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Simultaneous Activation of the Opioid Receptor (OR)/Sensory Neuron-Specific Receptor-4 (SNSR-4) Hetero-Oligomer by the Mixed Bivalent Agonist Bovine Adrenal Medulla Peptide 22 Activates SNSR-4 but Inhibits OR Signaling

Département de Biochimie et Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Québec, Canada (A.B., M.L., M.B.); and Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York (K.G., L.A.D.)

Received January 24, 2006; accepted May 8, 2006

	Abstract

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Hetero-oligomerization among G protein-coupled receptors has been proposed to contribute to signal integration. Because sensory neuron-specific receptors (SNSRs) and the opioid receptors (OR) share a common ligand, the bovine adrenal medulla peptide (BAM) 22, and have opposite effects on pain modulation, we investigated the possible consequences of OR/SNSR-4 hetero-oligomerization on the signaling properties of both receptor subtypes. Bioluminescence resonance energy transfer revealed that the human OR has similar propensity to homo-oligomerize and to form hetero-oligomers with human SNSR-4 when coexpressed in human embryonic kidney 293 cells. The hetero-oligomerization leads to a receptor form displaying unique functional properties. Individual activation of either OR or SNSR-4 in cells coexpressing the two receptors led to the modulation of their respective signaling pathways; inhibition of adenylyl cyclase and activation of phospholipase C, respectively. In contrast, the OR/SNSR-4 bivalent agonist BAM22, which could activate each receptor expressed individually, fully activated the SNSR-4-dependent phospholipase C but did not promote OR-mediated inhibition of adenylyl cyclase in OR/SNSR-4-coexpressing cells. Likewise, concomitant activation of the OR/SNSR-4 hetero-oligomer by selective OR and SNSR-4 agonists promoted SNSR-4 but not OR signaling, revealing an agonist-dependent dominant-negative effect of SNSR-4 on OR signaling. Furthermore, the OR selective antagonist naltrexone trans-inhibited the SNSR-4-promoted phospholipase C activation mediated by BAM22 but not by the SNSR-4-selective agonists, suggesting a bivalent binding mode of BAM22 to the OR/SNSR-4 hetero-oligomer. The observation that BAM22 inhibited the Leu-enkephalin-promoted cAMP inhibition in rat dorsal root ganglia neurons supports the potential physiological implication of such regulatory mechanism.

In addition to their ligand-sharing properties, the overlapping localization of SNSR-4 and OR within the same dorsal root ganglia compartments (lamina I and II) raises the possibility of functional interactions between the two receptors. This is particularly relevant when considering their opposite roles in pain perception (SNSR-4 being pronociceptive and OR antinociceptive) (Puttfarcken et al., 1988; Grazzini et al., 2004).

Interactions between subtypes of the same or even among distinct GPCR families have been described at different molecular levels. Receptor-oligomerization (Salahpour et al., 2000) as well as heterologous cross-talk between signaling pathways (Salahpour et al., 2000) have been shown to contribute to signal integration. Direct receptor-receptor interactions between the ₂-adrenergic receptor (Jordan et al., 2001), the µOR (George et al., 2000; Martin and Prather, 2001), or the OR (Jordan and Devi, 1999) have been shown to alter agonist-promoted receptor endocytosis or ligand binding properties of the OR. Signaling efficacy of the OR can also be regulated by a heterologous cross-talk with signaling pathways activated by other receptors or drugs. For example, specific activation of protein kinase C by phorbol ester has been shown to induce phosphorylation of the OR, which in turn results in the desensitization and internalization of the receptor (Xiang et al., 2001).

To date, no functional interaction between members of the OR and SNSR families have been reported. The present study was therefore initiated to examine the possibility that OR and SNSR-4 may interact when expressed in the same cells. Here, using bioluminescence resonance energy transfer (BRET) approaches, we report that the OR exhibits similar propensity to homo-oligomerize as to form hetero-oligomers with SNSR-4. The functional consequences of such hetero-oligomerization were then characterized in HEK293 cells stably expressing the human OR and SNSR-4. We found that SNSR-4 signaling promoted by the mixed, bivalent agonist BAM22 could be trans-inhibited by the OR-selective antagonist naltrexone. Moreover, simultaneous activation of the OR and SNSR-4 by BAM22 or by various combinations of selective agonists for each receptor dramatically decreased the efficacy of OR agonist to inhibit adenylyl cyclase activity, suggesting a dominant-negative effect of SNSR-4 on the OR activity. Together, these results demonstrate that hetero-oligomerization between OR and SNSR-4 generates a novel signaling unit with a unique pharmacological profile that may contribute to the signal integration involved in pain perception.

	Materials and Methods

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Materials. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, penicillin/streptomycin, L-glutamine, and G418 (Geneticin) were obtained from Wisent Inc. (St-Bruno, QC, Canada). Neurobasal medium, B27 supplement, L-glutamine, trypsin HEPES buffer, and Zeocin were purchased from Invitrogen (Carlsbad, CA). 3-Isobutyl-1-methylxanthine (IBMX), Leu-enkephalin (Leu-enk), naltrexone, and [D-Pen²,D-Pen⁵]enkephalin (DPDPE) were purchased from Sigma-Aldrich (St. Louis, MO). FuGENE 6 was obtained from Roche Diagnostics (Laval, QC, Canada). Coelenterazine H was from Molecular Probes (Eugene, OR), whereas DeepBlueC coelenterazine, [³H]adenine, [myo-³H]inositol, [³H]naloxone, and [³H]DPDPE were from PerkinElmer Life and Analytical Sciences (Boston, MA). cAMP antiserum and ¹²⁵I-cAMP were purchased from Biomedical Technologies (Stoughton, MA). Nerve growth factor was purchased from Harlan (Madison, WI). BAM22 was purchased from the American Peptide Co., Inc. (Sunnyvale, CA), whereas BAM8-22 was from Tocris Cookson, Inc. (Ellisville, MO). BAM1-12 and BAM2-22 were kindly provided by the AstraZeneca Research Center (Montréal, QC, Canada).

