Detergent-free Isolation of Functional G Protein ... - ACS Publications

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Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles Christel Logez,† Marjorie Damian,‡ Céline Legros,† Clémence Dupré,† Mélody Guéry,‡ Sophie Mary,‡ Renaud Wagner,§ Céline M’Kadmi,‡ Olivier Nosjean,† Benjamin Fould,† Jacky Marie,‡ Jean-Alain Fehrentz,‡ Jean Martinez,‡ Gilles Ferry,† Jean A. Boutin,† and Jean-Louis Banères*,‡ †

Pole d’expertise Biotechnologie, Chimie, Biologie, Institut de Recherches Servier, 125, chemin de Ronde, F-78290 Croissy-sur-Seine, France ‡ Faculté de Pharmacie, Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-Université Montpellier-ENSCM, 15 Avenue C. Flahault, F-34093 Montpellier, France § CNRS UMR7242, Institut de Recherche de l’ESBS, Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, 300 Boulevard Sébastien Brant, 67412 Ilkirch cedex, France S Supporting Information *

ABSTRACT: G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.

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amphipols (APols)5 or, more recently, lipid discs stabilized by different versions of lipoproteins called MSPs.6 These membrane mimics have been shown to be very advantageous to handle membrane protein in solution because of their increased stabilizing properties compared to detergents. They nevertheless have a major drawback; i.e., they still require an initial solubilization step in detergents to extract the protein from the membrane. A detergent-free approach for membrane protein manipulation has been recently described that is based on amphipathic styrene-maleic acid (SMA) copolymers. These copolymers solubilize membrane proteins directly from lipid bilayers. By doing so, they provide stable membrane patches where the protein is inserted into.7 Of importance, SMA-mediated extraction appears as a versatile method as it can afford functional proteins from membranes of diverse expression hosts.8−12 We explored here the potential of SMA copolymers

protein-coupled receptors (GPCRs) are one of the largest cell surface receptor families involved in many cellular signaling processes.1 For this reason, as well as for their importance as drug targets, the molecular aspects of their functioning have been extensively investigated. Although spectacular advances have been made through elucidation of the crystal structure of many different receptors,2 these structures only provide snapshots at the beginning and end of the activation process of GPCRs. Additional biophysical studies are still required to complete the description of the conformational space these receptors can explore. In most cases, biophysical studies rely on purified receptors. Detergents are commonly used to extract GPCRs out of their native membrane environment and stabilize them in solution. However, these compounds have major pitfalls that include insufficient mimicking of the lipid bilayer structure and denaturing effects that often lead to poor stability of the purified protein and/or to its altered structure and dynamics.3,4 Alternative media have been developed to circumvent the denaturing effects of detergents for membrane protein manipulation. Among them are amphipatic polymers called © XXXX American Chemical Society

Received: September 21, 2015 Revised: December 8, 2015

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DOI: 10.1021/acs.biochem.5b01040 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry to solubilize GPCRs and preserve their functional properties in vitro. To this end, we used two different systems of increasing complexity. The first one, proteoliposomes, was used to assess whether SMAs could afford GPCR-containing discs when starting from a simple model membrane where the lipid and protein composition is tightly controlled. The analysis was then extended to Pichia pastoris membranes to estimate the ability of the copolymer to solubilize receptors directly from a host frequently used to produce recombinant GPCRs.13 Two different receptors, the ghrelin receptor GHS-R1a and the melatonin receptor MT1R, were used as models for class A GPCRs. Ghrelin is a neuroendocrine peptide hormone that acts through its cognate GPCR GHS-R1a to control biological processes such as growth hormone secretion, food intake, or reward-seeking behaviors.14 GHS-R1a is thus a prominent pharmacological target with potential applications in conditions such as management of disease-induced cachexia, obesity or treatment of patients with diabetes, growth hormone deficiency, and drug or alcohol use disorders. Melatonin is a neurohormone synthesized by the pineal gland. It is the main messenger from the master clock, driving the body through the various cycles (diurnal and seasonal) by acting upon its receptors in peripheral tissues.15,16 In addition, melatonin receptors are new targets in depression.17 The molecular targets of melatonin are two GPCRs,18 the MT1 and MT2 receptors. A MT3 binding site has also been identified as quinone reductase 2.19 Besides being prototypes for rhodopsin-like GPCRs, GHSR1a and MT1R are thus major players in important biological processes and, as such, are targets for therapeutic compounds with applications in many different clinical spheres. Therefore, getting them as isolated functional proteins would be of paramount interest for both academic and drug screening purposes.

