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• W2053911700 abstract "Heptahelical receptors communicate extracellular information to the cytosolic compartment by binding an extensive variety of ligands. They do so through conformational changes that propagate to intracellular signaling partners as the receptor switches from a resting to an active conformation. This active state has been classically considered unique and responsible for regulation of all signaling pathways controlled by a receptor. However, recent functional studies have challenged this notion and called for a paradigm where receptors would exist in more than one signaling conformation. This study used bioluminescence resonance energy transfer assays in combination with ligands of different functional profiles to provide in vivo physical evidence of conformational diversity of δ-opioid receptors (DORs). DORs and αi1β1γ2 G protein subunits were tagged with Luc or green fluorescent protein to produce bioluminescence resonance energy transfer pairs that allowed monitoring DOR-G protein interactions from different vantage points. Results showed that DORs and heterotrimeric G proteins formed a constitutive complex that underwent structural reorganization upon ligand binding. Conformational rearrangements could not be explained by a two-state model, supporting the idea that DORs adopt ligand-specific conformations. In addition, conformational diversity encoded by the receptor was conveyed to the interaction among heterotrimeric subunits. The existence of multiple active receptor states has implications for the way we conceive specificity of signal transduction. Heptahelical receptors communicate extracellular information to the cytosolic compartment by binding an extensive variety of ligands. They do so through conformational changes that propagate to intracellular signaling partners as the receptor switches from a resting to an active conformation. This active state has been classically considered unique and responsible for regulation of all signaling pathways controlled by a receptor. However, recent functional studies have challenged this notion and called for a paradigm where receptors would exist in more than one signaling conformation. This study used bioluminescence resonance energy transfer assays in combination with ligands of different functional profiles to provide in vivo physical evidence of conformational diversity of δ-opioid receptors (DORs). DORs and αi1β1γ2 G protein subunits were tagged with Luc or green fluorescent protein to produce bioluminescence resonance energy transfer pairs that allowed monitoring DOR-G protein interactions from different vantage points. Results showed that DORs and heterotrimeric G proteins formed a constitutive complex that underwent structural reorganization upon ligand binding. Conformational rearrangements could not be explained by a two-state model, supporting the idea that DORs adopt ligand-specific conformations. In addition, conformational diversity encoded by the receptor was conveyed to the interaction among heterotrimeric subunits. The existence of multiple active receptor states has implications for the way we conceive specificity of signal transduction. Heptahelical receptors are versatile membrane proteins that play an important role in cellular communication. They do so by recognizing a large variety of extracellular ligands that convey information to the intracellular compartment by modifying receptor conformation upon binding. These structural modifications then trigger an array of biochemical changes that ultimately control vital processes within the cell. Traditionally, conformational changes induced by ligand binding have been thought to shift equilibrium between an active and an inactive conformation of the receptor. According to this classical view, all agonists would stabilize a single active state that equally effectively stimulates all signaling pathways controlled by the receptor (1Furchgott R. Bull. N Y Acad. Med. 1966; 42: 996-1006PubMed Google Scholar). In consequence, ligand ability to stabilize this unique active state would be the only determinant of ligand efficacy at all functional readouts. Furthermore, this model predicts that ligand rank order of efficacy across different readouts should be maintained, representing a progressive accumulation (or decrease) of the single active state of the receptor (2Kenakin T. Trends Pharmacol. Sci. 2004; 25: 186-192Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). However, recent data have challenged this view, suggesting that the complexity of heptahelical receptor signaling cannot be based on the accumulation of a single active receptor conformation, and efficacy cannot be restricted to only a quantitative dimension (3Pineyro G. Archer-Lahlou E. Cell. Signal. 