Supporting InformationRahmeh et al. 10.1073/pnas.1201093109SI TextConstruction of the Receptor Mutants. Site-directed mutagenesis ofthe human V2 receptor cDNA was directly performed ontopFASTBac1 vector (Invitrogen). Mutations were all generated byPCR-mediated mutagenesis using PrimeSTAR HS polymeraseaccording to the manufacturer’s instructions (TAKARA) andwere verified by restriction enzyme analysis and DNA sequencing.

Expression and Functionality of Flag-V2R. A Flag-tagged version ofV2R (Flag-V2R) was expressed in insect cells by using recombinantbaculovirus technology. Immunofluorescence experiments showedthat thereceptorwasproperlyexpressedat thecell surface(Fig.S1A).Saturation binding assays performed on whole cells with the ra-diolabeled agonist [3H]-AVP demonstrated that the Flag-V2R wasable to bind its ligandwith aKd value of 13.5± 0.7 nM (n=3), whichis close to that obtained in mammalian cells (1) (Fig. S1B). To ex-amine whether the Flag-V2R retained the ability to couple to Gsprotein, we determinedAVP-induced cAMPaccumulation inwholecells using a commercially available kit (cAMP dynamic2; Cis-BioInternational) as described previously (2), with some modifications(Fig. S1C). Briefly, 52 h after infection of Sf9 cells using the re-combinant baculovirus to be tested, 2 × 105 cells were distributed in200 μL of Insect-Xpress medium (Lonza) into a 96-well assay plate(Greiner Bio-One) and incubated at 27 °C. Five hours later, themedium was removed and replaced with 50 μL of incubation me-dium containing the agonist at the appropriate concentrations.

Receptor Expression, Solubilization, and Purification from Sf9 InsectCells. Sf9 insect cells were grown at 27 °C in suspension cultures inInsect-Xpress medium. As described previously (3), Sf9 cellswere infected by recombinant baculoviruses generated in thesecells using the Bac-to-Bac Baculovirus Expression system (In-vitrogen). For receptor purification, 1 L of Sf9 cell culture at adensity of 3 × 106 cells/mL was infected with appropriate virusesand harvested after 60 h by centrifugation (10 min at 5,000 × g).The cell pellets were kept at −80 °C until use.For solubilization, the cell pellets were lysed in lysis buffer [15

mM Tris·HCl (pH 7.4), 2 mM MgCl2, 0.3 mM EDTA, 10 μg/mLbenzamidine, 10 μg/mL PMSF, 5 μg/mL leupeptin]. Followingcentrifugation (10 min at 30,000 × g, 4 °C), different mixes ofdetergent were compared. The radiolabeled inverse agonist [3H]-SR121463 was used to measure the V2R fraction retaining li-gand-binding properties. We determined that a solubilizationbuffer [20 mM Tris·HCl (pH 7.4), 50 mM NaCl] containing 0.5%Fos-Choline-12 or Fos-Choline-14 (Anatrace) or 0.2% sodiumcholate (Sigma), 0.5% N-dodecyl-β-D-maltopyranoside (Ana-trace), and 0.05% cholesteryl hemisuccinate (Anatrace) main-tains the functional receptor. For all subsequent steps, the lysedcells were suspended in the appropriate solubilization buffer,subjected to 20 strokes of tight dounces using a 40-mL SZ tissuegrind tube (Kontes), and then stirred for 1 h at 4 °C. Aftercentrifugation (15 min at 30,000 × g) at 4 °C, the supernatant wasloaded onto an M1 Flag antibody affinity resin (Sigma) at a flowrate of about 1 mL/min. The resin was then washed with threecolumn volumes of low-salt washing buffer [solubilization bufferdiluted 10 times in 20 mM Tris·HCl, (pH 7.4), 50 mM NaCl]alternatively with a high-salt washing buffer (500 mM NaCl) (3times each). After a last washing step with the low-salt buffer, thereceptor was recovered with the elution buffer (washing buffercontaining 2 mM EDTA and 200 μg/mL Flag peptide). The totalprotein amount was estimated by UV absorbance spectroscopy.Reconstitution of detergent-purified V2R into NAPols and pu-

rification of complexes containing monomeric V2R are de-scribed in the main text and in Fig. 1B.

