking place when the two receptor types are coexpressed. The µ and opioid receptors have previously been fused to a pertussis toxin-insensitive mutant G subunit from the inhibitory G_i/o family. As a result, signaling through the fusion can be isolated from the cellular background, and the extent of activation of the mutant G can be measured in pertussis toxin-treated cells (Moon et al., 2001; Massotte et al., 2002).

In this work, we compared ligand binding properties and activation profiles of the pertussis toxin-insensitive G_i1 subunit when µ or opioid receptor-G fusions are expressed alone or coexpressed with wild-type or µ opioid receptors, respectively. Our data indicate that heterodimers are formed between µ and opioid receptors that retain their structural integrity. These µ- heterodimers seem to signal through a mechanism that involves a direct interaction between the nonfused receptor and the G subunit.

	Materials and Methods

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Materials
[³H]Diprenorphine (50 Ci/mmol) and [³H]naltrindole (35 Ci/mmol) were purchased from Amersham (Arlington Heights, IL), and [³⁵S]GTPS (1250 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Pertussis toxin and ligands were from Sigma-Aldrich (St. Louis, MO). Tic-deltorphin was obtained by solid-phase synthesis carried out on an Applied Biosystems 433A peptide synthesizer by using 9-fluorenylmethoxycarbonyl chemistry. N,N-(CH3)₂-Dmt-Tic-NH₂ was synthesized as reported in Salvadori et al. (1999). All materials for tissue culture were supplied by Invitrogen (Paisley, UK). Jet-PEI was from Polyplus-transfection (Illkirch, France). Anti-G_i1 M2 monoclonal antibodies were from NeoMarkers (Fremont, CA), and anti-FLAG polyclonal antibodies were from Sigma-Aldrich. Horseradish peroxidase-conjugated anti-mouse [F(ab')₂] fragments and ECL⁺ detection kit were from GE Healthcare (Chicago, IL).

Generation of µ and Opioid-C351I G_i1 Fusion Constructs and Cell Culture. Generation of the human µ opioid receptor fusion constructs to the pertussis toxin-insensitive G_i1 (hMOR-C351I G_i1) and generation of the fusion construct between the human opioid receptor and the pertussis toxin-insensitive G_i1 (hDOR-C351I G_i1) were described previously (Moon et al., 2001; Massotte et al., 2002). pcDNA plasmids encoding the Signal-FLAG-DOR and -MOR constructs were a generous gift from Dr. C. J. Evans (University of California, Los Angeles, CA). HEK 293 cells were maintained in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum and 2 mM glutamine. Stable cell lines expressing hMOR or hMOR-C351I G_i1 were generated previously (Massotte et al., 2002) and maintained in the presence of 100 µg/ml geneticin. Upon coexpression of the receptors, cells were transfected with Jet-PEI reagent according to the manufacturer's recommendations and collected after 48 h. Pertussis toxin treatment was performed for 20 h at a concentration of 100 ng/ml before cells were harvested.

Preparation of Membranes. Cells were collected, washed twice with phosphate-buffered saline, and stored at -80°C in phosphate-buffered saline containing 320 mM sucrose. Cell pellets were resuspended in ice-cold 50 mM Tris-HCl, 1 mM EDTA, pH 7.4; disrupted using a glass homogenizer; and centrifuged at 2000g for 10 min. The pellet was homogenized in ice-cold 50 mM Tris-HCl, 1 mM EDTA, pH 7.4, and centrifuged at 1000g for 5 min. Both supernatants were combined and ultracentrifuged at 100,000g for 40 min at 4°C. The pellet was resuspended in 50 mM Tris-HCl, 1 mM EDTA, 320 mM sucrose, pH 7.4, and then homogenized through a 26-gauge needle and stored in aliquots at -80°C before use.