Eukaryotic Expression Vectors. For BRET experiments, expression vectors encoding the human OR and SNSR-4 fused to either the green fluorescent protein (GFP²) (where GFP² is a variant of the wild-type GFP containing the following mutations: P64L, Y100F, S108T, M141L, S147P, S202F, I219V, and H213L) or Renilla reniformis luciferase (Rluc) were generated as follows: PCR fragments containing the entire coding sequences excluding the stop codons of the human OR or of the human SNSR-4 were subcloned into the humanized pGFP²-N2 vector (PerkinElmer Life and Analytical Sciences) in a way that fused the 3' end of the receptors onto the 5' end of the GFP²-cDNA using the restriction sites HinDIII and BamHI. This resulted in an in frame fusion of the receptors with GFP² separated by a seven-amino acids linker. The pcDNA3.1-OR-Rluc cDNA was obtained by subcloning a PCR fragment containing the human OR into the pcDNA3.1-Rluc vector using the restriction sites HinDIII and BamHI in a way that fused the receptor sequence onto the 5' end of Rluc. This resulted in an in frame fusion of the receptor with Rluc separated by a 4-amino acid linker. The pcDNA3.1-SNSR-4-Rluc cDNA containing the human SNSR-4 fused to the 5' end of Rluc with a 3-amino acid linker was kindly provided by AstraZeneca Research Center. The pcDNA3.1 GABA_BR1b-Rluc construct was generated as described previously (Villemure et al., 2005).

The cell lines stably expressing OR, SNSR-4, or the two receptors together were generated using the following cDNAs: The pcDNA4-SNSR-4 was created by subcloning a PCR fragment containing the entire coding sequence, including the stop codon of the human SNSR-4 into the pcDNA4 vector (Invitrogen) using the EcoRI and EcoRV restriction sites. The pcDNA3.1-OR-FLAG construct was generated as described previously (Petaja-Repo et al., 2000).

Cell Culture and Transfection. HEK293 cells were cultured in DMEM supplemented with 5% fetal bovine serum and 2 nM L-glutamine. For transient expression of recombinant proteins, cells were seeded at a density of 2 x 10⁵ cells in a six-well dish, cultured for 24 h, and then transfected with the appropriate vectors using FuGENE 6 reagent according to the manufacturer's protocol. Forty-eight hours after transfection, cells were washed twice with phosphate-buffered saline, detached with 5 mM EDTA in phosphate-buffered saline, and used immediately. HEK293 cell clones stably expressing human SNSR-4 (HEK293-SNSR-4) were obtained by selecting cells (50 µg/ml zeocin) transfected with the pcDNA4-SNSR-4 construct. The expression of SNSR-4 was controlled by measuring BAM22-induced accumulation of total phosphoinositols (IPn). HEK293 cells stably expressing the OR-FLAG fusion protein (HEK293-OR) were obtained as described previously (Petaja-Repo et al., 2000) by transfection of the pcDNA3.1-OR-FLAG construct and selection with 400 µg/ml G418. To obtain HEK293 cells stably coexpressing SNSR-4 and the human OR (HEK293-OR/SNSR-4), a cell clone already expressing the OR was transfected with the pcDNA4-SNSR-4 construct. After selection with 50 µg/ml zeocin, in the presence of 250 µg/ml G418, cell clones were isolated and coexpression of OR-FLAG and SNSR-4 monitored by ligand binding and BAM22-promoted IPn accumulation, respectively.

Dorsal Root Ganglia Cell Culture and Stimulations. Dorsal root ganglia were dissected from embryonic day 16 embryos of Long-Evans rats. Cells were obtained by mechanical dissociation in 0.25% trypsin and plated on poly-L-lysine/laminin coated 24-well plates (3 ganglia/well). Cells were grown in "culturing medium" consisting of neurobasal medium supplemented with B27, L-glutamine, and nerve growth factor. For the selection of neurons, cultures were cycled between culturing medium and medium containing fluorodeoxyuridine/uridine every 2 days for the fist week of culture. Two-week-old cultures were used for the study. Cells were washed with fresh neurobasal medium containing 20 mM HEPES, 0.1% BSA, and 250 µM IBMX. Cells were stimulated for 30 min at 37°C in the same buffer containing 20 µM forskolin along with the indicated agonists. The reaction was terminated by removing the medium and adding ice-cold 5% trichloroacetic acid to the cells. After 30-min incubation at 4°C, the solution was neutralized by the addition of 50 mM sodium acetate buffer, pH 6.2.

BRET Measurement. To monitor receptor-receptor interactions in living cells, BRET² assays were performed using a TopCount NXT (customized version purchased from BioSignal Packard, Inc., now PerkinElmer Life and Analytical Sciences) as described previously (Mercier et al., 2002). In brief, after catalytic degradation of the substrate DeepBlueC by the energy donor Rluc, light is emitted with a peak at 400 nm. The energy acceptor GFP² is excited by nonradiative energy transfer if GFP is located within a distance of less than 100 Å from the energy donor. As a result, fluorescence is re-emitted by GFP with a peak at 510 nm. The ratio of the light intensity emitted at 500 to 530 nm over 370 to 450 nm is defined as the BRET² signal. BRET signals were then corrected by subtracting the BRET background detected in cells expressing only the energy donor fusion protein. The expression level of the energy donor (Rluc) or acceptor (GFP) was controlled by measuring total luminescence and fluorescence for each BRET experiment, as described previously (Mercier et al., 2002). To increase the relative amount of OR fusion proteins expressed at the cell surface, we took advantage of the pharmacological chaperone effect of hydrophobic OR antagonist (Petaja-Repo et al., 2002) by treating OR-Rluc- or OR-GFP²-expressing cells with 1 µM naltrexone for 12 to 16 h before harvesting and washing the cells.

