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Proc Natl Acad Sci U S A. Jul 17, 2007; 104(29): 12199–12204.
Published online Jul 9, 2007. doi:  10.1073/pnas.0705312104
PMCID: PMC1913548

Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction


G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concentrations, NTS1 binds the agonist neurotensin with a Hill slope of ≈1; at higher receptor concentrations, neurotensin binding displays positive cooperativity with a Hill slope of ≈2. NTS1 monomers activate Gαqβ1γ2, whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.

Keywords: dimer, G protein activation, G protein-coupled receptor, monomer

Dimerization of G protein-coupled receptors (GPCRs) has been the subject of intense interest. Class C GPCRs, such as metabotropic glutamate receptors and γ-aminobutyric acid type B (GABAB) receptors, clearly form homo- and heterodimeric structures, essential both for trafficking of receptors to the cell surface and for ligand-induced activation of receptors and G protein coupling. Detailed models for receptor and G protein activation have been proposed that account for the multidomain structure of class C GPCRs (for review, see ref. 1).

In contrast, no conclusion has yet been reached as to the importance of dimerization for class A receptor function; the role of receptor monomers (2) or dimers (3, 4) in signal transduction is controversial. Models proposed to explain the mechanism of receptor-catalyzed G protein activation (57) assumed the interaction of G protein with a receptor monomer. More recently, class A GPCRs have been described as monomers (8) and dimers (see refs. 9 and 10) in living cells by resonance energy transfer methods. For rhodopsin, dimer particles and higher-order oligomers have been visualized in disk membranes by atomic force microscopy (11). Based on structural constraints, a model was suggested in which a receptor dimer provides an extensive contact area for the heterotrimeric G protein; the surface area of a GPCR monomer was deemed insufficiently large to anchor both the Gα and Gβγ subunits simultaneously (3, 4, 12). However, alternate models for the interaction of a monomeric rhodopsin with a G protein heterotrimer have also been proposed (2).

The concentration of rhodopsin in rod outer segment disk membranes is high (≈2.5 mM; for review, see ref. 13), and rhodopsin may therefore exist only as dimers, as seen by atomic force microscopy (11). Because a rod cell can respond to a single photon (14, 15), only one activated rhodopsin protomer, with the other protomer in its inactive state, seems to be sufficient for G protein activation in disk membranes. A similar situation has been observed for the leukotriene B4 receptor in detergent solution (16) (and for homo- and heterodimeric class C receptors; see ref. 17). The “functional unit” for signal transduction may well be a receptor dimer, but the molecular determinants for G protein recognition and activation may reside in one receptor protomer (18). It remains to be seen whether the interaction of rhodopsin with transducin serves as a model for all class A GPCRs.

Here, we ask whether the neurotensin (NT) receptor NTS1, a class A rhodopsin-like receptor, can activate G proteins as receptor monomers or whether receptors need to be in the dimeric state for G protein activation to occur. We show that purified NTS1 dimerizes in detergent solution in a concentration-dependent manner. NTS1 monomers activate the G protein Gαqβ1γ2, whereas receptor dimers catalyze nucleotide exchange less efficiently. Based on our experiments, NTS1 dimerization is not required for G protein activation, but NTS1 dimerization seems to alter the mode of the receptor–G protein interaction.


An assessment of the properties of NTS1 depends critically on an experimental setup that allows the unambiguous definition of receptor monomers and dimers. Here we present experiments in detergent solution, describing well defined preparations of receptor monomers and dimers and their ligand-binding and G protein-coupling properties.