NAPol Synthesis. TheNAPol used in the present study, NA11 (batchSS325), is amemberof themost advanced series (Pucci et al.,Frenchand European Patent; Sharma et al., in preparation; Denis-Billonet al., in preparation) of nonionic Apols (4–6). The synthesis ofNA11 is based on the free-radical homopolymerization of a diglu-cosylated amphiphilic monomer (7) in the presence of a thiol-basedtransfer agent (8). NA11 exhibits an average molecular massof ∼11.3 kDa and a number-average degree of polymerizationof ∼17.

[3H]-AVP Binding on Soluble V2R. The Flag-tagged monomericNAPol-reconstituted V2R was first quantified by UV spectro-photometryandby themicro-BCAprotein assay (ThermoScientificPierce). Binding of radiolabeled [3H]-AVP (56.9 mCi/mmol;PerkinElmer) to the soluble receptor was developed using anti-Flag M2 magnetic beads (Sigma Aldrich). V2R (5 nM, 1 pmol/tube) was incubated for 1 h at 22 °C in binding buffer [20 mMTris·HCl (pH 8.0), 100 mM NaCl] with increasing concentrationsof [3H]-AVP (from 100 nM to 5 μM) in the presence or absence ofAVP (50 μM) to define total and nonspecific binding. The mag-netic beads (10-μL slurry) were added at the beginning of the re-ceptor-AVP incubation period to allow proper binding betweenthe anti-Flag antibodies and the Flag tag. The samples were thenkept on ice for 20 min, and separation of bound and free radio-labeled [3H]-AVP was done by phase separation using a magneticparticle concentrator (Invitrogen). The supernatant was removed,and the magnetic bead pellet was resuspended in cold bindingbuffer (2 × 0.5 mL). The beads (1 mL) were directly diluted inscintillation fluid (4 mL), and radioactivity was determined byliquid scintillation counting. Kd and Bmax values were obtainedfrom the ligand binding data analyses performed with nonlinearleast-squares regression using Prism (GraphPad Software, Inc.).

[35S]-GTPγS Binding and Arrestin-2 Recruitment Assay. Gαs proteinwas produced and purified as described below and in Fig. S7. ThemonomericNAPol-reconstitutedV2R and purifiedGprotein weremixed (1:2.5 receptor/G protein ratio, 20:50 nM) in binding buffer[50 mM Tris·HCl (pH 7.4), 5 mM MgCl2, 5–10 nM [35S]-GTPγS(1,250 Ci/mmol; PerkinElmer Life Sciences), 1 μM GDP] with orwithout V2R ligands. Incubations were performed for 1 h at 22 °C.Nonspecific binding was determined in the presence of 100 μMGTPγS. Bound [35S]-GTPγS was separated from free [35S]GTPγSusing His-Select magnetic agarose beads (20-μL slurry of nickel-coated beads; SigmaAldrich) and amagnetic particle concentrator(Invitrogen). The supernatant was removed, and the magneticbead pellet was resuspended in cold binding buffer (2 × 0.5 mL).The beads (1 mL) were mixed with scintillation fluid (4 mL), andradioactivity was determined by liquid scintillation counting. The[35S]-GTPγS binding data were analyzed using Prism.The arrestin-2mutant L68C-R169Ewas produced inEscherichia

coli and purified by immobilized metal ion affinity chromatography(IMAC) as described by Gurevich and Benovic (9). The purifiedprotein was then labeled with monobromobimane at C68 as de-scribed by Sommer et al. (10). For the arrestin recruitment assay,arrestin-2 was added to the NAPol-reconstituted V2R (1:1 ar-restin/receptor molar ratio, 10 μM) in the absence or presence ofAVP, SR121463, or MCF14 (1:1 ligand/receptor molar ratio).After 2 h of incubation at 4 °C, fluorescence emission was recordedat 20 °C between 400 and 600 nm, with an excitation wavelength at

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380 nm. All measurements were done in triplicate. Raw data arepresented in Fig. S2.

Tryptophan Intrinsic Fluorescence. All tryptophan fluorescenceemission spectra were recorded at 20 °C between 305 nm and 395nm, with an excitation wavelength at 296 nm. A protein concen-tration of 200 nM was used in all measurements. For testing theeffects of V2R-specific drugs, NAPol-reconstituted V2R in thepresence or absence of the amount of ligands to be testedwasmixedand incubated for 15 min at room temperature before recordingfluorescence. Fluorescence intensity was corrected for backgroundfluorescence from buffer and ligands in all experiments. Thisbackground represents less than 2%, 16%, and 3% of the receptorsignal for AVP,MCF, and SR, respectively. All measurements weredone in triplicate on three independent samples, and individualspectra were acquired. An average of the intensity of tryptophanemission between 340 and 350 nm was then calculated and nor-malized to that of untreated V2R [Iλmax(ligand)/Iλmax(vehicle)].