Saturation and Competition Analysis. For each assay, 10 µg of membrane protein was incubated in 50 mM Tris-HCl, pH 7.4, with the appropriate ligands in a final volume of 500 µl for 30 min at 22°C. For saturation experiments [³H]diprenorphine was used in a 0.05 to 6.4 nM range. When µ and opioid receptors were coexpressed, [³H]naltrindole in a 0.05 to 4 nM range was used to determine the amount of receptors present. In all cases, naloxone was used at 2 µM to determine nonspecific binding. For all competition experiments, [³H]diprenorphine was used at 1 nM and the competing ligand in a 10^-5 to 10^-13 M range. In both cases, incubation was terminated by rapid filtration on GF/B microplate filters treated with 0.1% (v/v) polyethylenimine followed by three washes with ice-cold 50 mM Tris-HCl, pH 7.4, using a Filtermate Harvester (PerkinElmer Life and Analytical Sciences). Bound radioactivity was determined by scintillation counting. Scatchard and competition analyses were performed using Prism software (GraphPad Software Inc., San Diego, CA).

[³⁵S]GTPS Binding Studies. Stock [³⁵S]GTPS was diluted to 50 nM in 10 mM Tricine, pH 7.4, and 10 mM dithiothreitol. Aliquots were stored at -80°C. For each assay, 10 µg of membrane protein was incubated in 50 mM HEPES, pH 7.4, 5 mM MgCl₂, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% (w/v) bovine serum albumin, 10 µM GDP, 0.1 nM [³⁵S]GTPS, and the ligand, in a final volume of 200 µl for 2 h at 4°C. Nonspecific binding was determined in the presence of 10 µM GTPS, and basal binding was assessed in the absence of ligand. Incubation was terminated by rapid filtration on H₂O-presoaked GF/B filters followed by three washes with ice-cold 50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, and 50 mM NaCl using a Filtermate harvester. Bound radioactivity was determined by scintillation counting. EC₅₀ values were determined using Prism software.

Coimmunoprecipitation and Western Blot Analysis. Membrane preparations (500 µg) were solubilized in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10% CHAPS, complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) for 1 h at 4°C, immunoprecipitated with either 2 µg of anti-G_i1 or 2.5 µg of anti-FLAG antibodies for 1 h at 4°C, and isolated by incubation with G protein-Sepharose for 1 h at 4°C. Samples were washed three times with 50 mM Tris-HCl, pH 7.4; heated in loading buffer [62.5 mM Tris-HCl, pH 6.8, 5% (w/v) -mercaptoethanol, 2% (w/v) SDS, 10% (v/v) glycerol, and 0.1% (w/v) bromphenol blue] for 5 min at 95°C; and loaded onto a 8% SDS-polyacrylamide electrophoresis gel. Proteins were transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA). Following blocking in 5% (w/v) nonfat dry milk in 50 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.5% (v/v) Tween 20 (Tris-buffered saline/Tween 20; TBST) for 1 h, PVDF membranes were incubated overnight at 4°C with a 1:100 dilution of G_i1 antibody. PVDF membranes were washed three times for 10 min with 5% (w/v) nonfat dry milk in TBST, incubated for 2 h with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-mouse (Fab₂') fragment antibody in 5% (w/v) nonfat dry milk in TBST. PVDF membranes were washed three times for 10 min in TBST. Chemiluminescence was detected using ECL⁺ according to the manufacturer's instructions.

Statistical Analysis. Unless otherwise stated, data represent three independent experiments performed in duplicate (K_i values) or in triplicate ([³⁵S]GTPS). Data are presented as the mean ± S.E.M. Statistical analysis was performed using a two-way analysis of variance test.

	Results

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Coimmunoprecipitation. Coimmunoprecipitation has been previously used to demonstrate physical proximity between coexpressed µ and opioid receptors and hence to establish the existence of heterodimers (George et al., 2000; Gomes et al., 2000). Coimmunoprecipitation experiments were then performed to confirm µ- physical proximity under our conditions.