Radioligand Binding Assay. For ligand binding studies, 100,000 cells were seeded in 24-well dishes, coated with 0.1% poly-D-lysine. After 24 h, specific binding of [³H]naloxone (5-10 nM) or [³H]DPDPE (5 nM) was determined in the presence of increasing concentrations of the competing ligands. These radioligand binding assays were carried out on attached cells for 2 h on ice in DMEM with 100 mM HEPES, pH 7.4, and 0.1% of BSA. The amount of cell-bound radioactivity was measured after washing the cells twice with 2 ml of ice-cold binding buffer and lysing them with 0.1% SDS and 0.1 N NaOH.

Internalization Assay. To induce endocytosis of the OR, cells were stimulated at 37°C for 30 min with 1 µM BAM22 or Leu-enkephalin in DMEM containing 100 mM HEPES and 0.1% BSA. Cells were then washed extensively, and the ligands were allowed to dissociate for 10 min on ice in 2 ml of binding buffer. Receptor sequestration was measured by detecting both total and cell surface binding using 50 nM [³H]naloxone. Specific cell surface binding of [³H]naloxone was defined as the binding inhibited by 1 µM DPDPE, a hydrophilic ligand, whereas total specific binding sites were defined as the binding inhibited by 10 µM naltrexone, a hydrophobic ligand. The amount of total specific binding sites of untreated and agonist pretreated cells was similar, indicative of complete dissociation of the agonist used to induce receptor endocytosis. Receptor sequestration is given as the percentage of receptor binding sites that were no longer accessible by the hydrophilic ligand DPDPE after agonist stimulation.

cAMP Accumulation. To determine OR agonist-promoted inhibition of forskolin-induced cAMP accumulation in HEK293 cells, 200,000 cells were seeded in 12-well dishes coated with 0.1% poly-D-lysine 24 h before the experiment and labeled for 2 to 4 h in serum-free DMEM containing 2 µCi/ml [³H]adenine. Cells were stimulated for 30 min at 37°C in DMEM containing 100 mM HEPES, pH 7.4, 2.5 µM IBMX, 0.1% BSA, and 20 µM forskolin along with the different drugs at the indicated concentrations. The reaction was terminated by removing the medium and adding ice-cold 5% trichloroacetic acid to the cells. [³H]cAMP was then purified by sequential chromatography over DOWEX resin and aluminum oxide, and the cAMP accumulation was expressed as the total amount of [³H]cAMP in picomoles per hour per well. cAMP Radioimmunoassay. For dorsal root ganglia cells, cAMP levels were measured by incubating samples with anti-cAMP anti-serum and ¹²⁵I-cAMP in sodium acetate buffer at 4°C for 18 h and separating the antibody-bound radioligand by precipitation with 17.5% polyethylene glycol as described previously (Gomes et al., 2003). The radioactivity in the pellet was measured in a gamma counter (PerkinElmer Life and Analytical Sciences).

IPn Production. To determine the accumulation of IPn, 10⁵ cells/well were seeded 48 h before the experiment in 24-well dishes coated with 0.1% poly-D-lysine and labeled for 24 h with 1 µCi/ml [myo-³H]inositol. IPn production was then measured after stimulation of the cells for 1 h at 37 °C with the indicated concentrations of the various drugs in DMEM containing 100 mM HEPES, pH 7.4, 0.1% BSA, and 20 mM LiCl. Total IPn was isolated by chromatography over DOWEX (OH^-) as described previously (Berridge et al., 1983) and expressed as -fold over basal of nonstimulated cells.

Data Analysis. Data obtained in binding experiments and cAMP or IPn accumulation assays were analyzed using Prism 3.0 (Graph-Pad Software, San Diego, CA). Isotherms were plotted for one or two binding sites; the best fit was then used to calculate K_i or EC₅₀ values. Data obtained in the BRET titration experiments were plotted using a single binding site saturation isotherm. Statistical significance of the differences was assessed by two-tailed Student's t test.