V2R-Specific Labeling to Perform LRET Experiments. To study ligand-induced conformational changes, we measured LRET signals andcalculated distances between the C terminus and two domains ofthe V2R: the cytoplasmic end of TM6, near the third intracellularloop, and the cytoplasmic end of TM7, just before the in-tracellular H8. These domains are involved in G protein couplingand arrestin recruitment (11, 12).To label the distal C terminus of the V2R, we introduced the

previously characterized CCPGCC motif that can be specificallylabeled with FlAsH compounds (commercially known as LumioGreen labeling reagents). When the purified V2R-CCPGCC wasincubated with FlAsH without the reducing agent Tris(2-car-boxyethyl)phosphine (TCEP) or when V2R lacking the CCPGCCmotif was incubated with both FlAsH and TCEP, almost no la-beling was observed (Fig. S4A). This indicates that the endoge-nous cysteines of the receptor are not reactive toward the FlAsHcompound and that, as previously observed (3), a reducing agentlike TCEP is required for efficient labeling. Maximal labelingwas obtained by incubation of V2R-CCPGCC with both TCEP(100 μM) and FlAsH (3 equivalents).To label either the cytoplasmic end of TM6 or the TM7-H8

junction with a second fluorophore specifically, several naturalamino acids were substituted for cysteine one at a time. In ad-dition, because Cys358 located in the C-terminus end was po-tentially accessible to thiol-reactive compounds, this residue wasmutated to an alanine. To evaluate the accessibility of the in-troduced cysteines, we measured the labeling kinetics of V2Rcysteine mutants with fluorescein maleimide. This method waspreviously shown to assess the accessibility of cysteines inGPCRs reliably (13). We found that the cysteines at positions267 and 330 (Flag-V2R-A267C-C358A-FlAsH and Flag-V2R-S330C-C358A-FlAsH, respectively) were the most reactive (4- to6-fold more reactive than the WT V2R) (Fig. S4 B and C). Usingthese kinetics data to solve the association kinetics equationindicates that 85% of the most reactive cysteines are labeledwhen 0.5 equivalent of the thiol-reactive probes is used.Therefore, to measure LRET between fluorophores inserted at

the C terminus and at the end of the TM6 domain or at the TM7-H8 domain, the detergent-purified TM6 sensor and TM7-H8sensor were sequentially labeled with the acceptor fluorophoreFlAsH (C-terminal labeling), followed by the donor fluorophoreLumi4-Tb. The FlAsH/receptor labeling ratio ranged from 0.4 to0.6 mol of dye per mole of receptor, whereas that of Lumi4-Tb/receptor ranged from 0.5 to 0.7 (Fig. S5A). Considering theseacceptor/donor ratios, the probability of double-labeling a givenreceptor is only 30%. Consequently, it is expected that the LRETsignal would arise from the energy transfer in receptor speciescontaining only one donor and one acceptor.

Taken together, these results confirmed that V2R could beselectively labeled at the C terminus with FlAsH as an acceptorand at Cys267 or Cys330 at the cytoplasmic end of TM6 and TM7-H8, respectively, with Lumi4-Tb as a donor (Fig. 4).Donor/acceptor-labeled receptor or donor-labeled V2R was

reconstituted into NAPols, separated, and analyzed by size ex-clusion chromatography (SEC) as described above. A typical SECprofile of donor/acceptor-labeled V2R is shown in Fig. S5B. Themonodisperse monomeric peak was selected and used in all thefollowing LRET experiments.Wealsodeterminedanaffinityconstantofdonor/acceptor-labeled

receptor for [3H]-AVP of 733.8± 100 nM, a value similar to the onefound for unlabeled receptor (674 ± 90 nM). As observed for theunlabeled receptor, using the calculated Bmax value, we evaluatedthe active fraction of receptor at about 90% of the total protein.

Kinetics of Fluorescein Maleimide Labeling. Labeling studies werecarried out on Flag-purified receptor to determine the individualreactivity of the cysteines. The reactions were carried out in a1-mL quartz cuvette using receptor diluted in the elution buffer toa final receptor concentration of 250 nM. The reaction was ini-tiated by adding fluorescein maleimide to a final concentration of10 nM. The labeling reaction could be followed using a spectro-fluorometer (PTI) by monitoring covalent attachment to thecysteine as a function of the change in fluorescein fluorescenceintensity (λemission = 525 nm) over time. Rate constants for thelabeling reactions were determined by fitting the time traces toa monoexponential using Prism software.