resuspended in 50 mM Tris-HCl pH8. Monomeric GHS-R1a in MSP1E3-containing lipid discs was prepared as described.20 MT1R Expression and Membrane Isolation. MT1R was expressed in P. pastoris as previously described,24 and cells were lysed accordingly. The whole membrane fractions were then pelleted by ultracentrifugation (100000g for 45 min at 4 °C) and suspended with a dounce homogenizer in a cold membrane buffer (50 mM Tris-HCl pH 7.4, 0.5 M NaCl, 10% glycerol, 1 mM PMSF) to a final concentration of about 10 mg/mL as assessed by a BCA protein assay. CHO Membrane Preparation. CHO-K1 cell lines stably expressing the human MT1R were grown to confluence, harvested in PBS buffer (Gibco, Invitrogen) containing 5 mM EDTA, and centrifuged at 1000g for 20 min (4 °C). The resulting pellet was suspended in 5 mM Tris-HCl pH 7.4 containing 2 mM EDTA and homogenized using Kinematicapolytron. The homogenate was then centrifuged (20000g, 30 min, 4 °C), and the resulting pellet was suspended in 75 mM Tris-HCl pH 7.4 containing 2 mM EDTA and 12.5 mM MgCl2. Protein content was determined according to Bradford25 using the Bio-Rad kit (Bio-Rad). Aliquots of membrane preparations were stored in resuspension buffer (75 mM Tris-HCl pH 7.4, 2 mM EDTA, 12.5 mM MgCl2) at −80 °C until use. SMA Solubilization. The proteoliposomes or the membrane preparations in 50 mM Tris-HCl pH8 were incubated at 26 °C (GHS-R1a) or 4 °C (MT1R) with the SMA 2:1 copolymer (Malvern cosmeceutics). This 2:1 copolymer was used all along the present work unless otherwise stated. Comparable results were obtained in solubilization efficiency when using the 3:1 polymer (Sigma-Aldrich) instead (Supplementary Figure 1). For solubilization, the membrane solution was added progressively to a 5% (w:v) copolymer solution in 50 mM Tris-HCl pH 8 to reach a final concentration of 2.5% (w:v) SMA. After incubation, unsolubilized material was removed by ultracentrifugation at 100000g for 30 min. The resulting supernatant was directly loaded on a 1 mL HisTrap column (GE Healthcare) previously equilibrated with a 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole buffer. After extensive washing with the same buffer, the protein-containing discs were eluted with the same buffer containing 250 mM imidazole. The eluted fractions were concentrated to a volume of 500 μL, ultracentrifuged (100000g for 30 min), and loaded on a Superdex S200 HR column (10 × 300 mm; GE Healthcare) previously equilibrated in 50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA. Elution was carried out with the same buffer at a 0.2 mL/min flow rate. Fractions of 0.5 mL were collected. The average diameter of particles was estimated from calibration with a set of marker proteins of known diameter.26 GHS-R1a Labeling. GHS-R1a labeling with bimane was carried out by incubating the receptor protein with mBBr at 4 °C for 16 h. Unreacted dye was removed by dialysis.27 Fluorescein maleimide labeling of the ghrelin receptor in liposomes solubilized or not with 2% β-DDM was followed by monitoring covalent attachment of the probe to the cysteine (change in fluorescein emission intensity over time) until a plateau was reached. For LRET measurements, the ghrelin receptor mutant with a pAzF residue at position 71 (cytoplasmic end of TM1) and a unique reactive cysteine at position 255 (cytoplasmic end of TM6) was expressed and labeled with Lumi4-Tb maleimide and Alexa Fluor 488 after SMA-solubilization as described.28

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EXPERIMENTAL PROCEDURES Liposome Preparation. Asolectin (Sigma-Aldrich) was dissolved in chloroform and dried under a stream of nitrogen followed by overnight incubation under a vacuum. The lipids were then solubilized in a 50 mM Na-HEPES, 100 mM NaCl, pH 7.5 buffer. To this end, the buffer was added to the dried lipids, and the mixture was left at room temperature for 1 h. After vigorous vortexing, liposomes were obtained by extrusion through a polycarbonate filter (100 nm) using a mini-extruder device (Avanti Polar Lipids). GHS-R1a Expression, Purification, and Insertion into Liposomes. The ghrelin receptor was expressed, purified, and folded in vitro as described.20 After amphipol-to-detergent exchange, the receptor in β-DDM was directly used for insertion into preformed liposomes. Liposomes were first destabilized by addition of 4 mM β-DDM and incubation for 1 h at room temperature. This detergent concentration is just above the Rsat value, i.e. the detergent-to-lipid ratio at which the transition from a lamellar to a mixed lipid/detergent micelle begins.21 Rsat was first determined by following absorption changes at 500 nm of the liposomes as a function of detergent concentration.22 The optimal saturation point, Rsat, was found to be ca. 1:2 detergent-to-lipid molar ratio for β-DDM. The βDDM-solubilized receptor was then added at a protein-to-lipid ratio of 1:1000 (mol:mol), and the mixture was incubated for an additional hour. β-DDM was removed by incubating the mixture with SM-2 Biobeads (10 mg of beads per mg of detergent)23 overnight at 4 °C. Proteoliposomes were finally recovered by ultracentrifugation (100000g for 30 min) and B


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was monitored using a fluorescence spectrophotometer (Cary Eclipse, Varian) with the excitation wavelength set at 500 nm and the emission wavelength set at 511 nm. Reaction conditions were 100 nM Gαqβ1γ2 (GHS-R1a) or Gαi2β1γ2 (MT1R), 100 nM BODIPY FL GTPγS, and 20 nM receptor in lipid discs. Fluorescence was monitored for 40 min at 15 °C after addition of the ligands (1 μM final concentration). For arrestin recruitment assays, the phosphorylation-independent mutant of arrestin-3 (R170E) was produced in Escherichia coli, purified and labeled with the thiol alkylating fluorescent compound monobromobimane (mBBr) as described.20 For μAP2 recruitment assays, residues 160 to 435 of μ-AP2 were expressed in E. coli as an NH2-terminal hexahistidine fusion protein and labeled with mBBr.20 For arrestin and μ-AP2 recruitment assays, the partner proteins were first added to the receptor in lipid discs at 1:2 receptor:signaling protein molar ratio with protein concentrations in the 0.2 μM range. A 20 mM Tris-HCl, 250 mM NaCl, 0.5 mM EDTA, pH 7.4 buffer was used in all experiments. Incubation was carried out for 45 min at 4 °C. The different ligands were then added at a 1 μM concentration and incubated for 45 additional minutes at 4 °C. Fluorescence emission was recorded at 20 °C between 400 and 600 nm (Cary Eclipse, Varian) with an excitation wavelength set at 380 nm. [35S]-GTPγS Binding Assay with MT1R-Expressing CHO Membranes. The membranes and compounds were diluted in the binding buffer (20 mM Hepes pH 7.4, 100 mM NaCl, 3 mM MgCl2, 3 μM GDP) in the presence of 20 μg/mL saponin in order to enhance the agonist-induced stimulation.31 Incubation was started by adding 0.1 nM [35S]-GTPγS to the membranes and ligands in a final volume of 250 μL and allowed to continue for 60 min at room temperature. Nonspecific binding was assessed using nonradiolabeled GTPγS (10 μM). Reactions were stopped by rapid filtration through GF/B unifilters presoaked with distilled water, followed by three successive washes with ice-cold buffer. The data were analyzed using GraphPad PRISM software to yield the maximal effect expressed as a percentage of that observed with melatonin (1 μM = 100%). β-Arrestin Assay with MT1R-Expressing CHO Cells. βArrestin recruitment was monitored using PathHunter CHOK1MT1R beta-arrestin cell line from DiscoveRx. Cells were cultured at 37 °C, 5% CO2 in F12-GlutaMAX medium (ThermoFisher Scientific) supplemented with 10% heatinactivated fetal bovine serum. Engineered MT1R and βarrestin expression was maintained using antibiotic selection (Hygromycin B 300 μg/mL, Geneticin 800 μg/mL). For assays, cells were washed in DPBS, resuspended using Cell Dissociation Solution (Sigma-Aldrich) and seeded into CELLSTAR 384-well microplates (Greiner Bio-one) at 6500 cells per well in 20 μL PathHunter Cell Plating Reagent, then incubated at 37 °C, 5% CO2 for 24 h. After compound incubation (in 1.5% final concentration DMSO for a final volume of 25 μL) for 90 min at 37 °C, 5% CO2, PathHunter detection reagent was added and the Enzyme Fragment Complementation reaction was carried out for 120 min with shaking at room temperature. Chemiluminescent signal was measured on a MicroBeta TriLux (PerkinElmer).