	Results

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Hetero-Oligomerization between the OR and the SNSR-4 Monitored by BRET². The BRET technique has been successfully used to monitor receptor-receptor interactions in living cells (Angers et al., 2000; McVey et al., 2001; Ayoub et al., 2002). To determine whether the OR and the SNSR-4 can form hetero-oligomers, we fused both receptors on their C terminus to Rluc (energy donor) or GFP² (energy acceptor) and applied the BRET² technology. We took advantage of the quantitative features of BRET² and performed BRET titration experiments to determined BRET₅₀ values for both homo-oligomers and the OR/SNSR-4 hetero-oligomer. It has been proposed that BRET₅₀ values reflect the relative affinity of a receptor-receptor complex (Mercier et al., 2002) and therefore allow to compare the propensity of a receptor to form homo- or hetero-oligomeric complexes. As shown in Fig. 1, BRET titration curves for both homo- and hetero-oligomers were best fitted to a hyperbolic function, indicating the saturation of the energy donor by the energy acceptor as a result of specific interactions of the proteins fused to the BRET pair. In BRET titration experiments between the unrelated GABA_BR1-Rluc fusion protein and the SNSR-4-GFP fusion protein, no detectable BRET signal was observed (Fig. 1), providing further evidence for the specific interactions between the SNSR-4-Rluc or OR-Rluc fusion protein and the SNSR-4-GFP. To characterize the potential functional consequences of such hetero-oligomeric complexes between OR and SNSR-4, HEK293 cell lines stably expressing each of the receptor individually (HEK293-OR and HEK293-SNSR-4) or coexpressing the two receptors (HEK293-OR/SNSR-4) were generated. To limit the differences between cell lines, the HEK293-OR/SNSR-4 cell line was obtained by transfection of the SNSR-4 construct into the HEK293-OR cells used in the study (see Materials and Methods).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Detection of OR/SNSR-4 hetero-oligomers by BRET2. A constant cDNA amount of the energy donor OR-Rluc, SNSR-4-Rluc, or GABABR1-Rluc and increasing amounts of the energy acceptor OR-GFP2 or SNSR-4-GFP2 were transiently coexpressed in HEK293 cells, and 48 h after transfection, BRET signals were measured after the addition of the luciferase substrate DeepBlueC as described under Materials and Methods. BRET signals are plotted as a function of the expression ratio of receptor-GFP2 over receptor-Rluc (x100) obtained by measuring the total luminescence and fluorescence in each condition. The expression level of the receptor-Rluc fusion protein was not significantly affected (±20%) by the coexpression of the receptor-GFP2 fusion proteins. The BRET titration curves were drawn from data obtained in three independent experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Effects of SNSR-4 coexpression on agonist-promoted OR endocytosis. HEK293-OR and HEK293-OR/SNSR-4 cells were incubated with 1 µM Leu-enkephalin or BAM22 for 30 min at 37°C. After treatment with the corresponding drugs, cell surface receptors were quantified by assessing the proportion of [3H]naloxone (50 nM) binding sites accessible to the hydrophilic ligand DPDPE and are expressed on the y-axis as the percentage of total receptor number. Numbers above the columns indicate the extent of endocytosis in percentage after treatment with the corresponding drug. Results are expressed as the mean ± S.E. of three independent experiments carried out in triplicates. Asterisk indicates a significant (p < 0.05) difference of the OR endocytosis observed in OR/SNSR-4-versus OR-expressing cells.

Signaling Properties of the OR/SNSR-4 Hetero-Oligomer. Several studies have recently suggested the occurrence of receptor trans-activation within receptor hetero-oligomers (Galvez et al., 2001; Lee et al., 2002; Carrillo et al., 2003). Based on the distinct ligand binding profile and different G protein coupling selectivity of OR (binding of Leu-enkephalin and BAM1-12 and coupling to G_i/o that leads to adenylyl cyclase inhibition) and SNSR-4 (binding of BAM2-22 and BAM8-22 and coupling to G_q that promotes phospholipase C activation), the OR/SNSR-4 hetero-oligomer represents an excellent system to consider potential trans-activation. Hence, the ability of OR and SNSR-4 ligands to activate G_i/o and G_q was assessed in HEK293-OR, HEK293-OR/SNSR-4, and HEK293-SNSR-4 cells. As shown in Fig. 3A and as expected from the previously established selectivity of the compounds, saturating concentrations of the SNSR-4-selective agonists BAM2-22 and BAM8-22 activate IPn production in HEK293-SNSR-4 and HEK293-OR/SNSR-4 but not in the HEK293-OR cells. Saturating concentrations of the OR-selective agonists Leu-enkephalin and BAM1-12 inhibit forskolin-induced cAMP accumulation in HEK293-OR and HEK293-OR/SNSR-4 but not in HEK293-SNSR-4 cells (Fig. 3B). Furthermore, selective OR agonists Leu-enkephalin and BAM1-12 did not promote any IPn production in HEK293-OR cells (Fig. 3A), and the SNSR-4 agonists BAM8-22 and BAM2-22 did not inhibit forskolin-stimulated cAMP accumulation in HEK293-SNSR-4 cells (Fig. 3B), confirming the selective coupling of the OR to G_i/o and of the SNSR-4 to G_q. When considering cells expressing the OR/SNSR-4 hetero-oligomer, no evidence of receptor trans-activation was observed, and each receptor acted as an independent signaling unit, coupling exclusively to its cognate G protein upon selective activation. Indeed, as shown in Fig. 3A, the OR-selective agonists Leu-enkephalin and BAM1-12 did not activate the phospholipase C pathway in HEK293-OR/SNSR-4 cells. Likewise, no inhibition of the adenylyl cyclase activity could be promoted by the SNSR-4-selective agonists BAM8-22 and BAM2-22 in these cells (Fig. 3B).

View larger version (26K): [in this window] [in a new window]   Fig. 3. Signaling properties of OR/SNSR-4 hetero-oligomeric complexes. A, HEK293-SNSR-4, HEK293-OR, and HEK293-OR/SNSR-4 cells were stimulated for 60 min at 37°C with 1 µM concentrations of the SNSR-4-specific agonists BAM2-22 or BAM8-22 or the OR-specific agonists Leu-enk or BAM1-12. Phospholipase C activity was assessed by measuring the accumulation of [3H]IPn in cells prelabeled with [myo-3H]inositol and expressed as -fold over basal of non-stimulated cells. B, HEK293-SNSR-4, HEK293-OR, and HEK293-OR/SNSR-4 cells were stimulated for 30 min at 37°C with 20 µM forskolin and 1 µM concentrations of the SNSR-4-specific agonists BAM2-22 and BAM8-22 or the OR-specific agonists Leu-enk and BAM1-12. Adenylyl cyclase activity was assessed by measuring the accumulation of [3H]cAMP in cells prelabeled with [3H]adenine and expressed as the percentage of the agonist-promoted inhibition of forskolin-induced cAMP accumulation. Results are expressed as the mean ± S.E. of three independent experiments carried out in triplicates.