Initial experiments established that NTS1 coupled to Gαq in detergent solution (data not shown), as previously reported for membrane-bound NTS1 (see refs. 19 and 20). The interaction of purified NTS1 with purified G protein subunits critically depended on the amount of detergent present, and it was of utmost importance to control carefully the actual detergent levels in the experimental setup. High detergent conditions substantially reduced Gαq activation (see below). Therefore, we conducted the following characterizations at low detergent concentrations. To begin, we assessed the properties of NTS1, in the absence of G protein, by agonist-binding experiments. At low receptor protein concentrations (<1 nM), [3H]NT saturation binding (Fig. 1a) and homologous competition experiments (Fig. 1b) gave Hill slopes of ≈1, indicating that one receptor molecule binds one ligand. The Bmax values obtained from ligand binding closely matched the amount of receptor input [supporting information (SI) Table 1], estimated by the Amido black dye method (21). At increased receptor protein concentrations (>20 nM), we observed Hill slopes of ≈2 (Fig. 1 b and c and SI Table 1), which is indicative of positive cooperativity. For this to occur, NTS1 must exist as dimers or higher-order oligomers. We used size exclusion chromatography (SEC), along with light scattering (LS), refractive index (RI), and UV measurements, to confirm the dimeric state of NTS1. This technique allowed the quantitative assessment of the contributions of protein and detergent to the mass of the receptor–detergent complex. In the presence or absence of NT, the mass of the protein–detergent complex was found to be ≈210 kDa (Fig. 2 and SI Table 2) with an underlying protein mass of 77 kDa. The calculated molecular mass of the NTS1 monomer was 43.3 kDa. We conclude that NTS1 is dimeric and monodisperse in low-detergent buffer at micromolar protein concentrations. Our data would seem to exclude higher-order oligomers based on the hydrodynamic properties of NTS1 at micromolar concentrations (Fig. 2) and a Hill slope for NT binding that did not increase from ≈2 to 130 nM NTS1. The above pharmacological results (Fig. 1) combined with biochemical data (Fig. 2) are consistent with NTS1 undergoing a transition from monomer to dimer at receptor concentrations between 2 and 20 nM (Fig. 1c and SI Table 1). The Kd value is low compared with those determined for other membrane protein dimers (22, 23). However, a strong dependence of Kd value on detergent type and detergent concentration has been observed, with Kd values as low as 80 nM reported for the glycophorin A transmembrane domain (23).

Fig. 1.
Saturation and competition binding of NT to purified NTS1. (a) Saturation [3H]NT binding was measured in a reaction containing 0.34 nM receptor protein (estimated by the Amido black dye method). A one-site binding equation was used for curve fitting. ...
Fig. 2.
Mass analysis of purified NTS1 by SEC coupled with LS, RI, and UV measurements. The experiment shown was performed in buffer C (low detergent, no NT present). The Rayleigh ratio (left y axis) is proportional to the intensity of the scattered light. The ...

SEC experiments defined NTS1 as a dimer in the presence of low detergent concentrations (Fig. 2), whereas, under high-detergent conditions, NTS1 is monomeric (SI Fig. 4a and SI Table 3). Varying the detergent amounts would thus be one possible way of generating NTS1 in its monomeric or dimeric state. However, we could not assess whether the NTS1 monomer, produced in this way, catalyzed nucleotide exchange at Gαq, because high detergent concentrations severely impaired G protein activation (SI Fig. 4b). Therefore, to determine whether G protein activation requires receptors to be monomers or dimers, we assessed agonist saturation of NTS1-catalyzed GDP/guanosine 5′-[γ-thio]triphosphate (GTPγS) exchange in low-detergent buffer at receptor concentrations of 100 and 1 nM (Fig. 3 a and b), and our data demonstrated that NTS1 is predominantly dimeric or monomeric, respectively (see Fig. 1 and SI Table 1). Half-saturation of NTS1-catalyzed GDP/GTPγS exchange at high receptor concentrations (Fig. 3a) was reached at NT concentrations comparable with that obtained from homologous competition experiments. The positive cooperative binding of NT by NTS1 dimers appeared to be decreased in the presence of G protein. We conclude that agonist-occupied NTS1 dimers activate Gαq. As shown in Fig. 3b, NTS1 at low concentrations also catalyzed GDP/GTPγS exchange. Because NTS1-catalyzed nucleotide exchange at Gαq is critically dependent on the presence of Gβ1γ2 (SI Fig. 5) (see also refs. 24 and 25), both Gα and Gβγ docking sites must be present on the receptor monomer to allow G protein activation. This conclusion is supported by findings that transducin Gtβγ interacts directly with light-activated rhodopsin protein (see refs. 2628) and that the binding interactions of the Gtα and Gtβγ subunits with rhodopsin are synergistic (24).