Membranes from cells coexpressing the opioid receptor fused to a pertussis toxin-insensitive G protein (hDOR-C351I G_i1) with a FLAG-tagged µ opioid receptor (FLAG-MOR) or membranes from cells coexpressing the µ opioid receptor fused to a pertussis toxin-insensitive G protein (hMOR-C351I G_i1) with a FLAG-tagged opioid receptor (FLAG-DOR) were subjected to coimmunoprecipitation with a polyclonal anti-FLAG antibody. In both cases, detection using a monoclonal anti-G_i1 antibody revealed a band with a molecular mass of approximately 200 kDa (Fig. 1, a, lane 1, and b, lane 1, respectively). This band likely corresponds to a dimer of receptor-G protein fusion and was also detected by Western blot analysis when hDOR-C351I G_i1 or hMOR-C351I G_i1 fusions were immunoprecipitated using the anti-G_i1 antibody (Fig. 1, a, lane 5, and b, lane 3) (Massotte et al., 2002). Detection of such homodimeric forms suggests that µ and opioid receptors may be present in the cell as part of oligomeric complexes. Whether such high-order association results from direct specific interactions, is promoted by scaffolding proteins or simply results from the high density of receptors within given lipid microdomains remains to be established.

View larger version (36K): [in this window] [in a new window]   Fig. 1. The µ- opioid heterodimers can be detected by coimmunoprecipitation. a, membranes were prepared from cells transiently coexpressing FLAG-MOR and hDOR-C351I Gi1 (lane 1), from cells transiently expressing FLAG-MOR alone (lane 2) or from cells transiently expressing hDOR-C351I Gi1 alone (lane 3). In addition, a mixture of membranes expressing each construct separately was used as a control (lane 4). Membranes were solubilized, subjected to immunoprecipitation with a polyclonal anti-FLAG antibody, and immunoblotted with a monoclonal anti-Gi1 antibody. As a control, membranes prepared from cells transiently expressing hDOR-C351I Gi1 alone (lane 5) or mock-transfected (lane 6) were solubilized, subjected to immunoprecipitation, and immunoblotted with a monoclonal anti-Gi1 antibody. b, membranes prepared from cells transiently coexpressing FLAG-DOR and hMOR-C351I Gi1 (lane 1), or a mixture of membranes expressing each construct separately (lane 2) were solubilized, subjected to immunoprecipitation with a polyclonal anti-FLAG antibody and immunoblotted with a monoclonal anti-Gi1 antibody. As a control membranes prepared from cells expressing hMOR-C351I Gi1 alone (lane 3) or mock-transfected (lane 4) were solubilized, subjected to immunoprecipitation, and immunoblotted with a monoclonal anti-Gi1 antibody.

Similar opioid receptor dimers were detected earlier following immunoprecipitation on solubilized membranes coexpressing µ and opioid receptors (George et al., 2000; Gomes et al., 2000). Also dimers were observed when opioid receptors were coexpressed with kappa opioid or ₂-adrenergic receptors (Jordan and Devi, 1999; McVey et al., 2001) or when µ opioid receptors were expressed with _2A-adrenergic, sst2 somatostatin or neurokinin NK1 receptors (Pfeiffer et al., 2002, 2003; Jordan et al., 2003).

We verified the specificity of the µ- interaction by control experiments that included immunoprecipitation assays from membranes expressing either the receptor-G protein fusion alone, the nonfused FLAG-tagged receptor alone or a mixture of membranes expressing either the receptor-G protein fusion or the nonfused FLAG-tagged receptor alone. For these latter samples, PVDF membranes were stripped and reprobed with the monoclonal M2 anti-FLAG antibody to verify immunoprecipitation efficiency (data not shown).

Effect of Receptor Coexpression on High-Affinity Ligand Binding Sites. Coexpression of µ and opioid receptors was achieved by transfecting one type of receptor in cells stably expressing the other type. One receptor type was expressed as a receptor-G fusion together with a nonfused receptor molecule of the other type. In most experiments, the molar ratio between fused and nonfused receptors was of 1:1, but modifying this ratio to 2:1 in favor of any of the partners had no noticeable effect. To avoid possible cross-reactivity when the two opioid receptor types were coexpressed, we chose ligands with nanomolar affinities and high selectivity for either or µ opioid receptors.