Trans-Inhibition of SNSR-4 by the OR Antagonist Naltrexone. Naltrexone is a well established OR antagonist (Portoghese, 1993) that has previously been shown not to affect SNSR-mediated signaling (Lembo et al., 2002; Kirchmayer et al., 2003). To determine whether naltrexone could affect signaling of the OR/SNSR-4 hetero-oligomer, the effect of naltrexone was assessed on the BAM22-stimulated IPn production in HEK293-SNSR-4 and HEK293-OR/SNSR-4 cells. In line with a previous report (Lembo et al., 2002) and consistent with the notion that naltrexone does not bind SNSR-4, the dose-response curve of BAM22-stimulated IPn production in HEK293-SNSR-4 cells was unaffected by a cotreatment with naltrexone (Fig. 4A, left). In contrast, the efficacy of BAM22 to stimulate IPn production was clearly reduced in HEK293-OR/SNSR-4 cells when naltrexone was coadministered (Fig. 4A, right). Given that the activation of OR in HEK293-OR cells did not promote any detectable IPn production (Fig. 3A), these data indicate that naltrexone acted as an antagonist on the OR/SNSR-4-promoted IPn production stimulated by BAM22. The fact that naltrexone did not completely block the SNSR-4-promoted IPn production suggests either that naltrexone only partially blocks the hetero-oligomer activation by SNSR agonists or that a significant fraction of SNSR homo-oligomer is present in HEK293-OR/SNSR-4 cells. This later possibility is consistent with the finding that SNSR-4 seems to have greater propensity to form homo-than hetero-oligomers (Fig. 1). It is noteworthy that this trans-inhibition of the SNSR-4 signaling by naltrexone within the OR/SNSR-4 hetero-oligomer was restricted to the mixed, bivalent agonist BAM22. Indeed, the naltrexone treatment had no effect on the IPn production elicited by the SNSR-4-selective agonists BAM2-22 or BAM8-22, in HEK293-OR/SNSR-4 cells (Fig. 4B). These data suggest that coexpression of OR and SNSR-4 promotes a BAM22-mediated SNSR-4 signaling (IPn production) that is sensitive to the OR antagonist naltrexone, most likely as a result of hetero-oligomerization between the two receptors and the interaction of BAM22 with the two protomers. Because no selective SNSR-4 antagonist has been identified yet, the possible reciprocal antagonism of the OR-mediated inhibition of adenylyl cyclase by blockade of the SNSR-4 receptor could not be tested.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Naltrexone-sensitivity of BAM22-promoted production of IPn in HEK293-OR/SNSR-4 cells. A, HEK293-SNSR-4 and HEK293-OR/SNSR-4 cells were stimulated for 60 min at 37°C with increasing concentrations of BAM22 in the absence (open symbols) or presence (filled symbols) of 10 µM naltrexone. B, HEK293-SNSR-4 and HEK293-OR/SNSR-4 cells were stimulated with 1 µM BAM22, BAM2-22, or BAM8-22 in the presence or absence of 10 µM naltrexone. Phospholipase C activity was assessed as in Fig. 3 and expressed as -fold over basal of nonstimulated cells (A) or as the percentage of naltrexone-induced inhibition of agonist-promoted IPn production (B). Results are expressed as the mean ± S.E. of three independent experiments carried out in triplicates. Asterisk indicates a significant (p < 0.05) difference of the BAM22-promoted IPn accumulation in naltrexone-treated versus nontreated HEK293-OR/SNSR-4 cells.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Coactivation of the OR/SNSR-4 hetero-oligomer promotes phospholipase C activity but does not inhibit adenylyl cyclase activity. A, HEK293-OR and HEK293-OR/SNSR-4 cells were stimulated for 30 min at 37°C with 20 µM forskolin and 1 µM concentrations of the mixed, bivalent agonist BAM22 or the OR-specific agonists DPDPE, Leu-enk, or BAM1-12, in the absence (white columns) or presence of 1 µM concentrations of the SNSR-4-specific agonists BAM8-22 (gray columns) or BAM2-22 (black columns). Adenylyl cyclase activity was assessed as in Fig. 3 and expressed as picomoles of cAMP produced per hour per well. Results are expressed as the mean ± S.E. of three independent experiments carried out in triplicates. B, HEK293-SNSR-4 and HEK293-OR/SNSR-4 cells were stimulated for 60 min at 37°C with 1 µM concentrations of the mixed, bivalent agonist BAM22 or SNSR-4-selective agonist BAM8-22 in the absence (white columns) or presence of 10 µM concentrations of the OR-specific agonists DPDPE (gray columns) or Leu-enk (black columns). Phospholipase C activity was assessed as in Fig. 3 and expressed as -fold over basal of nonstimulated cells. Results are expressed as the mean ± S.E. of three independent experiments carried out in triplicates. Asterisk in A indicates a significant (p < 0.05) difference between single and coactivated receptors in HEK293-OR/SNSR-4 cells.