2007; 19: 8-19Crossref PubMed Scopus (44) Google Scholar, 4Urban J.D. Clarke W.P. von Zastrow M. Nichols D.E. Kobilka B. Weinstein H. Javitch J.A. Roth B.L. Christopoulos A. Sexton P.M. Miller K.J. Spedding M. Mailman R.B. J. Pharmacol. Exp. Ther. 2007; 320: 1-13Crossref PubMed Scopus (904) Google Scholar). Evidence supporting this assertion has been largely based on functional studies whose results cannot be adequately rationalized by accumulation of a single active state but are intuitively explained by assuming the existence of conformational diversity among active forms of the receptor (4Urban J.D. Clarke W.P. von Zastrow M. Nichols D.E. Kobilka B. Weinstein H. Javitch J.A. Roth B.L. Christopoulos A. Sexton P.M. Miller K.J. Spedding M. Mailman R.B. J. Pharmacol. Exp. Ther. 2007; 320: 1-13Crossref PubMed Scopus (904) Google Scholar). In particular, these studies show that agonists acting at the same receptor may display a different rank order of efficacies when tested at different functional readouts (5Berg K.A. Maayani S. Goldfarb J. Scaramellini C. Leff P. Clarke W.P. Mol. Pharmacol. 1998; 54: 94-104Crossref PubMed Scopus (446) Google Scholar, 6Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Pineyro G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (424) Google Scholar, 7Audet N. Paquin-Gobeil M. Landry-Paquet O. Schiller P.W. Pineyro G. J. Biol. Chem. 2005; 280: 7808-7816Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In addition to this indirect body of evidence, the possibility that different agonists may stabilize different conformations of the same receptor is supported by in vitro physical data. In particular, fluorescence and spectroscopy approaches have confirmed that ligands of different efficacies impose distinct structural constraints upon purified β2-adrenergic receptors, DORs, 4The abbreviations used are: DOR, δ-opioid receptor; BRET, bioluminescence resonance energy transfer; RLuc, Renilla luciferase; DPDPE, d-pen-2,5-enkephalin; TIPP, Tyr-l-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-Phe-Phe-OH; TICPΨ, Tyr-TicΨ[CH2NH]cyclohexylalanine-Phe-OH; ERK, extracellular signal-regulated kinase; pERK, phospho-ERK; GFP, green fluorescent protein; PTX, pertussis toxin; GTPγS, guanosine 5′-3-O-(thio)triphosphate. and muscarinic receptors (8Ghanouni P. Gryczynski Z. Steenhuis J.J. Lee T.W. Farrens D.L. Lakowicz J.R. Kobilka B.K. J. Biol. Chem. 2001; 276: 24433-24436Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 9Alves I.D. Salamon Z. Varga E. Yamamura H.I. Tollin G. Hruby V.J. J. Biol. Chem. 2003; 278: 48890-48897Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 10Li J.H. Han S.J. Hamdan F.F. Kim S.K. Jacobson K.A. Bloodworth L.M. Zhang X. Wess J. J. Biol. Chem. 2007; 282: 26284-26293Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The problem with these observations is that they have not allowed us to establish if ligand-specific receptor states exist in living cells and, if so, whether these different conformations may be discriminated by postreceptor signaling partners. In the present study, we sought to determine whether DORs occupied by different ligands would differ in the way they interact with heterotrimeric G proteins, the rationale being that if DORs were stabilized in ligand-specific conformations, then each of these receptor states should distinctively interact with its immediate signaling partners. Interaction between DORs and αβγ subunits was assessed using BRET assays, a technology that has been validated to study not only in vivo coupling of heptahelical receptors to αβγ subunits (11Gales C. Rebois R.V. Hogue M. Trieu P. Breit A. Hebert T.E. Bouvier M. Nat. Methods. 