Fig. 3.
Agonist-stimulated activation of Gαq by NTS1. (a) NT saturation of receptor-catalyzed GDP/GTPγS exchange at 100 nM NTS1. [35S]GTPγS binding was recorded in response to the indicated amounts of NT in the presence of Gαq ...

Next, we conducted experiments to analyze the Gαq saturation of NTS1-catalyzed GDP/GTPγS exchange (Fig. 3c). If we assume that the Gαq and the Gβ1γ2 docking sites of each of the receptor protomers in the dimer are identical to those of a receptor monomer and that, according to current models, one Gα subunit and one Gβγ dimer are present at the receptor dimer (29), then we would expect a similar Km value at high and low receptor concentrations. We found that the apparent Km value for the specific, NT-induced nucleotide exchange at a 1-nM receptor concentration was 530 nM, which was almost 2-fold lower than that at a 100 nM receptor concentration (Km value of 961 nM; t test, P < 0.05; Fig. 3 c and d). These results indicate that the G protein subunit interactions in the dimeric NTS1 are not equivalent to those in receptor monomers.


NTS1 dimerizes in detergent solution; however, its dimer interface is currently unknown. A molecular model of a rhodopsin dimer proposes protomer–protomer interactions through transmembrane helices 4 and 5 (3). A similar interface has been identified by cross-linking studies in membranes for opsin and other class A GPCRs (see, for example, refs. 30 and 31). Currently it is unknown whether productive interaction with G proteins only occurs when receptors dimerize through helices 4 and 5 or whether other dimer orientations are also permissible.

NTS1 dimerizes in a concentration-dependent manner, with a small percentage of dimers always present at low receptor concentrations. Therefore, a low residual NTS1 dimer population, rather than receptor monomers, could be responsible for efficient G protein coupling at a 1-nM receptor concentration. However, this seems unlikely because of the different binding characteristics of G protein to NTS1 at high and low receptor concentrations (Fig. 3).

NTS1 monomers catalyze nucleotide exchange at Gαq with higher affinity than NTS1 dimers (Fig. 3). This is in contrast to experiments with a purified leukotriene B4 receptor (16) and purified rhodopsin (32) that concluded that full G protein activation requires a dimeric or oligomeric receptor complex, even though the receptor monomer can activate G protein to some extent. Gαq activation by NTS1 monomers in the presence of Gβ1γ2 can readily be measured at low detergent concentrations (Fig. 3), but not under high detergent conditions (SI Fig. 4b). Rhodopsin was prepared as a mixture of monomers and dimers in the detergent n-dodecyl-β-d-maltoside [also called lauryl maltoside (LM)] at 0.15%, and as larger, nonhomogeneous particles in n-tetradecyl-β-d-maltoside (0.03%) and n-hexadecyl-β-d-maltoside (0.003%) (32). Therefore, elevated detergent concentrations, rather than monomer, dimer, or oligomer properties, may account for the reduced G protein coupling of rhodopsin in LM. Leukotriene B4 receptor monomers were prepared in n-hexadecyl-β-d-maltoside/asolectin in the presence of a large molar excess of a transmembrane peptide, TM6 (29), which, as noted, possibly decreased receptor–G protein coupling efficiency (16).

Lipid has been found to modulate the interaction between receptor and G protein (33). The leukotriene B4 receptor was prepared in lipid/detergent mixed micelles (n-hexadecyl-β-d-maltoside/asolectin) (16); the above mentioned rhodopsin preparations contained phospholipid ranging from 22 (in LM) to 44 (in n-hexadecyl-β-d-maltoside) lipid molecules per rhodopsin molecule (32). The lipid content of the NTS1 preparation and the potential effect of certain lipid types on G protein activation by NTS1 monomers and dimers await further analysis.

Our data, obtained for pure receptor and G protein subunits in defined detergent conditions, clearly describe an intrinsic dimerization of NTS1 with nanomolar affinity and agree with the conclusion that class A GPCRs dimerize, as seen with the leukotriene B4 receptor BLT1 (16, 29). The positive cooperative binding of NT by NTS1 dimers appears to be decreased in the presence of G protein (Fig. 3a), suggesting that G protein and agonist sites in the receptor dimer are linked in a negative cooperative manner. This observation with pure components in detergent solution may be a reflection of the long known GTP-induced shift of agonist affinity typical for GPCRs in membrane preparations (34).