The agonist DAMGO showed similar nanomolar affinity values and high selectivity for the fused (hMOR-C351I G_i1) and wild-type (hMOR) µ opioid receptors under our conditions (Table 1). Likewise, the agonists deltorphin II and SNC80 exhibited nanomolar affinities and high selectivity for the receptor in fusion (hDOR-C351I G_i1) or not (hDOR) (Table 1). Coexpression of hMOR-C351I G_i1 together with hDOR or coexpression of hDOR-C351I G_i1 together with hMOR did not significantly modify high-affinity K_i values for all ligands tested. These data suggest that the integrity of the binding sites was not altered upon receptor coexpression.

View this table: [in this window] [in a new window]   TABLE 1 Binding affinities for hDOR and hMOR expressed in HEK 293 cells alone or together with the fusion constructs to the pertussis-insensitive C351I Gi1 subunit (hDOR/Gi1 and hMOR/Gi1, respectively) Competition experiments were as described under Materials and Methods. Data are given as a mean ± S.E.M. from at least three independent experiments performed in duplicate.

Effect of Coexpression on Agonist-Induced Activation of the Receptor-G Fusion. Incorporation of the nonhydrolyzable GTP-analog [³⁵S]GTPS was used to monitor the impact of coexpression on agonist-induced stimulation of the fusion constructs. Indeed, cell treatment with pertussis toxin prevented [³⁵S]GTPS incorporation in endogenous G_i/o proteins and restricted agonist-induced activation to the G of the fusion construct (Massotte et al., 2002).

To avoid possible cross-reactivity when the two opioid receptor types were coexpressed, [³⁵S]GTPS experiments were performed at ligand concentrations that ensured their selectivity for one type of opioid receptor over the other (Table 1). When hMOR-C351I G_i1 was coexpressed together with hDOR, [³⁵S]GTPS incorporation was significantly decreased following the addition of 100 nM of the µ agonist DAMGO compared with hMOR-C351I G_i1 expressed alone. The decrease in activation of the fused G_i1 protein was similar when hDOR was transfected in cells stably expressing the hMOR-C351I G_i1 fusion (185 ± 16 versus 236 ± 14%; p < 0.05), or, conversely, when hMOR-C351I G_i1 was transfected in cells stably expressing hDOR (182 ± 12 versus 236 ± 14%; p < 0.05) (Fig. 2a). It should be noted that G protein activation was similar whether hMOR-C351I G_i1 was stably or transiently expressed (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Coexpression of a nonfused receptor affects signaling through the receptor-G fusion. a, [35S]GTPS incorporation induced by the µ agonist DAMGO (100 nM). Membranes were prepared from cells stably expressing hMOR-C351I Gi1 alone (black column), from cells stably expressing hMOR-C351I Gi1 and transfected with hDOR (striped column), or from cells stably expressing hDOR and transfected with hMOR-C351I Gi1 (gray column). b, [35S]GTPS incorporation induced by the agonists deltorphin II (100 nM) and SNC80 (10 nM). Membranes were prepared from cells stably expressing hDOR-C351I Gi1 alone (black column) or from cells stably expressing hMOR and transfected with hDOR-C351I Gi1 (striped column). Cells were treated with 100 ng/ml pertussis toxin for 20 h. Data are represented as mean ± S.E.M. The n value is indicated in each column. *, p < 0.05; ***, p < 0.001 compared with the fusion expressed alone. A schematic representation of the receptor pairs used in the experiment is represented above each panel.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Representative dose-response curves for agonist stimulated [35S]GTPS incorporation in the fused G subunit. a, DAMGO at hMOR-C351I Gi1 expressed alone () or together with hDOR (). hMOR-C351I Gi1 was stably expressed and hDOR was transiently expressed. Expression levels were, respectively, 1.1 and 1.4 pmol/mg hMOR-C351I Gi1 expressed alone or together with hDOR and 2.4 pmol/mg for hDOR. b, SNC80 at hDOR-C351I Gi1 expressed alone () or together with hMOR(). hDOR-C351I Gi1 was transiently expressed, and hMOR was stably expressed. Expression levels were, respectively, 1.2 and 1.7 pmol/mg for hDOR-C351I Gi1 expressed alone or together with hMOR and 1.8 pmol/mg for hMOR. Cells were treated with 100 ng/ml pertussis toxin for 20 h. Data are represented as the mean from one representative experiment. A schematic representation of the receptor pairs used in the experiment is represented above each panel.