2005; 2: 177-184Crossref PubMed Scopus (327) Google Scholar, 12Ayoub M.A. Maurel D. Binet V. Fink M. Prezeau L. Ansanay H. Pin J.P. Mol. Pharmacol. 2007; 71: 1329-1340Crossref PubMed Scopus (77) Google Scholar) but to monitor in vivo intermolecular interactions within the G protein heterotrimer (13Dupre D.J. Robitaille M. Ethier N. Villeneuve L.R. Mamarbachi A.M. Hebert T.E. J. Biol. Chem. 2006; 281: 34561-34573Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 14Gales C. Van Durm J.J. Schaak S. Pontier S. Percherancier Y. Audet M. Paris H. Bouvier M. Nat. Struct. Mol. Biol. 2006; 13: 778-786Crossref PubMed Scopus (347) Google Scholar). Results showed that DORs and α1β1γ2 subunits formed a constitutive complex, and BRET assays demonstrated that conformational changes imposed by different ligands were compatible with a multistate rather than a two-state model. Reagents—Buffer chemicals, protease inhibitors, DPDPE, morphine, naloxone, forskolin, 3-isobutyl-1-methylxanthine, PTX, anti-FLAG M2 affinity resin, and FLAG peptide were purchased from Sigma. [35S]GTPγS, [3H]adenosine, and coelanterazine were from PerkinElmer Life Sciences. SNC-80 was from Tocris Cookson, and TIPP and TICP were synthesized as previously described (15Schiller P.W. Weltrowska G. Berezowska I. Nguyen T.M. Wilkes B.C. Lemieux C. Chung N.N. Biopolymers. 1999; 51: 411-425Crossref PubMed Scopus (106) Google Scholar). G418, Dulbeccoʼns modified Eagleʼns medium, fetal bovine serum, glutamine, penicillin, and streptomycin were purchased from Wisent. DNA Constructs—Recombinant plasmids encoding for αi1-Luc constructs were prepared as previously described (14Gales C. Van Durm J.J. Schaak S. Pontier S. Percherancier Y. Audet M. Paris H. Bouvier M. Nat. Struct. Mol. Biol. 2006; 13: 778-786Crossref PubMed Scopus (347) Google Scholar), using flexible linkers (SGGGGS) to insert the coding sequence of humanized Renilla luciferase (RLuc; PerkinElmer Life Sciences) into the coding sequence of human Gαi1, either between residues Gly60 and Tyr61 (Gαi1-60Rluc), Leu91 and Lys92 (Gαi1-91Rluc), or Glu122 and Leu123 (Gαi1-122Rluc). The plasmid encoding γ2 with green fluorescent protein (GFP10) fused to its N terminus (11Gales C. Rebois R.V. Hogue M. Trieu P. Breit A. Hebert T.E. Bouvier M. Nat. Methods. 2005; 2: 177-184Crossref PubMed Scopus (327) Google Scholar) and vectors encoding FLAG, GFP2, and RLuc fused in frame at the C terminus of human DORs have been previously described (16Breit A. Gagnidze K. Devi L.A. Lagace M. Bouvier M. Mol. Pharmacol. 2006; 70: 686-696Crossref PubMed Scopus (55) Google Scholar). Generation of CD8-GFP2 and CD8-RLuc has also been reported (11Gales C. Rebois R.V. Hogue M. Trieu P. Breit A. Hebert T.E. Bouvier M. Nat. Methods. 2005; 2: 177-184Crossref PubMed Scopus (327) Google Scholar, 14Gales C. Van Durm J.J. Schaak S. Pontier S. Percherancier Y. Audet M. Paris H. Bouvier M. Nat. Struct. Mol. Biol. 2006; 13: 778-786Crossref PubMed Scopus (347) Google Scholar). Cell Culture and Expression of Heterologous Proteins—HEK293 cells were cultured in Dulbeccoʼns modified Eagleʼns medium supplemented with 5% fetal bovine serum and 2 nm l-glutamine. For transient expression of recombinant proteins, cells were seeded at a density of 3 × 106 cells in 100-mm Petri dishes, cultured for 24 h, and then transfected with vectors encoding BRET constructs for DORs and different G protein subunits along with untagged complementary heterotrimeric components. Transfections were done using FuGENE 6 reagent (Roche Applied Science) according to the manufacturerʼns protocol. Titration BRET assays were done as previously described (11Gales C. Rebois R.V. Hogue M. Trieu P. Breit A. Hebert T.E. Bouvier M. Nat. Methods. 2005; 2: 177-184Crossref PubMed Scopus (327) Google Scholar), using a fixed amount of donor-tagged proteins (RLuc) that was co-expressed with increasing amounts of vector coding for the acceptor (GFP). Untagged subunits complementary to G protein BRET constructs were also included, at DNA levels that would support membrane expression of the heterotrimer at all points of the titration curve. Titration curves allowed us to determine the relative amount of DNA constructs necessary to achieve a maximal BRET signal that was then used in transfections for single point assays. Forty-eight hours after transfection, cells were used in BRET, cyclase, or immunopurification assays. Clones stably expressing FLAG-tagged human DORs were generated as previously described (17Pineyro G. Azzi M. De Lean A. Schiller P. Bouvier M. Mol. Pharmacol. 