Fluorescence Labeling of Purified Receptors.The labelingwas doneasdescribed previously (3). Briefly, for the double labeling of V2R,purified receptors were reacted overnight at 16 °C in the dark with 3equivalents of FlAsH and TCEP (100 μM). To eliminate the un-reacted fluorophores and before proceeding to the next labelingstep, the FlAsH-labeled V2R was desalted by means of a gel fil-tration step (Sephadex G-50 fine). Lumi4-Tb maleimide (0.5equivalent) was then added to the mixture for 10 min at roomtemperature, and the double-labeled V2R was desalted by a gelfiltration step (Sephadex G-50 fine; Life technologies) beforereconstitution into NAPols. For single labeling of V2R with theLumi4-Tb maleimide, purified receptors were first incubatedovernight at 16 °C in the dark (same conditions as those of thedouble labeling but without FlAsH compound) and then desaltedbefore the addition of 0.5 equivalent of Lumi4-Tb maleimide for10 min at room temperature. The donor-labeled V2R was desaltedby a gel filtration step (Sephadex G-50 fine) before reconstitutioninto NAPols.The donor/receptor and acceptor/receptor ratios were de-

termined by dividing the bound dye concentration by the receptorconcentration [ε (V2R) = 73,295 cm−1·M−1, ε (FlAsH) = 41,000cm−1·M−1, and ε (Lumi4-Tb) = 25,000 cm−1·M−1].The FlAsH/receptor labeling ratio ranged from 0.4 to 0.6 mol

of dye per mole of receptor, and that of Lumi4-Tb/receptorranged from 0.5 to 0.7 (Fig. S5A). This incomplete labeling doesnot affect LRET measurements because the emission of theacceptor attributable only to energy transfer (defined as sensi-tized emission) is exclusively measured (14).

Characterization of V2R/NAPol Complexes by Negative-Stain Trans-mission EM. A freshly reconstituted V2R sample (3 μL) at a pro-tein concentration of ∼10 μg/mL was applied onto 400-mesh,glow-discharged, carbon-coated copper grids. Excess solutionwas blotted, and 4 μL of 1% uranyl formate was applied twice onthe grids for 1 min. The grids were then dried and kept in a des-iccator cabinet until observation. The grids were observed underlow-dose conditions with a Jeol 2200FS transmission electronmicroscope operating at 200 kV. Images were recorded at amagnification of 50,000× using a 4k × 4k slow-scan CCD camera

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with a defocus range ranging from 0.4 to 1.0 μm. Single-moleculeimages were extracted semiautomatically from images usingBoxer (15). The 2D image processing was performed usingIMAGIC V software (16). Briefly, the phase-contrast-transferfunction was corrected by phase-flipping, and images were au-tocentered using the CENTER-IMAGE with the average of rawimages as a reference. Some preferential views were selected byvisual inspection and chosen as references using the multi-reference alignment program. Images were then grouped intoclasses and averaged using the multistatistical alignment pro-cedure. The best class averages were selected visually and usedas new references for new alignment cycles.

SEC and PAGE Analyses. SEC was performed using an FPLCAKTA-purifier system. The column was previously equilibratedwith a buffer containing 20 mM Tris·HCl (pH 8.0) and 100 mMNaCl. Fractions were monitored at 280 nm, 335 nm, and 505 nm.Estimation of the protein molecular mass was established withstandards from GE Healthcare: ferritin (440 kDa), aldolase(158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa).Samples of 500 μL of NAPol-reconstituted V2R (10–20 μM)were loaded on a Superdex S200 10/300 column (Life technol-ogies) at a flow rate of 0.15 mL/min. Fractions of interest werepooled for performing the LRET experiments.Blue-native gels were performed using commercially available

gelsandnondenaturatingloadingbuffers(Expedeon,Inc.).Westernblot experiments were performed as previously described (17).

Construction of a 6× HIS-Tagged Gαs Protein and Expression in E. coli.The coding sequence for the short-splice variant of the α-subunit ofthe bovine stimulatory Gs protein (18, 19) was inserted into pET-28(+) prokaryotic vector (Novagen). The resulting pET-28(+)-Gαsvector contained a cDNA sequence encoding for an N-terminallymodified version of Gαs. The N-terminal added sequence, up-stream of the natural start codon ofGαs, corresponds to amino acidresiduesMGSS(H)6SSGLVPRGSH and contains a 6×HIS tag anda thrombin cleavage site (LVPRGS). Gαs was produced in Rosetta(DE 3) bacteria and kept frozen at −80 °C until use.