The diminished capacity of the NTS1 dimer, as compared with the monomer, to activate Gαq can be explained as follows. The NTS1 monomer is capable of initiating G protein signaling; hence, both Gαq and Gβ1γ2 docking sites must be present on the receptor monomer to allow G protein activation (see Results). Consequently, each protomer in the NTS1 dimer has Gαq and Gβ1γ2 binding sites; however, these sites may not be equivalent in each protomer. The reduced affinity of the NTS1 dimer to interact with Gαq could be due to an occlusion of one of the receptor protomer G protein subunit binding sites (either for Gαq or Gβ1γ2) within the dimer. This may lead to internal competition of G protein subunit binding and may explain why the G protein interaction in the dimeric NTS1 is not the same as that seen in the receptor monomer.

Materials and Methods

The detergents n-dodecyl-β-d-maltoside (LM), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and cholesteryl hemisuccinate Tris salt (CHS) were from Anatrace (Maumee, OH).

Preparation of NTS1, Gαq, and Gβ1γ2.

For details see SI Materials and Methods. NTS1 was produced in Escherichia coli as a maltose-binding protein fusion and purified as described (35). To obtain NTS1 devoid of the maltose-binding protein and affinity tag, the fusion protein was incubated with Tev protease to generate rT43NTR, with Ser-Gly-Ser at the N terminus and with the C terminus ending in Glu421-Asn-Leu-Tyr-Phe-Gln. We refer to rT43NTR as NTS1. SEC of NTS1 at low detergent concentrations preceded the ligand- and G protein-binding experiments. Cephalopod Gαq was purified from dark-adapted retinas of Sepia officinalis as described (36). The dimer of human Gβ1 and human Gγ2H6 (Gγ2 contains an N-terminal His6 tag) was expressed in the insect cell/baculovirus system and purified as described (37). We refer to Gβ1γ2H6 as Gβ1γ2.

Saturation and Competition Agonist-Binding Experiments.

Ligand-binding analyses with detergent-solubilized, purified receptors were done as described (35, 38, 39) with modifications. Receptors were incubated with [3H]NT (Perkin-Elmer, Wellesley, MA; specific activity of 70,599 cpm per pmol) and unlabeled NT on ice for 1 h in assay buffer [50 mM Mops, pH 7.5/7% (vol/vol) glycerol/1 mM EDTA/100 mM NaCl/1 mM DTT/40 μg/ml bacitracin/0.1% (wt/vol) BSA/0.01% LM/0.06% CHAPS/0.012% CHS]. Individual experiments were performed in duplicate. Separation of receptor–ligand complex from free ligand was achieved by centrifugation-assisted gel filtration using Bio-Spin 30 Tris columns (Bio-Rad, Hercules, CA) (equilibrated with assay buffer) according to the manufacturer's instructions. In homologous competition experiments (NT/[3H]NT) (Fig. 1b and SI Table 1), the [3H]NT amounts added were in the range of 1.2–3.2 nM. Samples were analyzed by liquid scintillation counting (LS 6500 counter, Beckman Coulter, Fullerton, CA). The receptor input (SI Table 1) was estimated by the Amido black dye method (21).

Data Evaluation of [3H]NT Binding Experiments.

Data were analyzed by nonlinear regression using Prism software, version 4 (GraphPad, San Diego, CA). A one-site binding equation was used to determine the Bmax and Kd values from saturation binding experiments at equilibrium (Fig. 1a and SI Table 1). In homologous competition (NT/[3H]NT) experiments at 0.95 nM receptor protein (Fig. 1b and SI Table 1), ligand depletion was considered to be comparatively low (<30%). A sigmoidal dose–response equation (variable slope) was used for curve fitting by using the concentrations of total NT added vs. specifically bound [3H]NT. The Bmax value at 0.95 nM receptor input (SI Table 1) was calculated by subtracting nonspecific [3H]NT binding (in the presence of 10 μM NT) from total [3H]NT binding (in the absence of NT). The amount of specifically bound [3H]NT was then corrected for fractional occupancy (Kd value of 1.14 nM). In homologous competition (NT/[3H]NT) experiments at receptor protein concentrations of ≥19 nM (Fig. 1b and SI Table 1), ligand depletion was substantial (54–73%). Therefore, we calculated the concentrations for free [3H]NT plus NT and specifically bound [3H]NT plus NT and used these values for curve fitting (sigmoidal dose–response, variable slope). The resulting EC50 values were used as Ki values, and the tops of the curves were taken as Bmax values. All Hill slope values reported in SI Table 1 (for saturation and competition experiments) were derived by using a sigmoidal dose–response equation (variable slope).