View this table: [in this window] [in a new window]   TABLE 2 Efficacy and potency of opioid agonists for the stimulation of [35S]GTPS binding in HEK 293 cells expressing hDOR or hMOR alone or together with hMOR-C351I Gi1 or hDOR-C351I Gi1, respectively Agonist efficacy was calculated as the maximal difference between [35S]GTPS binding in the presence and absence of agonist and is expressed as a percentage of basal. EC50 values were obtained from curve fitting of dose-response curves. Statistical analysis was performed using the two-way analysis of variance test. The asterisks (**, p < 0.01; ***, p < 0.001) refer to values significantly different from corresponding values for the receptor in fusion expressed alone.

Likewise, [³⁵S]GTPS incorporation after the addition of agonists was significantly reduced in the hDOR-C351I G_i1 fusion upon hMOR coexpression. Addition of 100 nM deltorphin II led to only 199 ± 16% activation over basal compared with 333 ± 22% when the fusion was expressed alone (p < 0.001; Fig. 2b). Stimulation by 10 nM SNC80 resulted in 168 ± 9% activation compared with 291 ± 18% when the fusion was expressed alone (p < 0.001). Dose-response curves using SNC80 also indicated a marked reduction of the maximal activation when hMOR was coexpressed with hDOR-C351I G_i1 (Fig. 3b; Table 2). As observed in hMOR-C351I G_i1, EC₅₀ values remained unchanged upon coexpression, suggesting once more that coexpression does not affect the structural integrity of the receptors (Table 2).

Expression levels for all constructs were typically 1 to 3 pmol/mg membrane protein and did not significantly vary upon coexpression. In addition, we verified that receptor expression levels and G protein activation were both similar whether cells stably expressing one receptor type were mock-transfected or not (data not shown). Therefore, differences observed between E_max values cannot be attributed to changes in receptor expression levels.

G Subunit Activation by Nonfused Coexpressed Receptors. We next examined whether the G subunit could be activated by coexpressed nonfused receptors. [³⁵S]GTPS incorporation was measured upon addition of an agonist at a concentration that ensured specificity for the nonfused receptor type. When hMOR-C351I G_i1 was coexpressed together with wild-type hDOR, the G subunit from the fusion was activated following 10 nM SNC80 or 100 nM deltorphin II addition (131 ± 7 and 132 ± 5%, respectively; p < 0.01) (Fig. 4a). In a control experiment, membranes from cells expressing hMOR-C351I G_i1 alone were mixed with membranes from cells expressing wild-type hDOR alone. No activation of the G subunit was observed upon deltorphin II (89 ± 2%) or SNC80 (94 ± 7%) addition (Fig. 4a). As expected, 100 nM deltorphin II or 10 nM SNC80 did not promote [³⁵S]GTPS incorporation in membranes from cells expressing hMOR-C351I G_i1 alone (94 ± 7 and 104 ± 5%, respectively) (Fig. 4a).

View larger version (29K): [in this window] [in a new window]   Fig. 4. Gsubunit activation by nonfused coexpressed receptors. a, [35S]GTPS incorporation was measured upon stimulation by the agonists deltorphin II (100 nM) or SNC80 (10 nM) on membranes prepared from cells stably expressing hMOR-C351I Gi1 alone (black column), membranes from cells stably expressing hDOR and transfected with hMOR-C351I Gi1 (striped column) or a mix of membranes individually stably expressing hDOR or hMOR-C351I Gi1 (gray column). b, [35S]GTPS incorporation was measured upon stimulation by the µ agonist DAMGO (100 nM) on membranes prepared from cells transiently expressing hDOR-C351I Gi1 alone (black column), membranes from cells stably expressing hMOR and transfected with hDOR-C351I Gi1 (striped column), or a mix of membranes individually expressing hMOR or hDOR-C351I Gi1 (gray column). Cells were treated with 100 ng/ml pertussis toxin for 20 h. Data are represented as mean ± S.E.M. The n value is indicated in each column. **, p < 0.01; ***, p < 0.001 compared with the fusion expressed alone. A schematic representation of the receptor pairs used in the experiment is represented above each panel.

