Direct observation of G-protein binding to the human delta-opioid receptor using plasmon-waveguide resonance spectroscopy.

Using a recently developed method (Salamon, Z., Macleod, H. A., and Tollin, G. (1997) Biophys. J. 73, 2791-2797), plasmon-waveguide resonance spectroscopy, we have been able, for the first time, to directly measure the binding between the human brain delta-opioid receptor (hDOR) and its G-protein effectors in real-time. We have found that the affinity of the G-proteins toward the receptor is highly dependent on the nature of the ligand pre-bound to the receptor. The highest affinity was observed when the receptor was bound to an agonist ( approximately 10 nm); the lowest when receptor was bound to an antagonist ( approximately 500 nm); and no binding at all was observed when the receptor was bound to an inverse agonist. We also have found direct evidence for the existence of an additional G-protein binding conformational state that corresponds to the unliganded receptor, which has a G-protein binding affinity of approximately 60 nm. Furthermore, GTP binding to the receptor.G-protein complex was only observed when the agonist was pre-bound. Similar studies were carried out using the individual G-protein subtypes for both the agonist and the unliganded receptor. Significant selectivity toward the different G-protein subtypes was observed. Thus, the unliganded receptor had highest affinity toward the Galphao (Kd approximately 20 nm) and lowest affinity toward the Galphai2 ( approximately 590 nm) subtypes, whereas the agonist-bound state had highest affinity for the Galphao and Galphai2 subtypes (Kd approximately 9 nm and approximately 7 nm, respectively). GTP binding was also highly selective, both with respect to ligand and G-protein subtype. We believe that this methodology provides a powerful new way of investigating transmembrane signaling.

Opioid receptors belong to the superfamily of GPCRs. 1 Their predicted topology is that of a polytopic integral membrane protein with seven membrane-spanning helical segments, an extracellular N terminus and an intracellular C terminus. These receptors and their endogenous ligands, opioid peptides such as endorphins and enkephalins, form a neuromodulatory system that is involved in stress-induced analgesia, that affects locomotive activity and regulates neuroendocrine physiology and autonomic functions such as respiration, blood pressure, and gastrointestinal motility. The opioid system has also been shown to play a role in learning and memory and possibly in the modulation of the immune system (1) and is an important factor in pain modulation and drug abuse. Studies to determine the G-protein subtypes that mediate the intracellular signaling of the opioid receptor systems have shown that functional coupling occurs to the G i -G o family of G-proteins (2)(3)(4). The human brain ␦-opioid receptor has been cloned (5), stably transfected in Chinese hamster ovary (CHO) cells, and characterized (6).
Due to their integral membrane nature and their low cellular concentrations, there is little information on the structures and functional mechanisms of the opioid receptors. Furthermore, classic pharmacological methods only give indirect information about the interaction of GPCRs with G-proteins, because they are based on downstream responses. The GTP␥S assay reflects a combination of both the affinity of the G-protein to the ligandbound receptor (7) and the ability of the agonist-bound state to initiate GDP dissociation from the G-protein (8). Thus, such measurements do not directly probe interactions between the various signal transduction partners and suffer the disadvantages of being very time consuming and dependent on the use of radiolabeled material. Another limitation of such experiments comes from the fact that cells usually express all the different G-protein subtypes making it very hard to understand the contribution of each individual subtype.
PWR spectroscopy is a newly developed innovative experimental methodology that enables in-depth characterization of protein-protein, protein-lipid, and protein-ligand interactions occurring either within or at the surface of anisotropic thin films under native conditions and without the use of labels (9 -14). By measuring plasmon resonances excited by lightpolarized both parallel (s) and perpendicular (p) to the deposited film, this technology allows direct measurements of the anisotropic optical properties of biomembranes and real-time characterization of changes in the mass density and molecular orientation of molecules contained therein and thus can be used to monitor the thermodynamics and kinetics of binding processes and the accompanying structural changes. In the present study we have incorporated purified detergent-solubilized hDOR molecules into a lipid bilayer deposited onto the silica surface of a PWR resonator (13,15) and have directly observed the interaction of a G-protein mixture with either the unliganded receptor or with receptor pre-bound with either the agonist DPDPE (c-[D-Pen 2 ,D-Pen 5 ]enkephalin), the antagonist naltrindole (NTI), or the inverse agonist TMT-L-Tic. We have found that the affinity of G-proteins to the hDOR and the ability to bind GTP are very much dependent on the identity of the ligand that is pre-bound to the receptor. Similar studies were also done using the individual G-protein subtypes, and a high level of selectivity was observed that is highly modulated by the liganded state of the receptor. The results yield new insights into GPCR function and demonstrate that PWR provides a new, simple, and direct approach to investigate transmembrane signaling.