GDP/GTPγS Exchange Assay.

The concentration of NTS1 obtained by SEC (see above) was ≈1 μM. Receptors were added to the nucleotide exchange reactions to a final concentration of 1, 95, or 100 nM by dilution, thereby ensuring that the final free detergent concentration was the same in all experiments. The receptor-catalyzed exchange of GDP for GTPγS on Gα was determined by modification of previously described procedures (40, 41). Reactions were carried out in 12 × 75 mm siliconized borosilicate glass test tubes in a total assay volume of 50 μl. Reaction mixtures were kept on ice throughout the procedure. Detergent-solubilized, purified NTS1 was added to G protein (Sepia Gαq and human Gβ1γ2) and agonist to give a volume of 30 μl. GDP/GTPγS exchange was initiated by the addition of 20 μl of [35S]GTPγS mix. The final component concentrations in each sample were 50 mM Mops (pH 7.5), 7% (vol/vol) glycerol, 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 3 mM MgSO4, 1.2% (wt/vol) BSA, 1 μM GDP, 4–8 nM [35S]GTPγS (Perkin-Elmer), 0.01% LM, 0.06% CHAPS, and 0.0012% CHS. The duration of incubations was as follows: 15 min for the determination of the effect of detergent on the agonist-stimulated GDP/GTPγS exchange by Gαq (NTS1 at 95 nM, Gαq at 100 nM, Gβ1γ2 at 500 nM, and NT at 1.12 μM; SI Fig. 4b), 10 min for NT saturation of Gαq activation at high receptor concentration (NTS1 at 100 nM, Gαq at 1 μM, and Gβ1γ2 at 1 μM; Fig. 3a), 2 h for NT saturation of Gαq activation at low receptor concentration (NTS1 at 1 nM, Gαq at 1 μM, and Gβ1γ2 at 1 μM; Fig. 3b), 5 min for the Gαq saturation of NTS1-catalyzed GDP/GTPγS exchange at high receptor concentration (NTS1 at 100 nM, Gβ1γ2 at 1 μM, and NT at 10 μM; Fig. 3c), and 1 h for the Gαq saturation of NTS1-catalyzed GDP/GTPγS exchange at low receptor concentration (NTS1 at 1 nM, Gβ1γ2 at 1 μM, and NT at 10 μM; Fig. 3c). The fractional contribution of the noncatalyzed nucleotide exchange at a given Gαq concentration was estimated in reactions containing NTS1 at 100 or 1 nM, in the presence of Gβ1γ2 at 1 μM (n = 1) or in the absence of Gβ1γ2 (n = 2) (similar results). Reactions were terminated by diluting the reaction mixture with 2 ml of ice-cold stop buffer (20 mM Tris·HCl, pH 8.0/100 mM NaCl/25 mM MgCl2) and were filtered over nitrocellulose membranes on a vacuum manifold. Filters were then washed six times with 2 ml of ice-cold stop buffer. The nitrocellulose membranes were dried overnight, and the radioactivity quantitated by liquid scintillation in a Beckman Coulter LS 6500 scintillation counter. For NT saturation experiments, initial rates of reaction were approximated throughout; i.e., <30% (Fig. 3a) or 15% (Fig. 3b) of [35S]GTPγS was consumed at the highest agonist concentration. In Gαq saturation experiments (Fig. 3c), <35% and 7% of [35S]GTPγS was consumed at 2 μM Gαq with 100 and 1 nM NTS1, respectively.

Data Evaluation of GDP/GTPγS Exchange Experiments.