These results indicate that activation of the G protein by the nontethered receptor takes place. Upon coexpression, spatial proximity between the nonfused receptor and the receptor-G fusion is required for activation of the G subunit by the nonfused receptor.

Modulation of G Activation by the Presence of an Antagonist or an Inverse Agonist Specific for the Fusion Protein. We then tested whether blocking the receptor of the receptor-G fusion would prevent G protein activation by the nonfused receptor. CTAP has been described previously as a µ opioid receptor antagonist (Sterious and Walker, 2003). When hMOR-C351I G_i1 was coexpressed together with hDOR, 100 nM CTAP did not activate the G protein fused to the µ opioid receptor (101 ± 4%) and was able to efficiently antagonize 100 nM DAMGO activation (107 ± 6 versus 185 ± 16%; p < 0.01) (Fig. 5a). However, 100 nM CTAP could not block [³⁵S]GTPS incorporation into the G subunit promoted by the agonists deltorphin II (143 ± 5 versus 132 ± 5% for 100 nM deltorphin II alone) or SNC80 (133 ± 10 versus 131 ± 7% for 10 nM SNC80 alone) (Fig. 5a).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Modulation of G activation by an antagonist or an inverse agonist specific for the receptor-G fusion. a, [35S]GTPS incorporation was measured upon stimulation by the agonists deltorphin II (100 nM) and SNC80 (10 nM) or the µ agonist DAMGO (100 nM) alone (striped columns) or in the presence of the antagonist CTAP (100 nM) (black columns) on membranes prepared from cells stably expressing hMOR-C351I Gi1 and transfected with hDOR. b, [35S]GTPS incorporation was measured upon stimulation by the agonists deltorphin II (100 nM) and SNC80 (10 nM) or the µ agonist DAMGO (100 nM) alone (striped columns), in the presence of the antagonist Tic-deltorphin (100 nM) (black columns), or in the presence of the inverse agonist N,N-(CH3)2-Dmt-Tic-NH2 (100 nM) (gray columns) on membranes prepared from cells stably expressing hMOR and transfected with hDOR-C351I Gi1. Cells were treated with 100 ng/ml pertussis toxin for 20 h. Data are represented as mean ± S.E.M. The n value is indicated in each column. **, p < 0.01; ***, p < 0.001 different from basal. ##, p < 0.01 effect of antagonist or inverse agonist. A schematic representation of the receptor pairs used in the experiment is represented above each panel.

Antagonists bind with similar affinity to both active and inactive forms of receptors, whereas inverse agonists are thought to favor inactive conformations (Strange, 2002). Recently, N,N-(CH3)₂-Dmt-Tic-NH₂ has been characterized as an inverse agonist with nanomolar affinity for the opioid receptor (Tryoen-Toth et al., 2005). Similarly to the antagonist Tic-deltorphin, N,N-(CH3)₂-Dmt-Tic-NH₂ was able to efficiently reverse [³⁵S]GTPS incorporation induced by 100 nM deltorphin II (109 ± 5 versus 199 ± 16; p < 0.001) or 10 nM SNC80 (110 ± 9 versus 168 ± 9%; p < 0.001) but could not reverse DAMGO-induced G protein activation (126 ± 5 versus 132 ± 3% for DAMGO alone) (Fig. 5b).

Therefore, an active conformation of the receptor in fusion does not seem required for G protein activation (see Discussion). In addition, data suggested that and µ ligands did not compete with each other for the same binding site but would rather bind to separate nonoverlapping binding pockets. This supports the hypothesis that the receptors retained their structural integrity upon coexpression as already inferred from the absence of changes in K_i and EC₅₀ values.

	Discussion

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An increasing number of GPCRs have been reported to form heterodimers with altered signaling and trafficking properties (Park et al., 2004). In this context, µ and opioid receptors seem of particular interest since heterodimers formed between these two receptors may modulate the response to opioids (Gomes et al., 2004). Therefore, we investigated the molecular interactions that lead to the activation of the first step of the signaling cascade when the two receptors are coexpressed.