2001; 60: 816-827PubMed Google Scholar) and transiently transfected with αi1-Luc ·GFP-γ2 along with untagged β1 subunits. BRET Measurements—Forty-eight hours after transfection, cells were washed twice and mechanically detached with phosphate-buffered saline and centrifuged 5 min at 300 × g, followed by resuspension in phosphate-buffered saline. Cells were then distributed into a 96-well microplate (white Optiplate; PerkinElmer Life Sciences) at a concentration of 50,000–100,000 cells/well, which allowed us to achieve luminescence levels suitable for BRET readings using different constructs. Treatments and BRET readings were done according to a previously established protocol that was optimized for assessing in vivo ligand effects on receptor interaction with heterotrimeric G proteins (11Gales C. Rebois R.V. Hogue M. Trieu P. Breit A. Hebert T.E. Bouvier M. Nat. Methods. 2005; 2: 177-184Crossref PubMed Scopus (327) Google Scholar, 14Gales C. Van Durm J.J. Schaak S. Pontier S. Percherancier Y. Audet M. Paris H. Bouvier M. Nat. Struct. Mol. Biol. 2006; 13: 778-786Crossref PubMed Scopus (347) Google Scholar). Briefly, intact living cells were suspended in phosphate-buffered saline, kept at room temperature, and incubated in the presence or absence of different ligands for 2 min, followed by the addition of the Rluc substrate, DeepBlueC coelenterazine (PerkinElmer Life Sciences) at a final concentration of 5 μm. Readings were obtained 2 min after coelanterazine addition, using a modified top count apparatus (TopCount NXTTM; PerkinElmer Life Sciences) that allows the sequential integration of the signals detected in the 370–450 and 500–530 nm windows using filters with the appropriate band pass (Chroma). The BRET2 signal was determined by calculating the ratio of the light emitted by GFP2 ·GFP10 (500–530 nm) over the light emitted by the Rluc (370–450 nm). BRET2 values were corrected by subtracting the BRET background signal (detected when the Rluc-tagged construct was expressed alone) from the BRET signal detected in cells coexpressing both Rluc- and GFP (net BRET). For titration experiments, the expression level of each tagged protein was determined by direct measurement of total fluorescence and luminescence on aliquots of the transfected cells. Total fluorescence was measured using a FluoroCount (PerkinElmer) with an excitation filter at 400 nm and an emission filter at 510 nm and the following parameters: gain, 1; photomultiplier tube, 1100 V; time, 1.0 s. After measuring fluorescence, the same cell samples were incubated with coelenterazine h (5 μm; 8 min; Nanolight Technology), and total cell luminescence was measured using a LumiCount (PerkinElmer Life Sciences) with the following parameters: gain, 1; photomultiplier tube, 900 V; time, 1 s. Immunopurification Assays and Western Blot Analysis—Cells were recovered in phosphate-buffered saline and treated with DPDPE or TICP (10 μm, 5 min) as described above. Following treatment, cells were suspended in lysis buffer (5 mm Tris, 3 mm MgCl2, 2 mm EDTA, 1 mm NaF, 1 mm Na3VO4, 5 μg/ml leupeptin, 5 μg/ml soybean trypsin inhibitor, and 10 μg/ml benzamidine) and homogenized using an Ultraturax homogenizer (IKA, Wilmington, NC). Following centrifugation at 300 × g for 5 min, the supernatant was centrifuged at 30,000 × g for 20 min, and the resultant pellet was resuspended in lysis buffer for a second round of centrifugation (30,000 × g; 20 min). The pellet obtained was then solubilized in 0.5% n-dodecylmaltoside, 25 mm Tris, pH 7.4, 140 mm NaCl, 2 mm EDTA, 1 mm NaF, 1 mm Na3VO4, 5 μg/ml leupeptin, 5 μg/ml soybean trypsin inhibitor, and 10 μg/ml benzamidine. Following agitation at 4 °C for 2 h, the solubilized fraction was centrifuged at 10,000 × g for 30 min, and the receptor was immunopurified from the supernatant fraction using an anti-FLAG M2 antibody resin. 