Data were analyzed by nonlinear regression using Prism software, version 4 (GraphPad). A sigmoidal dose–response equation (variable slope) was used for curve fitting of data from NT saturation of NTS1-catalyzed GDP/GTPγS exchange experiments at high and low receptor concentrations (Fig. 3 a and b). For the determination of Km values for Gαq saturation of NT-induced, receptor-catalyzed GDP/GTPγS exchange (Fig. 3 c and d), [35S]GTPγS binding in the absence of NT (i.e., the noncatalyzed nucleotide exchange at a given Gαq concentration) was subtracted from [35S]GTPγS binding in the presence of NT. The resulting data, reflecting specific NT-induced nucleotide exchange, were analyzed by using a one-site binding equation. In contrast to homologous competition (NT/[3H]NT) experiments, the degree of ligand depletion in GDP/GTPγS exchange assays at high receptor protein concentrations could not be directly assessed because no tracer was present. To compare nucleotide exchange experiments with homologous competition assays (Fig. 3 a and b), data are shown as total NT added (x axis) for both types of experiments. Note that the calculated EC50 values (GDP/GTPγS exchange) and IC50 values (homologous competition) are apparent values rather than true affinities at high receptor concentrations.

Determination of the Receptor Mass in Protein–Detergent Complexes by SEC with LS, RI, and UV Measurements (SEC LS/RI/UV).

SEC LS/RI/UV experiments have been used to obtain the molecular mass and oligomeric state of membrane proteins, and the amount of the protein-bound detergent (4245). The method is based on SEC used in series with UV absorption, laser LS, and differential RI detectors. The theory and applications of this method have been described (4648). LS and UV data were obtained by using a Superose 6 HR 10/30 column (GE Healthcare, Piscataway, NJ) with in-line multiangle LS (DAWN EOS, Wyatt Technology, Santa Barbara, CA), RI (Optilab DSP, Wyatt Technology), and UV detectors. The SEC column was equilibrated with buffer C [20 mM Tris·HCl, pH 7.4/7% (vol/vol) glycerol/1 mM EDTA/100 mM NaCl/1 mM DTT/0.01% LM/0.06% CHAPS/0.012% CHS], buffer C containing 23.9 μM NT (N6383, Sigma, St. Louis, MO), or buffer D [20 mM Tris·HCl, pH 7.4/7% (vol/vol) glycerol/1 mM EDTA/100 mM NaCl/1 mM DTT/0.3% LM/0.15% CHAPS/0.03% CHS]. The sample (180 μg of NTS1 at 1.8 mg/ml or 42 μM) was applied to the SEC column at a flow rate of 0.5 ml/min at room temperature. Before run C plus NT, a 4-fold molar excess of NT was added to the receptor. Before run D, LM was added to the sample to match the LM concentration in buffer D. The data were processed by using Astra software (Wyatt Technology), version The extinction coefficient ε for NTS1 at 280 nm was calculated from the amino acid sequence as 1,213 (ml·g−1·cm−1). The detergent was assumed to have negligible absorbance at 280 nm. A RI increment value (dn/dc value) of 0.185 (ml/g) was used for the protein component; a dn/dc value of 0.133 (ml/g) was used for the modifier (detergent) (49). BSA (A7638, Sigma) was used as a control (ε = 667 ml·g−1·cm−1).

SEC Experiments.

SEC was performed by using a Superose 6 HR 10/30 column (GE Healthcare), equilibrated with buffer C, buffer D, or buffer D containing 23.9 μM NT (N6383, Sigma). The sample (NTS1 at 2 mg/ml or 46 μM) was applied to the SEC column at a flow rate of 0.3 ml/min at 4°C, and 0.5-ml fractions were collected. Before run D plus NT, a 5-fold molar excess of NT was added to the receptor. Individual fractions were analyzed by SDS/PAGE using NuPAGE 4–12% Bis-Tris gels, MES SDS buffer, and SimplyBlue SafeStain, (Invitrogen, Carlsbad, CA) and Novagen (San Diego, CA) Perfect Protein Markers, 15–150 kDa.