Coexpression was achieved by transfecting one type of receptor in cells stably expressing the other type. This provided virtually identical cellular backgrounds that restricted the observed changes to the impact of coexpression. Coimmunoprecipitation experiments indicated that at least some µ and receptors are spatially close enough to allow heterodimerization to take place (Fig. 1). To address the link between receptor physical proximity and functional response, receptors were expressed either as receptor-mutant G fusions or as nonfused receptors. After cell treatment with pertussis toxin, only activation of the fusion protein is visualized. Ligands with nanomolar affinity and high selectivity were chosen to ensure specificity of the receptor activation. These experimental conditions placed the focus on heterodimers and their interactions with G proteins.

Under our conditions, receptor coexpression did not affect K_i values for high-affinity binding sites (Table 1), whereas altered affinities were reported for some ligands upon µ- opioid receptor coexpression in COS-7 cells (George et al., 2000). However a recent study clearly established that heterodimerization did not affect agonist binding affinities when the coexpressed partner was mutated in the third intracellular loop and therefore unable to couple to a G protein (Law et al., 2005). In heterodimers, agonist affinities at one receptor seem thus affected by the coupling state of the other receptor. In our case, one receptor was expressed as a receptor-G fusion together with a nonfused receptor. Cells were treated with pertussis toxin, and the nonfused receptor partner was therefore uncoupled from endogenous G proteins. Therefore, values for the high-affinity binding site of the receptor-G fusion remained unaffected by heterodimerization. Interestingly, unmodified ligand affinity and selectivity were also reported for heterodimers between an opioid receptor and a nonopioid one (Jordan et al., 2001; McVey et al., 2001; Pfeiffer et al., 2002, 2003; Chen et al., 2004).

Heterodimer formation may involve domain swapping between monomers (Dean et al., 2001). Alternatively, it may arise from a clinging process between monomers that establish tight contacts but retain their physical integrity as proposed for the vasopressin V2 receptor (Schulz et al., 2000). Under our conditions, both K_i and EC₅₀ values were not modified upon µ- receptor coexpression. In addition, the extent of G protein activation by nonfused receptors was not affected by the presence of antagonists specific for the receptor in fusion, suggesting no competition for a shared common receptor binding site. Those data argue against a model involving a domain exchange between receptors but rather support a tight contact association between monomers. This view is corroborated by mutagenesis studies that identified multiple determinants for ligand binding and specificity throughout the transmembrane core of the opioid receptors. Hence, a domain exchange between two different receptor types would affect the distribution of such determinants and therefore ligand binding affinities (Befort et al., 1996; Chavkin et al., 2001). Also expression of the opioid receptor did not alter µ opioid receptor trafficking making unlikely a chimeric heterodimer (Law et al., 2005).

View larger version (24K): [in this window] [in a new window]   Fig. 6. Models to depict receptor-G protein interactions in µ- opioid heterodimers. A receptor in fusion with a G subunit is coexpressed with a nonfused receptor to form a heterodimer. I, activation of one receptor affects the conformation of the other, which in turn activates its fused G subunit located in cis via intramolecular interactions. II, the length of the linker allows the G subunit of the fusion construct to be directly activated by the nonfused receptor as a result of their spatial proximity. Our experimental data best support this configuration. III, within the heterodimer, the fused G protein is only able to interact with the nonfused receptor. Activation of one receptor affects the conformation of the other, but the G subunit is no longer available to be activated by the fused receptor. The activation pathway is indicated by the arrow.

When an agonist specific for the nonfused receptor was added, significant activation of the G subunit in fusion was observed. Interestingly, results obtained using coexpression of hMOR with the opioid receptor-G fusion were the mirror image of those obtained when hDOR was coexpressed with the µ opioid receptor-G fusion (Fig. 4). This suggests that the nonfused receptor is able to promote activation of the G subunit tethered to the other receptor. Using a similar paradigm, Molinari et al. (2003) also detected G protein activation when a nonfunctional fusion between the opioid receptor and a G_s subunit was coexpressed with a nonfused ₂-adrenergic receptor.