Peptide Affinity Ligand Synthesis and Use in hDOR Purification-
The ligand resin was prepared with the following sequence attached: H-Tyr-D-Ala-Phe-Glu-Val-Val-Gly-␤-Ala-Gly-␤-Ala-Gly-resin, where the first four amino acids attached to the solid support function as a spacer arm and the rest of the sequence corresponds to Deltorphin II, a potent and selective ligand for the DOR. The N ␣ -Fmoc strategy of solid-phase peptide synthesis was used. The first Gly residue was coupled to Sepharose resin (CM-Sepharose, Amersham Biosciences) (0.09 -0.13 mmol/ml) using 10 eq of N ␣ -Fmoc-Gly, 10 eq of N-hydroxybenzotriazole, 10 eq of N,NЈ-diisopropylcarbodiimide, and 4 eq of Nmethylimidazole dissolved in a minimal amount of dimethyl formamide. The resin was reacted with the previous mixture in a rocking platform for 1 h. The N ␣ -Fmoc group was cleaved by treating the resin with 25% piperidine in dimethyl formamide during 30 min, and the absorbance at 302 nm was measured to determine the level of amino substitution achieved. These steps were repeated for all the amino acids in the sequence. After coupling all the amino acids, cleavage of the N ␣ -Fmoc group from the N terminus, and cleavage of the side-chain protecting groups was achieved using 95% trifluoroacetic acid, 2.5% thioanisole, and 2.5% anisole for 1.5 h. To test the quality of the peptide, a small part of the peptide was cleaved from resin using 0.5% NaOH in water during 30 min. The filtrate obtained was then submitted for mass spectral analysis that confirmed that the target peptide was synthesized in about 95% purity. The Deltorphin II resin was stored with buffer containing sodium azide and reused several times. The resin was washed with 5 column volumes of low salt (0.1 M KCl) detergent buffer and then incubated with the His tag-purified receptor for 2 h at 4°C. The affinity resin was then washed three times with 1 column of high salt detergent buffer (0.5 M KCl), three times with 1 column of no salt detergent buffer and three times with 1 column of high salt detergent buffer. The resin was then suspended in high salt detergent buffer containing 0.1 mM naltrindole for 1 h, and the receptor was eluted.
Receptor Purification and Characterization-A fully functional receptor, labeled at the C terminus with a myc epitope and His tag, was stably transfected into a Chinese hamster ovary cell line (CHO-K1), and the modified receptor was characterized (6). The receptor was purified using a modified version of a previously published method (13). Changes include use of 1% dodecyl maltoside instead of octylglucoside, the use of an antagonist (0.1 nM naltrindole, Sigma) during the solubilization and purification procedure and the use of ligand affinity chromatography as a second purification method. Details of the synthesis of this ligand affinity resin and its use, as well as the characterization of the solubilized receptor, can be found in the online Supplementary Material (Methods). For the present studies, the hDOR was preincubated with saturating amounts (at least one order of magnitude higher concentration than published binding affinities) of ligand, using agonist (DPDPE) (American Peptide Company, CA), antagonist (NTI, Sigma) or inverse agonist (TMT-L-Tic; synthesized in Dr. Hruby's laboratory following published procedures (16)). The receptor was incubated with the respective ligand for 1-2 h at 4°C.
A BCA (bicinchoninic acid) assay was performed to determine the protein concentration in the sample (Pierce). The purple reaction product was monitored at 560 nm using an enzyme-linked immunosorbent assay plate reader (Quant, Bio-Tek Instruments, Inc). Following purification, the quality of the receptor protein was assessed by determining the specific activity, i.e. the moles of functional receptor molecules (measured by ligand binding) per mole of receptor protein. Binding was performed by diluting the purified receptor to a final concentration of about 400 nM in low salt buffer and adding [ 3 H]naltrindole (PerkinElmer Life Sciences) to a final concentration of about 0.1 nM. The binding reaction was incubated for 1 h at room temperature. Unbound ligand was then separated from the ligand⅐receptor complex by ultrafiltration (YM 30.000, Centricon) using low salt buffer. A com-petition assay was then performed using DPDPE with concentrations ranging from 10 Ϫ3 to 10 Ϫ9 M. This ligand, solubilized in low salt buffer, was incubated with the solution containing the [ 3 H]naltrindole⅐receptor complex previously obtained at room temperature for 1 h. Samples were then placed in scintillation vials filled with scintillation liquid and measured in a counter (Beckman). Binding results were plotted using GraphPad Prism (San Diego, CA).