Spectroscopy. Steady state fluorescence emission spectra were recorded using a Cary Eclipse spectrofluorimeter (Varian) with an excitation wavelength at 380 nm (bimane) or 525 nm (fluorescein). The percentage of change in maximum bimane emission corresponds to the difference in intensity between bimane emission in a given condition and that of the ligand-free receptor (I − I(0))/I(0) × 100, where I is the maximum emission intensity in the condition considered and I(0) is the maximum emission intensity in the absence of ligand). SMAsolubilization assays were carried out by monitoring the changes in absorbance at 540 nm as a function of time with a Cary 400 spectrophotometer (Varian) equipped with a thermostated sample changer. A cuvette-based fluorescence lifetime spectrometer with a pulsed Xe lamp as the excitation source was used for the LRET measurements (exc: 337 nm). To avoid any artifact attributable to the incomplete labeling, the donor lifetimes in the presence of the acceptor were measured through the acceptor-sensitized emission at 515 nm. Sensitized emission was fitted to a two exponential decay function, and the goodness of the exponential fit was determined from the random residual distribution with a chi-square value being close to unity. Two lifetimes were calculated from the sensitized emission, a slow and a fast one, with the slow one corresponding to the inactive state of the receptor.28 Molecular fractions of the slow lifetime presented in Figure 4D were calculated from the preexponential factors and the excited state lifetime values as described in Heyduk and Heyduk.29 Finally, the hydrodynamic diameter of the particles was estimated from their diffusion coefficient in dynamic light scattering (λ = 633 nm) using the Stokes−Einstein equation. Functional Assays with the Purified Receptors. For the ghrelin receptor, competition ligand-binding assays were performed using fluorescence energy transfer with a purified receptor labeled with AlexaFluor-350 and a FITC-labeled ghrelin peptide.20 These competition experiments were carried out by adding increasing concentrations of the competing compound to a receptor/ghrelin peptide mixture (100 nM concentration range). Fluorescence emission spectra were recorded at 20 °C between 400 and 600 nm (Cary Eclipse, Varian) with an excitation at 346 or 488 nm. Buffer contributions were systematically subtracted. The FRET ratio corresponds to the ratio of the acceptor emitted fluorescence at 520 nm from excitation at two different wavelengths, 346 and 488 nm. For MT1R, competition ligand-binding assays were performed using 4 nM of [3H]-melatonin as the tracer, and competitor molecules were assayed in the range of 10−13 to 10−3 M. SMA-stabilized lipid discs (1 μg/mL) were incubated in 96-well plates for 2 h at 20 °C in binding buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, and 1 mM EDTA). After incubation, proteins were precipitated by supplementation with 0.1% gamma globulins and 25% PEG 6000 (Sigma-Aldrich) for 15 min. The reaction was stopped by rapid filtration through GF/B unifilters, followed by three successive washes with icecold buffer containing 50 mM Tris-HCl pH 7.4 and 8% PEG 6000. Binding data were analyzed with GraphPad PRISM Software. All data were normalized to the maximal effect triggered by the natural agonist (ghrelin or melatonin) and represent the mean ± SEM of two independent experiments. Functional Assays with the SMA-Stabilized Lipid Discs. GTPγS binding experiments with the receptor isolated in lipid discs were carried out using the BODIPY FL GTPγS analog whose fluorescence increases upon binding to the G protein.30 Association of BODIPY FL GTPγS to the G protein

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RESULTS GHS-R1a Insertion into Liposomes and SMA-Mediated Solubilization. To assess whether SMA could be used to solubilize functional purified GPCRs from a simple model C


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solubilization concentration Rsol recently reported.33 Solubilization was monitored by measuring the turbidity of the liposome solution as a function of time. As shown in Figure 1B, a fast and almost complete liposome solubilization was observed under the conditions used. Importantly, the copolymer did not solubilize the lipids and protein independently but instead cosolubilized both components of the proteoliposome in a single complex, as suggested by the comigration of the protein and fluorescent lipids in size-exclusion chromatography (Supplementary Figure 2). Physical Characterization of the SMA-Stabilized Lipid Discs. GHS-R1a-containing discs after solubilization of the proteoliposomes with the SMA copolymer were purified by Niaffinity chromatography. The homogeneity of these lipid discs was then assessed using size-exclusion chromatography. As shown in Figure 2, the lipid discs eluted from the SEC column

membrane system of well-defined lipid and protein composition, we first reconstituted the detergent-solubilized ghrelin receptor into liposomes. To this end, GHS-R1a was solubilized in β-DDM after refolding in APols.20 To reconstitute β-DDMsolubilized GHS-R1a into liposomes, we then used a method that is based on protein insertion into preformed vesicles as it yields liposomes with asymmetrically inserted proteins.22 Preformed liposomes destabilized by β-DDM were mixed with the β-DDM-solubilized receptor obtained after ligandaffinity chromatography20 under conditions that avoid complete solubilization of the liposomes (see Experimental Procedures). A 1000:1 lipid-to-protein molar ratio was used to avoid crowding effects. The orientation of the protein in the lipid vesicles was then assessed by monitoring the accessibility of a unique reactive cysteine in either the extracellular (C304)27 or the intracellular (C255)28 side of GHS-R1a to a membrane impermeable fluorescent probe (fluorescein maleimide). As shown in Figure 1A, no difference in the labeling ratio of C304

Figure 2. Physical characterization of ghrelin-containing SMAstabilized lipid discs. Size-exclusion chromatography (SEC) profile (Superdex S200 10 × 300 mm) of the ghrelin-containing lipid discs after purification on a Ni-NTA column. Inset: SDS-PAGE profile of the protein in the proteoliposomes (lane 1) and in the lipid disc peak (lane 2).