It is noteworthy that this trans-inhibition of the OR signaling by SNSR-4 agonists was found to be unidirectional because simultaneous activation of both receptors did not affect the SNSR-4-mediated production of IPn in HEK293-OR/SNSR-4 cells (Fig. 5B). Together, these findings suggest that simultaneous agonist binding to the two protomers of the OR/SNSR-4 hetero-oligomer leads to the exclusive SNSR-4-promoted G_q activation, to the expense of the OR-activated G_i/o.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Binding and signaling properties of the OR/SNSR-4 hetero-oligomer. A, competition binding studies with the OR-specific antagonist [3H]naloxone (10 nM) were performed on attached cells as described under Materials and Methods, and Ki values were calculated. HEK293-OR cells: Leu-enkephalin, 4.4 ± 0.3 nM; DPDPE, 3.2 ± 0.2 nM; and BAM1-12, 5.2 ± 0.4 nM. HEK293-OR/SNSR-4 cells: Leu-enkephalin, 4.2 ± 0.4 nM; DPDPE, 3.4 ± 0.2 nM; and BAM1-12, 5.1 ± 0.3 nM. Binding curves represent the mean ± S.E. of three independent experiments performed in duplicates. B, HEK293-OR and HEK293-OR/SNSR-4 cells were stimulated for 30 min at 37°C with 20 µM forskolin and increasing concentrations of the OR-specific agonists Leu-enkephalin, DPDPE, or BAM1-12. Adenylyl cyclase activity was assessed as in Fig. 3 and expressed as the percentage of the maximal accumulation induced by forskolin in the absence of any agonist. EC50 values are as follows. HEK293-OR cells: Leu-enkephalin, 8.6 ± 0.4 nM; DPDPE, 2.2 ± 0.2 nM; and BAM1-12, 4.8 ± 0.4 nM. HEK293-OR/SNSR-4 cells: Leu-enkephalin, 64.1 ± 1.4 nM; DPDPE, 25.5 ± 0.9 nM; and BAM1-12, 56.7 ± 2.3 nM. Dose-response curves represent the mean ± S.E. of three independent experiments performed in duplicates.

Selective SNSR-4 and OR Agonists Can Bind Simultaneously to the OR/SNSR-4 Hetero-Oligomer. The dramatically reduced efficacy of OR agonists in the presence of SNSR-4 agonists observed for HEK293-OR/SNSR-4 cells define SNSR-4 agonists as functional antagonists of OR within the OR/SNSR-4 hetero-oligomer. To determine whether this antagonism resulted from an inhibition of OR agonist binding to the oligomer, the ability of the selective SNSR-4 agonists BAM2-22 and BAM8-22 to inhibit [³H]DPDPE binding to HEK293-OR/SNSR-4 cells was assessed. As illustrated in Fig. 7, neither BAM2-22 nor BAM8-22 inhibited the binding of [³H]DPDPE. This contrasts with the potent inhibition of the [³H]DPDPE binding by the mixed OR/SNSR-4 agonist BAM22 and indicates that both OR and SNSR-4 protomers can bind their selective agonists simultaneously.

View larger version (13K): [in this window] [in a new window]   Fig. 7. SNSR-4-specific agonists do not inhibit [3H]DPDPE binding to HEK293-OR/SNSR-4 cells. Competition binding studies using the OR-specific agonist [3H]DPDPE (5 nM) and increasing concentrations of the mixed, bivalent agonist BAM22 or the SNSR-4-selective agonists BAM8-22 and BAM2-22 were performed on attached HEK293-OR/SNSR-4 cells as described under Materials and Methods. Results represent the mean ± S.E. of three independent experiments performed in duplicates.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Effects of protein kinase C activity on OR-promoted inhibition of adenylyl cyclase activity. HEK293-OR or HEK293-OR/SNSR-4 cells were stimulated for 30 min at 37°C with 20 µM forskolin and 1 µM concentrations of the various BAM peptides (white columns), costimulated with 50 µM concentrations of the protein kinase C-specific activator PMA (gray columns), or pretreated for 30 min at 37°C with 500 nM concentrations of the protein kinase C specific inhibitor bisindolylmaleimide I (BIM; black columns). Adenylyl cyclase activity was assessed as in Fig. 3 and expressed as picomoles of cAMP produced per hour per well. Results are expressed as the mean ± S.E. of three independent experiments carried out in triplicates. Asterisk indicates a significant (p < 0.05) difference of the agonist-promoted inhibition of forskolin-induced cAMP accumulation in cells treated with bisindolylmaleimide I or PMA versus nontreated cells.

View larger version (17K): [in this window] [in a new window]   Fig. 9. Signaling properties of OR and SNSR subtypes in rat dorsal root ganglion neurons. Neurons were stimulated for 30 min at 37°C with 20 µM forskolin and 1 µM concentrations of the SNSR-specific agonist BAM8-22 or the OR specific-agonist Leu-enk. Inhibition of adenylyl cyclase activity was assessed by measuring the amount of accumulated cAMP using a radioimmunoassay. Results are expressed as the mean ± S.E. of three independent experiments. Statistically significant differences between levels in cells treated with Leu-enk alone and those simultaneously treated with BAM8-22 are indicated (***, p < 0.001; *, p < 0.01).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we provide evidence based on BRET titration experiments that the OR and the SNSR-4 form hetero-oligomeric complexes when transiently coexpressed in HEK293 cells. The existence of such hetero-oligomers is further supported by the functional effects that stable coexpression of the two receptors had on each of the protomer properties. Although, protein kinase C-mediated cross-talk between the two receptors could contribute to some of the regulatory actions of the SNSR-4 on OR signaling, most of the observed alterations cannot be attributed to such cross-talk and most likely results from the hetero-oligomerization between the receptors.