Several configurations can be proposed to depict the mechanism of G protein activation in µ- heterodimers formed upon coexpression of a receptor-G fusion with a nonfused receptor (Fig. 6). Under our conditions, we observed a decrease in the amount of agonist-induced [³⁵S]GTPS incorporated in the G subunit of the fusion protein upon coexpression with a nonfused receptor (Figs. 2 and 3). Since the total number of receptor-G fusions is unchanged this suggests impaired signaling. This observation would support a mechanism in which the G subunit interacts physically with the nonfused receptor (Fig. 6, configuration II). In a similar approach, Carillo et al. (2003) reported that signaling was negatively affected by heterodimerization between an inactive form of the _1b adrenergic receptor and a histamine H1 receptor-G₁₁ fusion (Carrillo et al., 2003). Interestingly, the expression of increasing amounts of the inactive _1b adrenergic receptor further decreased signaling. This also led the authors to postulate that the histamine H1 receptor was unable to activate the G protein physically associated with itself.

Inverse agonists stabilize the receptor in an inactive state (Strange, 2002). Very few inverse agonists have been described for the opioid receptors. Among them, ICI 174864 serves as a reference but has a low affinity for the opioid receptor (Costa and Herz, 1989). Recently, N,N(CH3)₂-Dmt-Tic-NH₂ was identified as another potent -specific inverse agonist with nanomolar affinity (Tryoen-Toth et al., 2005). Under our conditions, N,N(CH3)₂-Dmt-Tic-NH₂ efficiently reversed agonist induced G protein activation through the receptor in fusion as expected (Fig. 5b). Alternatively, it had no effect on activation by the nonfused receptor as also observed with neutral antagonists (Fig. 5). These data support again a model in which the 19 amino acid linker of the fusion protein is indeed long enough to allow the G subunit some degree of motion resulting in G subunit interaction by the nonfused receptor (Fig. 6, configuration II).

An alternative mechanism can also be postulated by which activation of the nonfused receptor induces a conformational change that is transmitted from the active receptor to the G subunit located in trans via the transmembrane domains of the receptor in fusion (Fig. 6, configuration I). In such a configuration, decreased G activation of the fusion construct would also be observed upon coexpression with a nonfused receptor. However, this model is not supported by inverse agonist data (Fig. 5). According to such an allosteric mechanism, G protein activation should then be prevented by an inverse agonist of the fused receptor that would constrain it in an inactive state.

Likewise, no G protein activation would be observed if the G subunit from the fusion construct was physically interacting with the nonfused receptor and G protein activation would occur via an allosteric process. In this case, the conformational change resulting from the activation of the nonfused receptor would be transferred to a neighbor, uncoupled receptor (Fig. 6, configuration III).

In summary, we propose that coexpressed µ and opioid receptors are able to form heterodimers by contact interactions between receptors retaining their structural integrity. Our results also indicate that the resulting heterodimers are functional. Further investigation is now required to examine a possible impact on in vivo signaling.

	Acknowledgements

 
We thank Pascal Eberling for Tic-deltorphin synthesis and Drs. Katia Befort and Larry Toll for critical reading and helpful discussions.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.101220.

ABBREVIATIONS: GPCR, G protein-coupled receptor; Tic, 1,2,3,4-tetrahydroisoquinoline; GTPS, guanosine 5'-O-(3-thio)triphosphate; DOR, opioid receptor; MOR, µ opioid receptor; HEK, human embryonic kidney; h, human; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate; PVDF, polyvinylidene difluoride; TBST, Tris-buffered saline/Tween 20; DAMGO, [D-Ala², N-Me-Phe⁴,Gly⁵-ol]-enkephalin; SNC80, (+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; TM, transmembrane; Tic-deltorphin, H-Tyr-Tic-Phe-Phe-Val-Val-Gly-NH₂; ICI 174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; CTAP, D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH₂.

¹ Current affiliation: Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1.

Address correspondence to: Dr. Dominique Massotte, Unité Mixte de Recherche 7104, Institut de Génétique et Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries BP 10142, F-67404 Illkirch cedex, France. E-mail: massotte{at}igbmc.u-strasbg.fr

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