as a single, major peak with the occurrence of a minor peak in the void volume. The latter peak certainly corresponds to residual aggregated species. This elution profile indicates that the disc preparations were homogeneous, with essentially one single kind of particles whose diameter was estimated to be in the 13 nm range (Supplementary Figure 3). This size is in the same range than that reported for discs obtained from empty liposomes fully solubilized with an excess SMA copolymer.32,33 The ghrelin receptor in SMA containing lipid discs was as stable as it was in lipid discs stabilized with the MSP1E3 lipoprotein and was significantly more stable than in β-DDM (Supplementary Figure 4). Moreover, no noticeable aggregation of the SMA-stabilized lipid discs was observed up to receptor concentrations in the 0.4 μM range (Supplementary Figure 5). Pharmacological Characterization of the Ghrelin Receptor in SMA-Stabilized Lipid Discs. We then assessed whether GHS-R1a in SMA-stabilized lipid discs was functional besides being stable. To this end, we first analyzed the ligand binding properties of the purified receptor and compared them to those we previously measured with the same receptor expressed in HEK293T cells.34 The chemical structure of the ligands used is given in Supplementary Figure 6A. As shown in Table 1, very similar pKi values were obtained in both cases, indicating that the receptor embedded in lipid discs is functional as far as ligand binding is considered. Moreover, essentially all the receptors in the lipid discs were ligandcompetent, as indicated by the molar binding ratio of 0.96

Figure 1. Ghrelin insertion into liposomes and solubilization with the SMA copolymer. (A) Fluorescein maleimide labeling of a unique reactive cysteine in the intracellular (C255) or extracellular (C304) part of the receptor in intact proteoliposomes or in proteoliposomes solubilized with β-DDM. (B) Time-dependent changes in the absorbance at 540 nm of a ghrelin-containing proteoliposome solution after addition of 2.5% SMA (w:v) and incubation at 26 °C.

was observed whether the liposome was solubilized or not with β-DDM. In contrast, no labeling of the receptor could be achieved with the reactive cysteine in the cytoplasmic end of the TM6 segment of GHS-R1a if the liposome was not first solubilized with β-DDM (Figure 1A). This implies that, in the intact liposome, only the extracellular side of GHS-R1a is accessible to the membrane impermeable reagent. Altogether, these data demonstrate that insertion into the liposome was asymmetric with essentially all receptors in the same inside-in orientation. SMA-stabilized lipid discs preparations were then obtained by directly adding the copolymer to the proteoliposomes. The SMA concentration used, i.e., 2.5% (w:v), was that previously reported to be efficient for solubilization of empty liposomes.32 This concentration is significantly higher than the D


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To this end, we used a fluorescence-based assay where a phosphorylation-independent mutant of arrestin-3 was labeled with the fluorescent probe bimane.20 In this case, recruitment of arrestin to the receptor is accompanied by an increase in bimane emission intensity that results from a change in the environment of the probe. As shown in Figure 3B, ghrelin, MK0677 and JMV1843 induced a significant change in bimane emission intensity, indicating that they triggered arrestin recruitment. In contrast, no effect was observed in the presence of JMV2959 and JMV3002, as well as with the SPA inverse agonist. Again, the ligand-dependent arrestin recruitment profile of GHS-R1a in SMA-stabilized lipid discs was fully related to that previously reported in both HEK293T cells35 and MSP1E3-stabilized lipid discs.20 Associated with the fact that the purified ghrelin receptor in SMA-stabilized lipid discs recruited the adaptor protein μ-AP2 also (Supplementary Figure 8), these data directly demonstrate that the ghrelin receptor embedded in SMA-stabilized lipid is fully functional with regard to the first steps of signal transduction, i.e., ligand binding, G protein activation, and recruitment of protein partners. Structural Characterization of the Ghrelin Receptor in SMA-Stabilized Lipid Discs. We then assessed whether the receptor embedded in lipid discs could undergo specific ligandinduced conformational changes. To this end, a receptor mutant with a unique cysteine residue in the extracellular part of TM7 (C304) was first assembled into SMA-stabilized lipid discs as described above and subsequently labeled with the conformational reporter bimane.27 Bimane is a particularly welladapted probe because of its small size and sensitivity to the polarity of its immediate environment.36 Any change in receptor conformation that affects the environment of the probe is therefore accompanied by a change in bimane emission. As shown in Figure 4A, the emission properties of the bimane probe attached to the ghrelin receptor in SMAstabilized lipid discs were sensitive to the activation state of the receptor. Indeed, bimane emission intensity and maximum wavelength were significantly different for the unliganded and ghrelin-liganded GHS-R1a, indicating that both receptor states vary in their conformational features. Furthermore, the emission intensity was also different depending whether the ligand was a full- (ghrelin), a partial- (JMV3002), or an inverseagonist (SPA) (Figure 4B). This indicates that each of these ligand stabilize a different, well-defined conformation directly related to its pharmacological class. Of importance, the changes in fluorescence intensity triggered by the different ligands with the receptor in SMA-stabilized lipid discs were essentially similar to those we previously measured with the same receptor in lipid discs stabilized with the MSP1E3 lipoprotein (Figure 4B). We then used intramolecular LRET to further explore the activation process of the ghrelin receptor assembled into SMAstabilized lipid discs and establish that the copolymer does not introduce additional restrictions to the protein other than those of the membrane itself. In this case, the ghrelin receptor was labeled with a Tb donor and an Alexa Fluor 488 acceptor in the cytoplasmic ends of TM6 and TM1, respectively (Figure 4C).28 As shown in Figure 4D, the population of the short lifetime corresponding to the inactive state of the receptor28 was very similar whether SMA- or MSP-stabilized discs were considered, both in the absence of ligand, in the presence of the full-agonist ghrelin, or in the presence of the inverse agonist SPA. In contrast, the proportion of the short component was

Table 1. pKi of GHS-R1a Ligands Determined from Competitions Assays Using the Purified Receptor in Lipid Discs or HEK293T Cellsa SMA-stabilized lipid discs ghrelin MK0677 JMV1843 JMV2959 JMV3002 SPA a