Resonance energy transfer approaches similar to the BRET assay are becoming tools of choice to monitor receptor-receptor interactions in living cells (Angers et al., 2000; Ayoub et al., 2002; Ramsay et al., 2002). The strength and limitations of the technique were extensively discussed in a previous report (Breit et al., 2004). In the present study, we report similar propensity of the OR to homo-oligomerize and to form hetero-oligomers with the SNSR-4, suggesting that a significant amount of OR/SNSR-4 hetero-oligomers arises when coexpressed. This is not unique to this receptor pair because similar high-affinity hetero-oligomerization has been reported for several closely related GPCR subtypes (Ayoub et al., 2002; Mercier et al., 2002; Ramsay et al., 2002; Terrillon et al., 2003; Breit et al., 2004).

Coexpression of two receptor subtypes often changed agonist-promoted receptor trafficking, probably as a result of direct receptor-receptor interactions (Jordan and Devi, 1999; AbdAlla et al., 2000; George et al., 2000; Rocheville et al., 2000b; Pfeiffer et al., 2001; Stanasila et al., 2003; Xu et al., 2003; Breit et al., 2004). In particular, coexpression of the endocytosis resistant OR has been shown to inhibit OR endocytosis (Jordan and Devi, 1999). In line with these previous studies, coexpression of the SNSR-4 significantly reduced BAM22-promoted OR endocytosis. Similar to the OR, SNSR-4 does not undergo agonist-promoted endocytosis (data not shown), suggesting that endocytosis-resistant receptors may dominate over the endocytosis-prone OR. The lack of BAM22-promoted OR endocytosis in the coexpressing cells could result from a cross-talk between the SNSR-4 and the OR because of the BAM22-promoted activation of the SNSR-4/protein kinase C signaling pathway. This is, however, very unlikely because the agonist-promoted OR endocytosis induced by the OR-selective agonist Leu-enkephalin was also inhibited, indicating that the mere expression of SNSR-4 is sufficient to block OR endocytosis. Inhibition of the OR endocytosis independent of agonist-promoted SNSR-4 activity strongly suggests that direct receptor-receptor interactions are responsible for the reduced OR endocytosis, pointing out to an important role of hetero-oligomerization in the regulation of agonist-promoted endocytosis of GPCRs.

The analysis of HEK293-OR/SNSR-4 cells revealed changes in the sensitivity of the protomers to agonists and antagonists. For example, phospholipase C activity promoted by the nonselective agonist BAM22 was blocked by the OR antagonist naltrexone, whereas the SNSR-4-selective agonists, BAM2-22 and BAM8-22 were not affected by the same treatment. This trans-inhibition of the SNSR-4-specific signaling pathway by a OR antagonist provides strong evidence that, after coexpression of both receptors, a signaling unit arose that was clearly distinguishable from OR (agonist-promoted IPn accumulation) and SNSR-4 (no sensitivity toward naltrexone). Given the fact that BAM22 is not able to penetrate the cell membrane, these results indicate that OR/SNSR-4 hetero-oligomeric complexes are located at the cell surface and functionally coupled to the G_q/phospholipase C signaling pathway. So far, trans-inhibition has only been described in cells coexpressing the dopamine-2 and the somatostatin-5 (Rocheville et al., 2000a) or the -adrenergic and the angiotensin-II type 1 (Barki-Harrington et al., 2003) receptor subtypes. However, in contrast with these previous reports, the OR antagonist naltrexone could not trans-inhibit the signaling promoted by the SNSR-4-selective agonists BAM2-22 or BAM8-22 but could only inhibit the phospholipase C activation promoted by the mixed OR/SNSR-4 agonist BAM22, suggesting that the inhibition is linked to the potential of BAM22 to bind simultaneously to OR and SNSR-4 through its N and C terminus, respectively. Such bivalent ligand binding mode has already been described for synthetic OR ligands that can bind to the OR/OR hetero-oligomer (Portoghese et al., 1986) and revealed unique properties of this receptor complex (Bhushan et al., 2004; Daniels et al., 2005). Assuming a comparable bivalent binding mode between BAM22 and each of the protomers within the OR/SNSR-4 hetero-oligomer, naltrexone-induced competition of BAM22 from the OR could in turn lead to the displacement of the agonist from the SNSR-4, thus inhibiting phospholipase C activation.

Coexpression of two receptors has previously been described to inactivate one of the two receptors. For example, coexpression of the angiotensin AT₂ subtype blocked the agonist-promoted activation of the AT₁ receptor (AbdAlla et al., 2001). A second report demonstrated that the presence of the somatostatin SST₃ receptor inhibited the agonist-promoted activation of the SST_2A (Pfeiffer et al., 2001). Of particular interest for the present study is the observation that coexpression of the OR and OR blocked the agonist-promoted activation of both receptors (Jordan and Devi, 1999). However, simultaneous activation of the OR and OR rescued the agonist-promoted receptor activity of both receptors, indicating positive synergistic effects after coactivation of these opioid receptor subtypes. In contrast, coactivation of the µOR/-adrenergic-2A hetero-oligomer initiated less G coupling compared with single-activated protomers within the hetero-oligomer (Jordan et al., 2003). In the case of the OR/SNSR-4 hetero-oligomer, both receptors maintained their signaling integrity (activation of G_q and G_i/o for SNSRs and OR, respectively) when specifically activated. However, simultaneous activation of the two protomers by the mixed agonist BAM22 or by two receptor-specific agonists led to a selective activation of phospholipase C without any inhibition of the adenylyl cyclase pathway, indicating that the dual activation of the hetero-oligomer led to the activation of G_q to the exclusion of G_i/o. Such a protomer-exclusive signaling after receptor coactivation is unique for the OR/SNSR-4 hetero-oligomer and indicates a dominant-negative effect of the SNSR-4 signaling on the OR signaling. This intriguing regulation of hetero-oligomeric receptor activity could be due to three different mechanisms:

Competitive Antagonism. Agonist binding of one receptor protomer inhibits the agonist binding to the second protomer. In the case of the OR/SNSR-4 hetero-oligomer, such competitive antagonism cannot account for the decreased efficacy of OR agonists because [³H]DPDPE binding to the OR was not inhibited by SNSR-4-specific agonists (Fig. 7). These findings further indicate that each of the protomer can bind agonist simultaneously within a hetero-oligomeric complex and that there is no apparent allosteric regulation between the agonistic binding sites of the two protomers.