Extraction/Purification of the Recombinant 6× HIS-Tagged GαsProteinR. All steps were carried out at 0–4 °C. The cell pellet wasthawed and resuspended in 10mL of ice-cold lysis buffer consistingof 50 mM Tris·HCl (pH 8.0), 20 mM β-mercaptoethanol, anda mixture of protease inhibitors [benzamidine (10 μg/mL), leu-peptin (5 μg/mL), and PMSF (10 μg/mL)]. The suspension waslysed by sonication. The lysates were then ultracentrifuged at100,000 × g for 30 min. The supernatant was kept on ice, and thepellet was lysed, sonicated, and ultracentrifuged again. The su-pernatants from ultracentrifugation steps 1 and 2 were pooled.Imidazole (5 mM) and NaCl (100 mM) were added to the wholesupernatant (20 mL), which was incubated overnight at 4 °C with3 mL of 50% nickel-nitrilotriacetic acid superflow slurry (Qiagen)preequilibrated with binding buffer (lysis buffer supplemented with100 mM NaCl and 5 mM imidazole). The resin was washed with30 mL of binding buffer supplemented with 10 mM imidazole and500 mM NaCl (wash 1). Contaminant proteins were then elimi-nated using 10 mL of binding buffer containing 100 mM imidazoleand 10% glycerol (vol/vol) (wash 2). The purified recombinant Gαsproteinwas finally elutedwith 3mLof binding buffer supplementedwith 200 mM imidazole and 10% glycerol (vol/vol). [The elutedfraction was analyzed using 12% (vol/vol) SDS/PAGE, and therecombinantGαs protein was visualized by Coomassie blue staining(Fig. S7)]. The purified recombinant Gαs protein (5 μM) was di-alyzed overnight at 4 °C in 20 mM Tris·HCl (pH 7.4), 100 mMNaCl, 1 mMMgCl2, and 15% glycerol (vol/vol) using Slide-A-Lyserdialysis cassettes with a 10-KDa MWCO cutoff (Pierce). GDP (10μM) and EDTA (2 mM) were added to the purified dialyzedsample, and the Gαs protein stock was kept frozen at −80 °C.

Functionality of the purifiedGαs protein was checked by receptor-dependent agonist-stimulated [35S]-GTPγS binding.We estimatedthe active G protein fraction to constitute only a small percentageof the sample preparation, which could explain its poor coupling tothe purified V2 receptor.

LRET Spectroscopy. LRET is a spectroscopic technique in whicha long-lived lanthanide donor transfers energy to an acceptorin a distance-dependent manner. This technique overcomesproblems of the conventional FRET approach (short lifetimesof commonly used donor fluorophores, interfering fluorescencefrom the donor and from direct excitation of the acceptor,and orientational constraints of fluorophores) (14). It is basedon measurements of luminescence emission decay and calcu-lations of lifetimes (τ constants) that give access to measure-ment of distances. This technique has previously been used tomeasure Å-scale conformational changes in membrane pro-teins (20, 21).The study presented here represents a unique application of

LRET in mapping the structure and conformational changesassociated with receptor activation in the GPCR family. Thelabeling methods used here can, in principle, be applied tomonitor conformational changes in other domains of GPCRs.

Analysis of LRET Data.Luminescence emission decay was measuredon a Rubystar (BMG–Labtech) using the TR-FRET mode (ex-citation of the donor at 340 nm, emission at 520 nm for acceptor,and emission at 620 nm for donor). Background fluorescencefrom buffer and direct excitation of the acceptor do not con-tribute at all to the measured signals; the only signal is comingfrom excitation of the donor. The terbium donor-only emissionat 620 nm and FlAsH-sensitized emission at 520 nm were fittedto exponential decay functions. To avoid any artifact measure-ments attributable to the incomplete labeling strategy we used inthis study (mix of donor-only, donor plus acceptor, and acceptor-only labeled proteins), the donor lifetimes in the presence of theacceptor were measured through the acceptor-sensitized emis-sion, which only arises from energy transfer. Donor-only datawere fit to one exponential, and donor-acceptor data were fit totwo exponentials. Before fitting the FlAsH-sensitized emissiondata, we systematically removed the first 50-μs windows thatwere contaminated by the detector-ringing phenomenon. Energytransfer was calculated by comparing the donor-only lifetime(τD) with the lifetime of the acceptor-sensitized emission (τAD)using Eq. S1:

ET ¼ 1  � ðτAD=τDÞ: [S1]

For acceptor-donor, we calculated two lifetimes called τAD fastand τAD slow.Of note, the detector-ringing problem makes measurement of

the short lifetime component τAD fast more delicate. We thusdecided only to illustrate the drug effect on the slow componentsin the main text. The drug effect on the τAD fast can be found inTables S1–S4.To determine ligand-induced changes in LRET, samples were

incubated for 1 h at 4 °C in presence or absence of drugs (10 μM).Samples from three separate preparations were used for testingeach drug. Of note, the τD was not significantly affected by drugtreatment (<1% change). We then calculated the percentage ofchanges in τAD using Eq. S2:

ðτADðV2R  þ   ligandÞ � τADðV2R  þ   vehicleÞ=τADðV2R  þ   vehicleÞÞ × 100: [S2]

The distance was calculated using Eq. S3:

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R ¼ R0ðE �1T �1Þ1=6; [S3]

where ET is the energy transfer calculated using Eq. S1 and R0 isthe distance at which E = 50%. The calculated R0 for the Tb-FlAsH pair is 39 Å (CisBio Bioassays).The drug effects on lifetimes and distances are shown in Tables

S1–S4 for the TM6 and TM7-H8 sensors. The proportions ofslow and fast populations were calculated as previously described(21, 22). The amplitudes of sensitized acceptor decays werecorrected to reflect the true relative populations of donors (αDi),which give rise to the observed sensitized acceptor decays usingHeyduck’s equation (23):

αDi ¼ ðAi=ðkTiÞÞX

ðAi=ðkTiÞÞ;.

[S4]

where Ai is the observed sensitized acceptor amplitudes and kTiis the rate constant of energy transfer calculated by using Eq. S5:

kT ¼ ð1=τADÞ � ð1=τDÞ: [S5]

The drug effect on donor species distribution is shown in TablesS1–S4 for the TM6 and TM7-H8 sensors.

Statistical Analysis.Statistical significanceof thedifferencesbetweenindependent groups was determined by the Mann–Whitney test(unpaired nonparametric test). The data were not sampled fromGaussian distributions; thus, we used this unpaired nonparametrictest to compare two groups both for the LRET and for the tryp-tophan intrinsic fluorescence spectroscopy data. Tryptophan fluo-rescence set samples were as follows: two-tailed P value: P= 0.002for AVP, P = 0.0007 for SR121463, and P = 0.0012 for MCF14(n= 6). LRET set samples were as follows: two-tailed P value (n=6). For theTM6 sensor,P (τAD fast) = 0.01 forAVP;P (τAD fast) =0.02 for MCF14; and P (τAD slow) = 0.002 for AVP, MCF14, andSR121463. For the TM7-H8 sensor, P (τAD fast) = 0.001 forAVP; P (τAD fast) = 0.004 for SR121463; P (τAD slow) = 0.004for AVP; and P (τAD slow) = 0.002 for SR121463.

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Fig. S1. Expression of Flag-V2R and AVP-induced cAMP accumulation in Sf9 cells. (A) Sf9 cells (mean diameter about 18 μm) were infected with the Flag-tagged V2R recombinant baculovirus. After 60 h of infection, nonpermeabilized cells were incubated with Alexa 488-labeled M1 antibody for 15 min. Themedium was then removed, and cells were observed using an inverted microscope (IX70; Olympus). Images were acquired under visible light (Upper) or undera mercury lamp illumination with 460- to 490-nm excitation and 520-nm emission filters using an Apochromat (ACH) 0.25-N.A. objective with a magnificationof 10×. (B) Representative Scatchard plot of [3H]-AVP binding to Sf9 cells infected by recombinant baculovirus as described above. Kd = 13.5 ± 0.7 nM. Threeindependent experiments were performed. B/F is the ratio between bound and free radioactivity. (C) cAMP accumulation experiments were performed in cellsexpressing Flag-V2R, Flag-A267C-C358A-FlAsH, and Flag-S330C-C358A-FlAsH. Receptors were stimulated by increasing concentrations of AVP ranging from 10−10

to 10−6 M. The cAMP produced was expressed in nanomolar concentration and reported as a percentage of the maximal cAMP accumulation measured for eachconstruct. Data represent the mean ± SEM of three experiments performed in triplicate. Calculated EC50s are 13.7 ± 2 nM, 7.5 ± 0.5 nM, and 17 ± 3 nM for WTV2R, Flag-A267C-C358A-FlAsH, and Flag-S330C-C358A-FlAsH, respectively.