Supplementary Material

Supporting Information:


We thank In-Ja Byeon [Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)] for help with initial LS experiments; Klaus Peter Hofmann (Institut für Medizinische Physik und Biophysik, Berlin, Germany) for discussions; and Jürgen Wess (NIDDK), Nick Ryba (National Institute of Dental and Craniofacial Research, Bethesda, MD), Paul Randazzo (National Cancer Institute, Bethesda, MD), Dave Sibley [National Institute of Neurological Disorders and Stroke (NINDS)], Kenton Swartz (NINDS), and Joe Mindell (NINDS) for critically reading the manuscript. Part of the experiments by J.F.W., J. Grodnitzky, and R.G. were conducted at the Laboratory of Molecular Biology, NIDDK. This research was supported by the Intramural Research Program of the National Institutes of Health, NINDS, National Institute on Deafness and other Communication Disorders, NIDDK, and by the National Institutes of Health Intramural AIDS Targeted Antiviral Program.


G protein-coupled receptor
size exclusion chromatography
light scattering
refractive index
guanosine 5′-[γ-thio]triphosphate
cholesteryl hemisuccinate Tris salt.



While this manuscript was under revision, Whorton et al. (50) and Bayburt et al. (51) reported efficient activation of G protein by GPCR monomers (β2-adrenergic receptor and rhodopsin) in lipid disks. In addition, Bayburt et al. (51) described experiments with lipid disks containing two rhodopsin molecules.


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0705312104/DC1.