20 μl of antibody-coupled resin equilibrated in solubilization buffer and supplemented with 0.1% bovine serum albumin (w/v) were used to purify the receptor overnight at 4 °C under gentle agitation. The next morning, the resin was pelleted and washed twice with 500 μl of solubilization buffer and four times with 500 μl of modified solubilization buffer (containing 0.1% instead of 0.5% n-dodecyl-maltoside (w/v)). The receptor was then eluted by incubating the resin for 10 min at 4 °C with 100 μl of modified solubilization buffer containing a FLAG peptide (150 μg/ml). This elution was repeated three times, and the eluates were combined and concentrated by membrane filtration over Microcon-30 concentrators (Millipore). SDS sample buffer was then added, and samples were used for SDS-PAGE. SDS-PAGE was performed using a 4% stacking gel and 10% separating gel. Proteins resolved in SDS-PAGE were then transferred (50 mA, 16 h; Bio-Rad Mini-Trans Blot apparatus) from gels onto nitrocellulose (GE Healthcare). The amount of endogenous or Luc-tagged Gαi1 or Gβ that was recovered with immunopurified DORs was assessed using 1:1000 polyclonal antibodies raised against Gαi1 or Gβ, followed by secondary anti-rabbit horseradish-conjugated antibodies (1:40,000; Amersham Biosciences). The total amount of receptor loaded for each sample was detected by probing the samples with anti-FLAG M2 antibody (1:5000), followed by secondary anti-mouse horseradish-conjugated antibodies (1:40,000; Amersham Biosciences). cAMP Accumulation Assays—cAMP accumulation assays were carried out according to a previously described protocol (18Pineyro G. Azzi M. deLean A. Schiller P.W. Bouvier M. Mol. Pharmacol. 2005; 67: 336-348Crossref PubMed Scopus (18) Google Scholar), and [3H]ATP and [3H]cAMP were separated by sequential chromatography on Dowex exchange resin and aluminum oxide columns. cAMP produced was estimated by calculating the ratio of [3H]cAMP/[3H]ATP plus [3H]cAMP in each sample. [35S]GTPγS Binding Assays—The procedure for [35S]GTPγS binding has been detailed in a previous report (18Pineyro G. Azzi M. deLean A. Schiller P.W. Bouvier M. Mol. Pharmacol. 2005; 67: 336-348Crossref PubMed Scopus (18) Google Scholar). Data Analysis—Statistical comparisons were done by one-way analysis of variance using Dunnettʼns correction to compare drug effects with basal conditions and Fisherʼns “least significance difference” adjustment in order to assess differences among drugs. Modification of drug effects by PTX was analyzed by covariance using basal values as co-regressor. Except for Fig. 2, all figures present data as differences or percentage changes with respect to basal, but in all cases statistical analyses were carried out on raw net BRET ratio, cAMP/cAMP + ATP ratio, or pERK/total ERK ratio. Signaling Efficacy of DOR Ligands—In a first series of experiments, we sought to establish the signaling profile of peptidic (DPDPE, TIPP, and TICP) and nonpeptidic (SNC-80, morphine, and naltrindole) DOR ligands that would then be tested in BRET assays. Consistent with previous reports (6Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Pineyro G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (424) Google Scholar, 7Audet N. Paquin-Gobeil M. Landry-Paquet O. Schiller P.W. Pineyro G. J. Biol. Chem. 2005; 280: 7808-7816Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), ligand efficacy was influenced by the signaling pathway in which drugs were tested (Fig. 1). Naltrindole, classically considered a neutral antagonist (19Quock R.M. Burkey T.H. Varga E. Hosohata Y. Hosohata K. Cowell S.M. Slate C.A. Ehlert F.J. Roeske W.R. Yamamura H.I. Pharmacol. Rev. 1999; 51: 503-532PubMed Google Scholar), was without effect in the ERK cascade but displayed partial efficacy in the cAMP pathway. DPDPE and SNC-80 inhibited cAMP production and induced ERK phosphorylation, behaving as highly efficacious agonists in both cascades, whereas morphine and TIPP displayed partial efficacy at both readouts. TICP, on the other hand, had a dual profile, sharing agonist capacity to induce ERK activation but opposing agonist ability to inhibit cAMP production. Comparison of ligand rank order of efficacy in cyclase (Fig. 1A) and ERK (Fig. 1B) cascades also