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sity

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.)

Fig. S2. Typical fluorescence spectra of bimane-labeled arrestin (position 68) in the absence of V2R (black trace) after incubation with V2R (gray trace) andafter agonist treatment (AVP, blue trace). For the arrestin recruitment assay, bimane-labeled arrestin-2 was added to the NAPol-reconstituted V2R (1:1 arrestin/receptor molar ratio, 10 μM) in the absence or presence of AVP (1:1 ligand/receptor molar ratio). After 2 h of incubation at 4 °C, fluorescence emission wasrecorded at 20 °C between 400 and 600 nm with excitation at 380 nm. All measurements were done in triplicate. a.u., arbitrary unit.

Rahmeh et al. www.pnas.org/cgi/content/short/1201093109 5 of 10

a b c

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AVP (log M)

%o

fmax

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mre

spo

nse

Fig. S3. Dose-dependent effects of ligands AVP (A), MCF14 (B), and SR121463 (C) on NAPol-reconstituted V2R. Intrinsic tryptophan fluorescence changes ofNAPol-reconstituted V2R were measured after treatment with different concentrations of drugs as described in SI Text. The percentage of the maximumresponse was plotted against the drug concentrations.

b c

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a

Fig. S4. FlAsH labeling of V2R and kinetics of V2R cysteine labeling. (A) Purified Flag-V2R-CCPGCC was incubated in the presence of 3 equivalents of FlAsHwith or without 100 μM TCEP (thick and dashed traces, respectively). V2R lacking a CCPGCC site (Flag-V2R) was incubated with 3 equivalents of FlAsH and 100μM TCEP (dotted trace). Fluorescence emission was recorded between 515 and 555 nm, with excitation at 508 nm. Emission spectra were scanned using 20 μMdesalted receptor. Representative spectra are shown and have been monitored on three independent preparations. (B) Intensity of fluorescein fluorescenceemission resulting from the reaction between the indicated V2R constructs and fluorescein maleimide was continuously recorded at 520 nm, normalized to themaximal (max) fluorescence intensity, and plotted against the time (s). (C) Labeling rate of mutants (K mutant) was calculated as described in Materials andMethods and normalized to that of Flag-V2R (K Flag-V2R). Data represent the mean ± SEM of at least three independent experiments performed in triplicate.

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11 12 13 14 15 16 17

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.U.)

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.)

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Fig. S5. (A) Double-labeling of V2R by FlAsH and Lumi4-Tb. The detergent-purified Flag-A267C-C358A-FlAsH was sequentially labeled with FlAsH followed byLumi4-Tb. Absorbance of the double-labeled receptor was recorded from 250 to 600 nm. The concentrations of V2R, FlAsH, and Lumi4-Tb were determinedusing the Beer–Lambert law and calculation of molar extinction coefficients (SI Text). The fluorophore/receptor ratios were deduced from the calculatedconcentrations. Several spectra from different preparations were recorded, and a representative spectrum with emission peaks at 280 nm (protein), 335 nm(Lumi4-Tb), and 505 nm (FlAsH) is shown. A.U., arbitrary unit. (B) SEC of double-labeled NAPol-reconstituted V2R. The labeled receptors were reconstituted inNAPols and then separated by SEC using an S200 10/300 High Resolution (HR) column (Life technologies) at a flow rate of 0.15 mL/min as described inMaterialsand Methods. Absorbance of Lumi4-Tb and FlAsH was monitored at 335 nm and 505 nm, respectively. We used the enriched monomeric fractions eluted atabout 15.5 mL for all our LRET experiments.

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Mix of Donor only and acceptor only labeled V2R

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ore

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Fig. S6. Absence of intermolecular LRET. Acceptor-sensitized emission of the double-labeled V2R (0.1 μM) reconstituted in NAPols (black trace) and of a mix ofthe donor-only NAPol-reconstituted V2R (0.1 μM) and acceptor-only NAPol-reconstituted V2R (0.1 μM) (gray trace). Luminescence emission decay was measuredon a Rubystar using the TR-FRET mode with excitation of the donor at 340 nm and emission at 520 nm for the acceptor.