7.02 7.24 5.93 7.06 7.77 6.84

± ± ± ± ± ±

0.96 1.02 1.55 1.08 1.13 0.95

HEK293T 7.95 8.07 6.31 7.26 7.85 6.64

± ± ± ± ± ±

1.79 1.54 1.17 1.11 1.45 0.73

pKi data from HEK293T cells are those reported in Leyris et al.34.

ghrelin peptide per GHS-R1a inferred from the stoichiometric titration assay in Supplementary Figure 7. We subsequently analyzed receptor-catalyzed G protein activation using the purified Gαqβ1γ2 trimer and the fluorescent GTPγS analogue Bodipy FL GTPγS.20 As shown in Figure 3A,

Figure 3. The ghrelin receptor in SMA-stabilized lipid discs activates G proteins and recruits arrestin. (A) BODIPY FL GTPγS binding to the α subunit of Gαqβ1γ2 triggered by empty discs or GHSR-1acontaining discs in the absence or in the presence of ligands. (B) Changes in emission intensity of bimane-labeled arrestin-3 induced by empty lipid discs or GHSR-1a-containing discs in the absence or in the presence of ligands. All data are presented as the mean value ± SEM of two independent measurements.

the unliganded receptor displayed a significant activity at promoting GDP/GTP exchange, as is the case of this receptor in eukaryotic cells or in MSP-stabilized lipid discs.20 In the presence of its full agonists ghrelin, JMV1843 or MK0677, a significant increase in G protein activation was observed, whereas the partial agonists JMV3002 and JMV295935 triggered a slight but nevertheless significant increase in Gαq activation (Figure 3A). Finally, the inverse agonist SPA essentially abolished basal activity (Figure 3A). As is the case for ligand binding, these data demonstrate that the ghrelin receptor embedded in SMA-stabilized lipid discs is fully functional with regard to G protein activation. Finally, we analyzed whether the purified receptor in lipid discs recruited purified arrestin in the presence of its agonists. E


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Figure 4. Ligands trigger conformational changes of GHS-R1a in SMA-stabilized lipid discs. (A) Fluorescence emission spectra of bimane attached to C304 in GHS-R1a-containing discs in the absence of ligand or in the presence of ghrelin. (B) Percent change in bimane maximum emission intensity induced by functionally different ligands. (C) Schematic representation of the system used for the LRET experiments. (D) Amplitude of the short lifetime component in the sensitized emission decays for the ghrelin receptor in SMA discs, in MSP1E3 discs or in β-DDM in the absence of ligand, in the presence of ghrelin, or in the presence of SPA. Data are presented as the mean value ± SEM of two independent measurements.

significantly higher when the receptor was assembled in βDDM. All together, the bimane emission and LRET experiments indicate that activation-related changes in ghrelin receptor conformation are identical whether the lipid discs are stabilized by the SMA copolymer or the MSP lipoprotein. Besides validating the SMA-solubilization procedure, these data suggest that GHS-R1a conformational dynamics is dependent on its direct lipid environment and that lipids have a similar effect on receptor dynamics whether the bilayer is stabilized by the SMA copolymer or the MSP1E3 lipoprotein. GPCR Solubilization from P. pastoris Membranes. The data reported above demonstrate that a functional GPCR can be extracted directly from a simple membrane model system, liposomes, in the absence of any detergent. However, this procedure still requires manipulation of the protein prior to insertion into liposomes. To assess whether SMA could be utilized also to extract a GPCR directly from a cellular membrane, we then used the melatonin receptor MT1R expressed in P. pastoris. This expression system was selected as it has been repeatedly used for high-yield expression of GPCRs.13 The MT1R was used here instead of GHS-R1a because it is expressed in high yields in P. pastoris membranes and it has been extensively characterized in this system.24 Indeed, the human MT1R is routinely expressed to amounts close to 12 pmol/mg,24 which roughly corresponds to about 0.5 mg of ligand-binding active receptors per liter of culture. The membrane fractions of P. pastoris analyzed here were thus prepared from this MT1R-expressing clone. The same strain expressing no recombinant receptor was used as a control. SMA solubilization was carried out by incubating the membrane preparation with the polymer at 4 °C. Solubilization was carried out at 4 °C instead of 26 °C, as we did with proteoliposomes,

to avoid any possible degradation of the receptor protein when using crude membrane extracts. Although decreasing the temperature was associated with a decrease in the kinetics of solubilization (Supplementary Figure 9), similar yields in lipid discs were obtained at both temperatures. Only increasing the temperature up to 40 °C resulted in a more efficient solubilization (Supplementary Figure 10A). However, under such conditions, the receptor was essentially unable to activate its cognate G protein (Supplementary Figure 10B), probably due to protein unfolding at this temperature. In the same way, increasing the amount of SMA copolymer increased the kinetics of solubilization (Supplementary Figure 11) without significantly affecting solubilization efficiency (Supplementary Figure 12). Finally, slightly slower solubilization kinetics were also observed upon increasing the salt concentration (Supplementary Figure 13). After removal of the unsolubilized material by ultracentrifugation, MT1R-containing discs were purified by Niaffinity chromatography. The homogeneity of our preparation was then assessed with SEC. As shown in Figure 5, the SEC profile was significantly different from that obtained after solubilization of liposomes. In particular, a major peak was observed in the void volume of the column, indicating the occurrence of large aggregates that likely correspond to large lipid structures and/or unsolubilized membranes fragments. Besides the peak in the void volume of the column, however, another peak was observed in the SEC profile at an elution volume of ca. 11.5 mL, corresponding to particles with a diameter in the 14 nm range (Supplementary Figure 3) and containing MT1R (see inset to Figure 5). Pharmacological Characterization. We then assessed the functional features of the MT1R receptor in lipid discs after F


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observed with S20928, indicating that this ligand is neutral with regard to both Gαi activation and arrestin recruitment (Figure 6C). As shown in Figure 6D, these ligands triggered arrestin recruitment to the MT1 receptor expressed at the membrane of CHO cells with an efficacy closely related to that measured with the purified receptor. The close correlation between the ligand-dependent arrestin recruitment profiles obtained with MT1R in lipid discs and CHO cells is a further demonstration of the relevance of the purified receptor with regard to its downstream signaling properties. Altogether, all these data directly demonstrate that the MT1 receptor embedded in SMA-stabilized lipid discs is fully functional as far as ligand binding, G protein activation and arrestin recruitment are considered.