revealed that DOR ligands did not maintain their ordinal positions in the two assays, an observation that has been classically associated with the existence of ligand-specific receptor states (4Urban J.D. Clarke W.P. von Zastrow M. Nichols D.E. Kobilka B. Weinstein H. Javitch J.A. Roth B.L. Christopoulos A. Sexton P.M. Miller K.J. Spedding M. Mailman R.B. J. Pharmacol. Exp. Ther. 2007; 320: 1-13Crossref PubMed Scopus (904) Google Scholar, 7Audet N. Paquin-Gobeil M. Landry-Paquet O. Schiller P.W. Pineyro G. J. Biol. Chem. 2005; 280: 7808-7816Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). We reasoned that if this interpretation of functional data were correct, then DORs occupied by different ligands should distinctively interact with intracellular signaling partners. To assess this possibility, we required an experimental approach that would allow us to monitor DOR interaction with signaling proteins under conditions similar to the ones used in cyclase and mitogen-activated protein kinase readouts. We therefore turned to BRET, since this technology offers the possibility of monitoring protein-protein interactions within living cells.TABLE 4Correlation analysis of ligand-induced change at BRET pairs evaluating conformational change within the G protein heterotrimers and ligand efficacy to induce modulation of cAMP accumulation and ERK phosphorylationR2pCorrelation for cAMP signaling and αiLuc·γ2-GFP constructs αi1-Luc60·γ2-GFP0.9540.008 αi1-Luc91·γ2-GFP0.7980.017 αi1-Luc122·γ2-GFP0.4420.150Correlation for ERK signaling and αiLuc·γ2-GFP constructs αi1-Luc60·γ2-GFP0.3440.226 αi1-Luc91·γ2-GFP0.5070.112 αi1-Luc122·γ2-GFP0.6590.049 Open table in a new tab FIGURE 3BRET changes promoted by DPDPE and TICP are consistent with conformational reorganization of preformed DOR ·αi1β1γ2 complexes. HEK 293 cells were transfected as in Fig. 2, and net BRET signals generated by DOR-GFP and specified αi1-Luc partners (A), GFP-γ2 and DOR-Luc (B), or GFP-γ2 and specified αi1-Luc constructs (C) were assessed in the presence or absence of DPDPE or TICP (10 μm; 2 min). Results were expressed as the difference between measures obtained in the presence or absence of ligand and correspond to mean ± S.E. of at least six experiments carried out in duplicates. *, p < 0.05; **, p < 0.01. D, BRET titration assays were carried out as in Fig. 2, in the presence or absence of DPDPE. BRET50 values represent the calculated ratio of donor/acceptor molecules producing 50% of the energy transfer observed at saturation. E, following transfection with DOR-FLAG, αi1-Luc91, or vector, in combination with β1γ2, cells were exposed or not to saturating concentrations of DPDPE or TICP as above. Following treatment, receptors were immunopurified, and the product was separated by SDS-PAGE. The amount of αi1-Luc91 or endogenous αi1 subunits recovered with the receptor was then assessed by immunoblot. DOR interaction with transfected or endogenous αi1 subunits was assessed by calculating the immunoreactivity ratio αi1/FLAG present in each sample. Results were expressed as percentage of basal values and represent mean ± S.E. of four experiments. Blots for αi1-Luc91 and endogenous αi1 were scanned from separate films. F, cells stably expressing DOR-FLAG were transfected with αi1β1γ2 or vector and exposed or not to DPDPE or TICP. Following DOR immunopurification, the amount of endogenous or overexpressed β1 subunits recovered with the receptor was assessed by immunoblot. Results are expressed as in E and correspond to four experiments. Blots for endogenous and overexpressed β1 subunits were scanned from separate films.