1. Pin J-P, Kniazeff J, Liu J, Binet V, Goudet C, Rondard P, Prézeau L. FEBS J. 2005;272:2947–2955. [PubMed]
2. Chabre M, le Maire M. Biochemistry. 2005;44:9395–9403. [PubMed]
3. Liang Y, Fotiadis D, Filipek S, Saperstein DA, Palczewski K, Engel A. J Biol Chem. 2003;278:21655–21662. [PMC free article] [PubMed]
4. Park PS, Filipek S, Wells JW, Palczewski K. Biochemistry. 2004;43:15643–15656. [PMC free article] [PubMed]
5. Cherfils J, Chabre M. Trends Biochem Sci. 2003;28:13–17. [PubMed]
6. Iiri T, Farfel Z, Bourne HR. Nature. 1998;394:35–38. [PubMed]
7. Rondard P, Iiri T, Srinivasan S, Meng E, Fujita T, Bourne HR. Proc Natl Acad Sci USA. 2001;98:6150–6155. [PMC free article] [PubMed]
8. Meyer BH, Segura JM, Martinez KL, Hovius R, George N, Johnsson K, Vogel H. Proc Natl Acad Sci USA. 2006;103:2138–2143. [PMC free article] [PubMed]
9. Mercier JF, Salahpour A, Angers S, Breit A, Bouvier M. J Biol Chem. 2002;277:44925–44931. [PubMed]
10. Urizar E, Montanelli L, Loy T, Bonomi M, Swillens S, Gales C, Bouvier M, Smits G, Vassart G, Costagliola S. EMBO J. 2005;24:1954–1964. [PMC free article] [PubMed]
11. Fotiadis D, Liang Y, Filipek S, Saperstein DA, Engel A, Palczewski K. Nature. 2003;421:127–128. [PubMed]
12. Filipek S, Krzysko KA, Fotiadis D, Liang Y, Saperstein DA, Engel A, Palczewski K. Photochem Photobiol Sci. 2004;3:628–638. [PubMed]
13. Daemen FJM. Biochim Biophys Acta. 1973;300:255–288. [PubMed]
14. Baylor DA, Lamb TD, Yau KW. J Physiol. 1979;288:613–634. [PMC free article] [PubMed]
15. Hecht S, Shlaer S, Pirenne MH. J Gen Physiol. 1942;25:819–840. [PMC free article] [PubMed]
16. Damian M, Martin A, Mesnier D, Pin JP, Banères JL. EMBO J. 2006;25:5693–5702. [PMC free article] [PubMed]
17. Hlavackova V, Goudet C, Kniazeff J, Zikova A, Maurel D, Vol C, Trojanova J, Prezeau L, Pin JP, Blahos J. EMBO J. 2005;24:499–509. [PMC free article] [PubMed]
18. Dell'Orco D, Seeber M, Fanelli F. FEBS Lett. 2007;581:944–948. [PubMed]
19. Gailly P, Najimi M, Hermans E. FEBS Lett. 2000;483:109–113. [PubMed]
20. Hermans E, Maloteaux JM. Pharmacol Ther. 1998;79:89–104. [PubMed]
21. Schaffner W, Weissmann C. Anal Biochem. 1973;56:502–514. [PubMed]
22. Butler PJ, Ubarretxena-Belandia I, Warne T, Tate CG. J Mol Biol. 2004;340:797–808. [PubMed]
23. Fisher LE, Engelman DM, Sturgis JN. J Mol Biol. 1999;293:639–651. [PubMed]
24. Clark WA, Jian X, Chen L, Northup JK. Biochem J. 2001;358:389–397. [PMC free article] [PubMed]
25. Phillips WJ, Wong SC, Cerione RA. J Biol Chem. 1992;267:17040–17046. [PubMed]
26. Kelleher DJ, Johnson GL. Mol Pharmacol. 1988;34:452–460. [PubMed]
27. Kisselev OG, Downs MA. Biochemistry. 2006;45:9386–9392. [PubMed]
28. Phillips WJ, Cerione RA. J Biol Chem. 1992;267:17032–17039. [PubMed]
29. Banères J-L, Parello J. J Mol Biol. 2003;329:815–829. [PubMed]
30. Guo W, Shi L, Filizola M, Weinstein H, Javitch JA. Proc Natl Acad Sci USA. 2005;102:17495–17500. [PMC free article] [PubMed]
31. Kota P, Reeves PJ, Rajbhandary UL, Khorana HG. Proc Natl Acad Sci USA. 2006;103:3054–3059. [PMC free article] [PubMed]
32. Jastrzebska B, Fotiadis D, Jang GF, Stenkamp RE, Engel A, Palczewski K. J Biol Chem. 2006;281:11917–11922. [PMC free article] [PubMed]
33. Bubis J. Biol Res. 1998;31:59–71. [PubMed]
34. Rodbell M, Krans HM, Pohl SL, Birnbaumer L. J Biol Chem. 1971;246:1872–1876. [PubMed]
35. White JF, Trinh LB, Shiloach J, Grisshammer R. FEBS Lett. 2004;564:289–293. [PubMed]
36. Hartman JL, IV, Northup JK. J Biol Chem. 1996;271:22591–22597. [PubMed]
37. Wildman DE, Tamir H, Leberer E, Northup JK, Dennis M. Proc Natl Acad Sci USA. 1993;90:794–798. [PMC free article] [PubMed]
38. Grisshammer R, Averbeck P, Sohal AK. Biochem Soc Trans. 1999;27:899–903. [PubMed]
39. Tucker J, Grisshammer R. Biochem J. 1996;317:891–899. [PMC free article] [PubMed]
40. Hellmich MR, Battey JF, Northup JK. Proc Natl Acad Sci USA. 1997;94:751–756. [PMC free article] [PubMed]
41. Jian X, Sainz E, Clark WA, Jensen RT, Battey JF, Northup JK. J Biol Chem. 1999;274:11573–11581. [PubMed]
42. Albright RA, Ibar JL, Kim CU, Gruner SM, Morais-Cabral JH. Cell. 2006;126:1147–1159. [PubMed]
43. Hayashi Y, Matsui H, Takagi T. Methods Enzymol. 1989;172:514–528. [PubMed]
44. Wei Y, Li H, Fu D. J Biol Chem. 2004;279:39251–39259. [PubMed]
45. Yernool D, Boudker O, Folta-Stogniew E, Gouaux E. Biochemistry. 2003;42:12981–12988. [PubMed]
46. Folta-Stogniew E, Williams KR. J Biomolecular Techniques. 1999;10:51–63. [PMC free article] [PubMed]
47. Kendrick BS, Kerwin BA, Chang BS, Philo JS. Anal Biochem. 2001;299:136–146. [PubMed]
48. Wen J, Arakawa T, Philo JS. Anal Biochem. 1996;240:155–166. [PubMed]
49. Strop P, Brunger AT. Protein Sci. 2005;14:2207–2211. [PMC free article] [PubMed]
50. Whorton MR, Bokoch MP, Rasmussen SGF, Huang B, Zare RN, Kobilka B, Sunahara RK. Proc Natl Acad Sci USA. 2007;104:7682–7687. [PMC free article] [PubMed]
51. Bayburt TH, Leitz AJ, Xie G, Oprian DD, Sligar SG. J Biol Chem. 2007;282:14875–14881. [PubMed]

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