Figure 5. Physical characterization of MT1R-containing SMAstabilized lipid discs. Size-exclusion chromatography (SEC) profile (Superdex S200 10 × 300 mm) of the MT1R-containing lipid discs after purification on a Ni-NTA column. Inset: Western blot analysis of the proteins in the MT1R-containing discs (elution peak at 11.5 mL) using an antiFlag antibody.

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DISCUSSION We have shown here that SMA copolymers can solubilize GPCRs in lipid discs starting from a simple lipid model system such as liposomes or from the more complex membranes of yeast. This solubilization step preserves the functional properties of the receptors not only with regard to ligand binding, as reported with the adenosine receptor,12 but also with regard to agonist-specific G protein activation and arrestin recruitment. Moreover, agonist-induced changes in receptor conformation are preserved after SMA solubilization, directly indicating that the copolymer does not introduce any constraint on the receptor protein. These receptor-containing lipid discs are therefore valuable models to further analyze the molecular events underlying the first steps in GPCR-mediated signaling with any biophysical method that requires purified and functional receptors in a native lipid environment. Detergents have been widely used to manipulate GPCRs in the context of biophysical studies.38 However, they have deleterious effects on receptor folding. There are many causes of detergent-associated membrane protein inactivation. These include loss of physical constraints provided by the membrane environment, competition with protein/protein and protein/ lipid interactions, and trapping of any lipid or cofactor that may be necessary to the protein stability and/or function.3 On the basis of these considerations, alternative media have been designed to circumvent the deleterious effects of detergents. In this context, we show here that SMA copolymers can be used to solubilize GPCRs directly from a lipid environment. Associated with previous data demonstrating that this procedure applies to other membrane proteins as well,7−9,11,12 our observations emphasize the potential of these copolymers in the context of biophysical characterization of integral membrane proteins such as GPCRs. We used two independent model membrane systems of increasing complexity. The first one, proteoliposomes, was obtained after insertion of the detergent-solubilized ghrelin receptor into preformed liposomes. This procedure allowed asymmetric insertion of the receptor in the kinetically most favorable orientation that corresponds, in the case of GHS-R1a, to the inside-in one. Such an asymmetric insertion could be linked to the distribution of charged residues of the ghrelin receptor that significantly differs between the intra- and extracellular sides of the protein. Although not crucial for the experiments reported here that are based on subsequent solubilization of the proteoliposomes, this asymmetric insertion is of interest for further studies on the functional behavior of GHS-R1a in a complex lipid environment as it solves the critical issue of protein orientation. The SMA copolymer then

SEC fractionation (elution peak at 11.5 mL). To this end, we first analyzed the ligand binding properties of the purified receptor and compared them to those measured from the starting material of the SMA-solubilization, i.e., MT1Rcontaining P. pastoris membranes. As shown in Table 2, almost Table 2. pKi of MT1R Ligands Determined from Competitions Assays Using the Purified Receptor in SMAStabilized Discs or in Pichia pastoris Membrane Fractionsa SMA-stabilized lipid discs melatonin SD6 S20928 S21278

8.29 8.94 5.24 5.88

± ± ± ±

0.49 0.43 0.38 0.58

Pichia pastoris membrane 8.88 9.68 5.93 5.34

± ± ± ±

0.02 0.01 0.04 0.01

a

pKi data from P. pastoris membranes are those reported in Logez et al.24.

identical pKi values were obtained in both cases for all ligands (see chemical structures of the ligands in Supplementary Figure 6B), indicating that the receptor embedded in lipid discs is functional as far as ligand binding is considered. We subsequently analyzed receptor-catalyzed G protein activation using the purified G protein partner of MT1R, Gαi2β1γ2.37 As shown in Figure 6A, in the presence of the natural agonist melatonin, a significant increase in G protein activation was observed. A similar effect was observed with another full agonist SD6, whereas the partial agonist S21278 at saturating concentrations triggered receptor-catalyzed G protein activation to an extent lower than that triggered by melatonin or SD6 (Figure 6A). Finally, no change in G protein activation was observed with the neutral antagonist S20928 (Figure 6A). As shown in Figure 6B, an almost identical liganddependent [35S]-GTPγS binding profile was obtained with membranes of CHO cells expressing MT1R. As is the case for ligand binding, these data demonstrate that the MT1R embedded into SMA-stabilized lipid discs is fully functional with regard to G protein activation. Finally, we analyzed whether the purified MT1 receptor in SMA-stabilized lipid discs recruited the phosphorylationindependent mutant of arrestin-3 labeled with bimane. As shown in Figure 6C, melatonin induced arrestin recruitment to the MT1R-containing discs, whereas S21278 and SD6 appeared as partial agonists at saturating concentrations with regard to arrestin recruitment. Finally, no arrestin recruitment was G


Article

Biochemistry

Figure 6. The melatonin receptor in SMA-stabilized lipid discs activates G proteins and recruits arrestin as it does in CHO cells. (A) BODIPY FL GTPγS binding to the α subunit of Gαi2β1γ2 triggered by MT1R-containing discs in the presence of ligands. (B) [35S]-GTPγS-binding in MT1Rexpressing CHO cell membranes in the presence of different ligands. (C) Changes in emission intensity of bimane-labeled arrestin-3 induced by MT1R-containing discs in the presence of ligands. (D) Arrestin-recruitment in MT1R-expressing CHO cells in the presence of different ligands. Data with purified receptors are presented as the mean value ± SEM of two independent measurements; data in CHO cells are presented as mean value ± SEM of three independent measurements.