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kDa

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Fig. S7. Purification of the recombinant 6× HIS-tagged Gαs protein. The Gαs protein was overexpressed in bacteria and purified by immobilized metal affinitychromatography. Proteins from the various purification steps were separated onto 10% (vol/vol) SDS/PAGE and analyzed by Coomassie blue staining. Arepresentative experiment is illustrated. Lane 1, molecular mass markers; lane 2, flow-through; lane 3, wash 1; lane 4, wash 2; and lane 5, eluate. The apparentmolecular mass of Gαs protein is consistent with the theoretical value at around 45 kDa.

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ityFlag-V2R-C358A-FlAsH TM6 SENSOR

TM6 SENSORTM7/H8 SENSOR

a b

c d

Fig. S8. LRET raw data. (A) FlAsH-sensitized emission obtained for the Flag-V2R-C358A-FlAsH construct. Ligand-dependent sensitized emission changes of theTM6 sensor (B and C) and the TM7-H8 sensor (D). FlAsH-sensitized emission values in the presence of vehicle (black trace), MCF (green trace), and SR (blue trace)are represented using a linear scale. The residual values represent the goodness of the fits. Each trace is the fluorescence decay measurement of a single well.Each measurement was analyzed independently, and the lifetime was calculated as the average of three independent measurements for each experiment.

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Table S1. Calculated lifetimes and distances between V2R C terminus and TM6 for the basal state and after drug treatment

τAD fast, μs τAD slow, μs τD, μs Distance fast, ÅDistanceslow, Å

Corrected donorspecies fast, %

Corrected donorspecies slow, %

Basal state 92 ± 3 783 ± 5 2,238 ± 20 23.01 ± 0.2 35.08 ± 0.1 22 ± 0.3 78 ± 0.3SR121463 94 ± 6 688 ± 22 2,241 ± 48 23.13 ± 0.3 34.11 ± 0.3 25 ± 0.7 75 ± 0.7AVP 108 ± 4 986 ± 2 2,212 ± 33 23.7 ± 0.2 37.5 ± 0.1 18 ± 1 82 ± 1MCF14 119 ± 10 966 ± 19 2,243 ± 42 24.03 ± 0.4 37.1 ± 0.2 19 ± 0.7 81 ± 0.7Leuprolide 91 ± 4 740 ± 17 2,203 ± 42 23.06 ± 0.2 34.8 ± 0.2 24 ± 3 76 ± 3

Table S2. Calculated lifetimes and distances between V2R C terminus and TM7-H8 for the basal state and after drug treatment

τAD fast, μs τAD slow, μs τD, μs Distance fast, Å Distance slow, ÅCorrected donorspecies fast, %

Corrected donorspecies slow, %

Basal state 37 ± 2 333 ± 10 2,210 ± 8 19.8 ± 0.2 29.2 ± 0.2 43 ± 1 57 ± 1SR121463 56 ± 5 433 ± 17 2,179 ± 14 21 ± 0.3 30.7 ± 0.2 40 ± 1 60 ± 1AVP 58 ± 3 422 ± 20 2,159 ± 38 21.4 ± 0.2 30.8 ± 0.3 39 ± 2 61 ± 2MCF14 38 ± 6 320 ± 23 2,215 ± 15 19.3 ± 0.6 28.9 ± 0.4 42 ± 2 58 ± 2

Table S3. Calculated lifetimes and distances in Flag-V2R-C358A-FlAsH receptor for the basal state

τAD fast, μs τAD slow, μs τD, μs Distance fast, Å Distance slow, ÅCorrected donorspecies fast, %

Corrected donorspecies slow, %

Basal state 73 ± 16 435 ± 57 2,100 ± 28 23.3 ± 0.8 31.15 ± 0.9 40 ± 4 60 ± 4

Table S4. Ligand efficacy and effects on the V2R sensors

Gs pathwayArrestinpathway

Changes inTryptophan

fluorescence, %

LRET [changesin τAD slow

(TM6-C terminus)], %

LRET [changesin τAD fast

(TM6-C terminus)], %

LRET [changesin τAD slow

(H8-C terminus)], %

LRET [changesin τAD fast

(H8- C terminus)], %

SR121463 Inverse agonist Partial agonist −21*** −12 ± 3** No effect +31 ± 6** +46 ± 14**AVP Full agonist Full agonist +8** +26 ± 0.7** +21 ± 7* +27 ± 7** +50 ± 10**MCF14 Full agonist Antagonist +10** +23 ± 3** +30 ± 12* No effect No effect

Mann–Whitney test: *P < 0.05; **P < 0.01; ***P < 0.001.

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