Heterologous Cross-Talk. Activation of a signaling pathway by one receptor subtype desensitizes the second subtype. It has been reported that pretreatment of OR expressing cells with PMA desensitized the agonist-promoted OR activity and induced receptor internalization (Xiang et al., 2001). In our study, protein kinase C-dependent OR desensitization (Fig. 8), but no internalization (data no shown) could be observed after PMA costimulation. However, the amplitude of the PMA induced-inhibition of OR signaling (Fig. 8) was much smaller than that observed by the coactivation by either the bivalent ligand BAM22 or the concomitant activation with both OR- and SNSR-selective agonists (Fig. 5A), making it unlikely that cross-talk desensitization mediated by protein kinase C could completely explain the trans-inhibition observed. Thus, although we cannot totally exclude that the protein kinase C-mediated cross-talk is partly involved in the inhibition of the G_i/o coupling to the OR after simultaneous coactivation of the SNSR-4, we suggest that other regulatory mechanisms also contribute to this inhibitory process.

G Protein Competition. The coupling of one receptor protomer to its cognate G protein inhibits the coupling of a second G protein to the hetero-oligomer. It has been proposed that rhodopsin and the leukotriene B₄ receptor form oligomeric complexes that interact with only one trimeric G protein (Baneres and Parello, 2003; Filipek et al., 2004). A similar interaction of an oligomeric OR/SNSR-4 complex with a single G protein would exclude simultaneous coupling of G_i/o and G_q to the hetero-oligomer. Assuming a preferential coupling of the hetero-oligomer to G_q within this model, simultaneous activation of both receptors would lead to the G_q coupling of the SNSR-4-protomer to the expense of the G_i/o coupling. This process would lead to the substitution of G_i/o by G_q, resulting in the activation of the phospholipase C and the depression of the adenylyl cyclase pathway. In the case of a single OR agonist, there would be no competition between G_i/o and G_q, explaining the high efficacy of OR agonists to inhibit the cyclase after single activation. Consistent with this model, the OR/SNSR-4 hetero-oligomer exhibits a lower ability to couple to G_i/o compared with the OR, even in the absence of SNSR-4 agonists, as indicated by the decreased potency of OR agonists to activate the OR/SNSR-4 hetero-oligomer despite their unchanged affinity to bind this complex (Fig. 6). Given the detectable amount of spontaneous SNSR-4 activity in OR/SNSR-4 coexpressing cells (data not shown), the decreased affinity of the OR/SNSR-4 hetero-oligomer in the absence of SNSR-4 agonist could be explained by the competition of G_i/o and G_q as a result of basal SNSR-4 activity.

The colocalization of OR and SNSR-4 in dorsal root ganglia raises the intriguing possibility that hetero-oligomerization between these receptors may also occur in cells naturally expressing both receptors. Hetero-oligomerization between OR and SNSR-4 in cells regulating pain perception would allow the OR to mediate its antinociceptive effects when specifically activated by enkephalins. However, in a situation that requires the sensation of pain, coactivation of OR and SNSR-4 would not only enhance the pain sensation by the pronociceptive effects of the SNSR-4 but also by the inhibition of the analgesic effects of the OR. Such an agonist-dependent dominant-negative effect of the SNSR-4 on OR would increase both the amplitude and the fine-tuning of pain sensation that may be advantageous in an evolutionary context. In this article, we provide data indicating that SNSR stimulation attenuates OR signaling in cultured rat dorsal root ganglia neurons. Although not proving the existence of OR/SNSR-4 hetero-oligomers in native tissues, the recapitulation of the hetero-oligomer signaling property observed in HEK293 cells, in the dorsal root ganglia, supports the existence of such oligomers and warrants further investigation that would directly investigate these hetero-oligomers in dorsal root ganglias and their potential role controlling pain perception.

	Acknowledgements

 
We are grateful to Drs. T. Groblewski and S. Ahmad (AstraZeneca Research Center) for the fruitful discussions and for providing various BAM peptides.

	Footnotes

 
This work was supported by grants from the Canadian Institute for Health Research and from the Heart and Stroke Foundation of Canada. A.B. was supported by a postdoctoral fellowship from AstraZeneca Research Center, and K.G. and L.A.D. received National Institutes of Health grants NS026880 and DA008862, respectively. M.B. holds a Canada Research Chair in Signal Transduction and Molecular Pharmacology.

ABBREVIATIONS: SNSR, sensory neuron-specific G protein-coupled receptor; GPCR, G protein-coupled receptor; BAM, bovine adrenal medulla; OR, opioid receptor(s); BRET, bioluminescence resonance energy transfer; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; IBMX, 3-isobutyl-1-methylxanthine; Leu-enk, Leu-enkephalin; DPDPE, [D-Pen²,D-Pen⁵]-enkephalin; GFP, green fluorescent protein; Rluc, Renilla reniformis luciferase; PCR, polymerase chain reaction; IPn, total fraction of phosphoinositols; BSA, bovine serum albumin; PMA, phorbol 12-myristate-13-acetate.

Address correspondence to: Dr. Michel Bouvier, Département de Biochimie, Université de Montréal, H3C 3J7 Montréal, QC, Canada. E-mail: michel.bouvier{at}umontreal.ca

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