inserted in lipid discs stabilized by the SMA copolymer or by the MSP1E3 lipoprotein. These data directly establish that the copolymer does not introduce additional restrictions to the protein other than those of the membrane itself. Of importance, the purified receptor in SMA-stabilized lipid discs also activates G proteins and recruits partner proteins such as arrestin or μAP2. As these proteins have been shown to interact with the intracellular loops (G protein, arrestin) and/or the C-terminal domain (arrestin, μ-AP2) of the receptor, these data indicate that the polymer does not affect the organization of the extramembrane domains either. The absence of any direct effect of the copolymer on both the transmembrane and the intra- and extracellular domains of the receptor suggests that the SMA copolymer does not interact directly with the protein, in contrast to other polymeric species such as amphipols, but rather stabilizes the lipid bilayer around the receptor protein. As lipids have been repeatedly shown to have an impact on the conformational dynamics of GPCRs,39 the similarity in fluorescence signature between the receptor in SMA- and MSPstabilized discs suggest that the copolymer cosolubilizes lipids in sufficient amount so that the receptor dynamics are preserved. This is confirmed by the ability of the purified receptor to activate G proteins and recruit arrestins. Indeed, the lipid environment has been shown to play a critical role in both

efficiently cosolubilized lipid:protein complexes from proteoliposomes using standard conditions previously reported for empty liposomes. These conditions correspond to an SMA concentration significantly higher than the Rsol value recently determined.33 Rsol is the concentration value above which full solubilization of liposomes occurs to give rise to lipid particles of well-defined size. The similarity in the solubilization behavior of liposomes and proteoliposomes suggests that the presence of the receptor at the lipid-to-protein ratios used in this work does not significantly affect the mechanism of solubilization of the lipid bilayer by the SMA copolymer. Although it requires purified protein, the approach based on the insertion of GPCRs in liposomes of controlled lipid composition followed by SMA solubilization has major advantages for the analysis of the influence of the lipid nature on receptor dynamics. This approach could also be an attractive way to investigate receptor oligomerization as it should be possible to coassemble within the same liposomes a receptor with its potential partners and then trap the protein complexes with SMAs. Both the bimane and LRET measurements indicate that the ghrelin receptor in SMA-stabilized lipid discs undergoes specific conformational rearrangements associated with the binding of ligands from distinct pharmacological classes. Moreover, these conformational changes were similar whether the receptor was H


Article

Biochemistry

ligands.12 In addition, we showed here that receptors isolated in SMA-stabilized lipid discs maintain their functional properties with regard to G protein activation and arrestin recruitment. This implies that the SMA copolymer preserves the lipid environment and structural features required for all these processes. Such an observation indicates that purified receptors could be used for primary screening purposes to unambiguously identify compounds that bind to the receptor and selectively activate one or the other of the signaling pathways associated with this receptor. This could help circumvent issues of allosteric modulation, receptor dimerization, and ligand functional selectivity that still create challenges in selecting suitable assay formats. In closing, the data reported here with two model receptors, GHS-R1a and MT1R, associated with the recent report that SMA can solubilize the adenosine receptor expressed in HEK cells also,12 are evidence that SMA-stabilized lipid discs are a highly promising and convenient alternative to detergents pharmacological characterization of purified GPCRs. The potential use of these discs in many biophysical studies may lead to significant advances in the elucidation of GPCR dynamics and of how the conformational landscape of a receptor is modulated not only by ligands and signaling proteins but also by the membrane environment.

processes. When considering the influence of lipids on receptor dynamics, it is important to note that the LRET data with the ghrelin receptor in lipid discs stabilized by either SMAs or MSPs were significantly different from those obtained with the receptor assembled in β-DDM. In particular, the inactive state of the ghrelin receptor appeared to be favored in the detergent compared to the lipid discs. This observation clearly indicates that the membrane environment has an influence on receptor conformational exchange and that detergent molecules are probably not the best-adapted system to analyze these dynamics, as they seem to modify the equilibrium between active and inactive states of the receptor. Whether this difference is due to a direct effect of the lipids on GHS-R1a conformational transitions or the consequence of the assembly in detergent because, for instance, of a fast chemical exchange of detergent molecules at the surface of the protein,40 is an open question at the present stage of the analysis. SMA-based solubilization was tested, in a second stage, with a more complex membrane system, i.e., membranes from yeast. Compared to what we obtained with proteoliposomes, solubilization of P. pastoris membranes resulted in lower solubilization yields and more heterogeneous SEC profiles with the occurrence of residual membrane fragments eluting in the void volume of the column, although the SMA copolymer concentrations used were well-above the Rsol value.33 In our hands, increasing the lipid-to-SMA ratio or changing the ionic strength did not significantly increased solubilization yields, although it affected significantly the kinetics of solubilization. Only increasing the solubilization temperature up to 40 °C resulted in significantly higher solubilization ratios, but this also triggered receptor misfolding. One possibility, as recently proposed,32 would be that the lipid composition of the yeast membrane precludes efficient solubilization either through a direct effect associated with the phase transition of the lipids or through an indirect effect on membrane thickness. Further work, such as that recently published for the solubilization of liposomes with SMAs,33 will likely be necessary to rationalize the solubilization process of these complex membranes by the SMA copolymer and by doing so allow optimal yields in receptor-containing particles to be achieved. Interestingly, the species obtained after solubilization of proteoliposomes and yeast membranes were of similar size, i.e., in the 13−14 nm range. Particles of similar size were also obtained after solubilization of P. pastoris membranes expressing the adenosine receptor.12 This could mean that the disc size is not primarily dependent on the kind of starting membrane system used. In contrast, the size of the trapped protein could impact the geometrical features of the discs as incorporation into SMA-stabilized lipid discs of proteins and/or complexes of different sizes led to particles of varied size ranging from 12 up to 24 nm in diameter.32 The yields in purified receptor-containing discs obtained from P. pastoris membranes are significantly lower than those achieved with proteoliposomes. This could preclude their use in biophysical approaches that require large protein amounts such as NMR. SMA-stabilized discs obtained from such complex expression systems nevertheless appear as a most valuable alternative to other stabilizing media for structural studies requiring native receptors in low amounts (e.g., single molecule fluorescence methods) or in the context of GPCR pharmacological characterization. It had been previously shown that the adenosine receptor solubilized from P. pastoris or HEK cell membranes in SMA-stabilized discs was able to bind its