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 2Spontaneous signals generated by different BRET pairs. A, HEK 293 cells were transfected with recombinant plasmids for DOR-GFP, the indicated αi1-Luc constructs, and untagged β1γ2 subunits. Spontaneous interaction between DORs and αi1 subunits was measured by assessing net BRET values in the absence of ligand. Values correspond to mean ± S.E. of 5–9 experiments carried out in duplicates. Inset, HEK 293 cells expressing or not DOR-FLAG were transfected with αi1-Luc91β1γ2 or vector, and DORs were immunopurified as described under “Experimental Procedures.” The amount of αi1-Luc91 (∼75 kDa) or endogenous αi1 (∼39 kDa) subunits recovered with each purification product was assessed by immunoblot. Results correspond to a representative example of four independent experiments. Blots for αi1-Luc91 and endogenous αi1 were scanned from separate films. B, BRET titration assays were performed by measuring net energy transfer in HEK 293 cells transfected with increasing concentrations of DOR-GFP or CD8-GFP and a fixed amount of αi1-Luc91, in combination with untagged β1γ2 subunits. C, basal interaction between the βγ complex and DORs was assessed by measuring the spontaneous BRET signal generated by HEK 293 cells expressing GFP-γ2 and DOR-Luc in combination with untagged αi1 and β1 subunits. Interaction between the βγ complex and αi1 subunits was assessed by co-transfecting αi1-Luc60, αi1-Luc91, or αi1-Luc122 with GFP-γ2, β1 subunits, and DOR-FLAG into HEK 293 cells. Values correspond to mean ± S.E. of 4–9 experiments carried out in duplicates. Inset, HEK 293 cells expressing or not DOR-FLAG were transfected with αi1β1γ2 or vector, and DORs were immunopurified as described under “Experimental Procedures.” The amount of endogenous or overexpressed β1(∼32 kDa) subunits recovered with each purification product was assessed by immunoblot. Results correspond to a representative example of four independent experiments. D, BRET titration assays were performed by measuring net energy transfer in HEK 293 cells transfected with increasing amounts of GFP-γ2 and a fixed amount of DOR-Luc or CD8-Luc, in combination with untagged αi1 and β1 subunits.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 1Functional responses of DOR ligands in adenylyl cyclase and ERK pathways. A, HEK293 cells expressing DOR-FLAG were treated with saturating concentrations (10 μm) of indicated ligands and cAMP accumulation assays performed in the presence of 25 μm forskolin as detailed under “Experimental Procedures.” Drug effects are expressed as percentage change with respect to the total amount of cAMP produced in the absence of ligand (percentage change in cAMP accumulation = (cAMPligand – cAMPno ligand)/cAMPno ligand × 100) and correspond to mean ± S.E. of seven experiments carried out in triplicates. B (top), HEK293 cells expressing DOR-FLAG were exposed to saturating concentrations of the indicated ligands for 5 min, following which ERK signaling was assessed by immunoblot. ERK phosphorylation was normalized to the amount of protein loaded per lane by expressing the data as phospho-ERK/total ERK ratio. Drug effects were expressed as percentage of the basal ratio (percentage of basal = ((pERK/total ERKligand)/(pERK/total ERKno ligand) × 100)) and represent mean ± S.E. of at least six experiments. B (bottom), representative immunoblots. pERK and total ERK bands observed in the presence and absence of the indicated drugs. Each drug was paired to its corresponding experimental control from the same blot. To achieve this pairing, lanes containing information not presented in the study were removed by splicing. Examples for different drugs were not necessarily all from the same blot, but they were all matched for total ERK contents and time of film exposure. Statistical analysis is detailed under “Experimental Procedures.” *, p < 0.05; **, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Characterization of BRET Constructs and Signal Specificity—BRET is a naturally occurring phenomenon resulting from the nonradiative transfer of energy between a luminescent donor and a fluorescent acceptor (20Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar). In BRET2 assays, RLuc catalyzes oxidation of" @default.
• W2053911700 created "2016-06-24" @default.
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• W2053911700 creator A5055103031 @default.
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• W2053911700 date "2008-05-01" @default.
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• W2053911700 title "Bioluminescence Resonance Energy Transfer Assays Reveal Ligand-specific Conformational Changes within Preformed Signaling Complexes Containing δ-Opioid Receptors and Heterotrimeric G Proteins" @default.
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