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01040. Supplementary Figure 1. Effect of the polymerization degree of the SMA copolymer on Pichia pastoris membrane solubilization. Supplementary Figure 2. SMA copolymers co-solubilize proteins and lipids. Supplementary Figure 3. The SMA-solubilized lipid discs are homogeneous. Supplementary Figure 4. The SMAsolubilized lipid discs are stable. Supplementary Figure 5. The SMA-solubilized lipid discs can be concentrated. Supplementary Figure 6. Chemical structure of the GHSR1a (A) and MT1R (B) ligands used throughout this work. Supplementary Figure 7. The ghrelin receptor in lipid discs is ligand-competent. Supplementary Figure 8. GHSR-1a in lipid discs binds μ-AP2. Supplementary Figure 9. Effect of the temperature on Pichia pastoris membrane solubilization by the SMA copolymer. Supplementary Figure 10. Effect of the temperature on Pichia pastoris membrane solubilization by the SMA copolymer and on receptor stability. Supplementary Figure 11. Effect of the amount of polymer on Pichia pastoris membrane solubilization. Supplementary Figure 12. Effect of the amount of polymer on Pichia pastoris membrane solubilization. Supplementary Figure 13. Effect of the salt concentration on Pichia pastoris membrane solubilization by the SMA polymer. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

C.L., C.L., C.D., O.N., B.F., G.F., and J.A.B. are employees of the pharmaceutical company Les Laboratoires SERVIER. This work was supported by the Centre National de la Recherche I


Article

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(12) Jamshad, M., Charlton, J., Lin, Y. P., Routledge, S. J., Bawa, Z., Knowles, T. J., Overduin, M., Dekker, N., Dafforn, T. R., Bill, R. M., Poyner, D. R., and Wheatley, M. (2015) G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent. Biosci. Rep. 35, No. e00188. (13) Bornert, O., Alkhalfioui, F., Logez, C., Wagner, R. (2012) Overexpression of membrane proteins using Pichia pastoris. In Current Protocols in Protein Science; Coligan, J. E., Eds.; Chapter 29, Unit 29.22. (14) Sivertsen, B., Holliday, N., Madsen, A. N., and Holst, B. (2013) Functionally biased signalling properties of 7TM receptors opportunities for drug development for the ghrelin receptor. British journal of pharmacology 170, 1349−1362. (15) Wurtman, R. J., and Axelrod, J. (1965) The Formation, Metabolism, and Physiologic Effects of Melatonin in Mammals. Prog. Brain Res. 10, 520−529. (16) Reiter, R. J. (1991) Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr. Rev. 12, 151−180. (17) Millan, M. J., Brocco, M., Gobert, A., and Dekeyne, A. (2005) Anxiolytic properties of agomelatine, an antidepressant with melatoninergic and serotonergic properties: role of 5-HT2C receptor blockade. Psychopharmacology 177, 448−458. (18) Zlotos, D. P., Jockers, R., Cecon, E., Rivara, S., and WittEnderby, P. A. (2014) MT1 and MT2 melatonin receptors: ligands, models, oligomers, and therapeutic potential. J. Med. Chem. 57, 3161− 3185. (19) Nosjean, O., Ferro, M., Coge, F., Beauverger, P., Henlin, J. M., Lefoulon, F., Fauchere, J. L., Delagrange, P., Canet, E., and Boutin, J. A. (2000) Identification of the melatonin-binding site MT3 as the quinone reductase 2. J. Biol. Chem. 275, 31311−31317. (20) Damian, M., Marie, J., Leyris, J. P., Fehrentz, J. A., Verdie, P., Martinez, J., Baneres, J. L., and Mary, S. (2012) High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs. J. Biol. Chem. 287, 3630−3641. (21) Rigaud, J. L., Pitard, B., and Levy, D. (1995) Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins. Biochim. Biophys. Acta, Bioenerg. 1231, 223−246. (22) Rigaud, J. L., and Levy, D. (2003) Reconstitution of membrane proteins into liposomes. Methods Enzymol. 372, 65−86. (23) Rigaud, J. L., Mosser, G., Lacapere, J. J., Olofsson, A., Levy, D., and Ranck, J. L. (1997) Bio-Beads: an efficient strategy for twodimensional crystallization of membrane proteins. J. Struct. Biol. 118, 226−235. (24) Logez, C., Berger, S., Legros, C., Baneres, J. L., Cohen, W., Delagrange, P., Nosjean, O., Boutin, J. A., Ferry, G., Simonin, F., and Wagner, R. (2014) Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes. PLoS One 9, e100616. (25) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248−254. (26) Kunji, E. R., Harding, M., Butler, P. J., and Akamine, P. (2008) Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography. Methods 46, 62−72. (27) Mary, S., Damian, M., Louet, M., Floquet, N., Fehrentz, J. A., Marie, J., Martinez, J., and Baneres, J. L. (2012) Ligands and signaling proteins govern the conformational landscape explored by a G proteincoupled receptor. Proc. Natl. Acad. Sci. U. S. A. 109, 8304−8309. (28) Damian, M., Mary, S., Maingot, M., M’Kadmi, C., Gagne, D., Leyris, J. P., Denoyelle, S., Gaibelet, G., Gavara, L., Garcia de Souza Costa, M., Perahia, D., Trinquet, E., Mouillac, B., Galandrin, S., Gales, C., Fehrentz, J. A., Floquet, N., Martinez, J., Marie, J., and Baneres, J. L. (2015) Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex. Proc. Natl. Acad. Sci. U. S. A. 112, 1601−1606. (29) Heyduk, T., and Heyduk, E. (2001) Luminescence energy transfer with lanthanide chelates: interpretation of sensitized acceptor decay amplitudes. Anal. Biochem. 289, 60−67.

Scientifique (CNRS), Université de Montpellier, grants from the Agence Nationale de la Recherche (PCV08-323163; ANR13-BSV8-0006) and from Laboratoires Servier. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We wish to thank Eric Trinquet (Cisbio Bioassays) and Sebastien Granier (IGF, Montpellier) for fruitful discussions on LRET measurements. We are also grateful to Bernard Mouillac, Gérald Gaibelet and Hélène Orcel (IGF, Montpellier) for discussions on arrestin production and labeling as well as for their advice on G protein preparations.

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ABBREVIATIONS APol: amphipol; DLS: dynamic light scattering; FITC: fluorescein isothiocyanate; GHS-R1a: growth hormone secretagogue receptor type 1; GPCR: G protein-coupled receptor; mBBr: monobromobimane; LRET: time-resolved luminescence resonance energy transfer; MSP: membrane scaffolding protein; MT1R: melatonin receptor type 1; pAzF: p-azido-L-phenylalanine; SEC: size-exclusion chromatography; SMA: styrenemaleic acid; SPA: substance P analogue; TM: transmembrane; β-DDM: n-dodecyl-β-D-maltopyranoside

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