Lipid Bilayer Formation, hDOR Incorporation, and G-protein Addition-In this study we used self-assembled solid-supported lipid membranes (17,18). The method of preparation uses the same principles that govern the spontaneous formation of a freely suspended lipid bilayer membrane (called a black lipid membrane) (19), as previously reported (13,15). The lipid films were formed on the silica surface of the PWR resonator from the following membrane-forming solutions: 7 mg/ml egg PC and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol (Avanti Polar Lipids) (75:25 mol/mol) in squalene/butanol/methanol (0.05:0.95:0.5, v/v). The incorporation of the human ␦-opioid receptor into this lipid bilayer was accomplished by introducing the detergentsolubilized hDOR into the aqueous compartment under conditions that dilute the detergent to below the critical micelle concentration, which allows the membrane protein to spontaneously incorporate into the lipid bilayer. In these experiments we were interested in studying the ternary complex, i.e. ligand, receptor, and G-protein. However, we do not have simultaneous access to both sides of the receptor, and we think that neither the G-protein nor most of the ligands are able to cross the bilayer. Since binding to both the G-proteins and ligands can occur after receptor incorporation, it appears that the receptor inserts bi-directionally into the lipid bilayer. Thus, we have studied the ternary complex by prebinding the receptor with the ligand before incorporation into the lipid bilayer. In this way, some of the ligand-bound receptors will have their G-protein binding sites accessible to the external aqueous medium. Small aliquots of G-proteins consisting of a purified mixture of the predominant forms of pertussis toxin-sensitive G-proteins, associated with the DOR, from bovine brain (Calbiochem) containing G␣ o (ϳ4 -5 M), G␣ i1 (ϳ1-2 M), G␣ i2 (ϳ1-2 M), G␣ i3 (Ͻ1 M), and the ␤␥ subunit complex (ϳ8 M) were incrementally added to the equilibrated proteolipid system. The purified individual G-protein subtypes were also obtained from Calbiochem. After saturation was reached, GTP␥S (Sigma) was added and the PWR spectral changes were monitored.
Plasmon-Waveguide Resonance Spectroscopy-The method is based upon the resonant excitation by polarized light from a CW He-Ne laser ( ϭ 632.8 nm or ϭ 543.5 nm), passing through a glass prism under FIG. 1. Side view of a PWR experimental device containing a glass prism coated with a silver layer overcoated with a SiO 2 film. Also shown in the diagram are a deposited lipid bilayer held in place by a Teflon spacer via a plateau-Gibbs border. In these experiments a receptor bound to a ligand is inserted into the bilayer and G-protein is added. The excitation and measuring systems are also shown. The prism, the detector, and the aqueous compartment of the device are mounted onto a rotating table allowing the angle ␣ to be varied from 35 to 70°with 1-millidegree steps.
total internal reflection conditions, of collective electronic oscillations (plasmons) in a thin metal film (Ag) deposited on the external surface of the prism, which is overcoated with a dielectric layer (SiO 2 ). The resonant excitation of plasmons generates an evanescent electromagnetic field localized at the outer surface of the dielectric film, which can be used to probe the optical properties of molecules immobilized on this surface (9,20,21). Resonance is achieved either by varying the incident light wavelength () at a fixed angle (␣), or by varying ␣ at a fixed (the latter protocol was used in our experiments). Because the resonance coupling generates electromagnetic waves at the expense of incident light energy, the intensity of totally reflected light is diminished at a specific angle. Thus, the angular dependence of the reflectance corresponds to a PWR spectrum. The resonance can be excited with light polarized with the electric vector either parallel (p) or perpendicular (s) to the incident plane thereby allowing for characterization of the molecular organization of anisotropic systems such as biomembranes containing integral proteins (11,14,21). The experimental arrangement for PWR measurements is shown in Fig. 1. Under the experimental conditions employed in this work the optical parameters obtained with the p-polarization refer to the perpendicular direction, and those obtained with s-polarization refer to the parallel direction, relative to the bilayer membrane surface. PWR spectra can be described by three  ). B, PWR spectra measured using s-polarized exciting light; the spectra correspond to the same sequence of events described in A. C, binding curves obtained from plotting the shifts in the resonance position minimum of the PWR spectra obtained after several incremental additions of aliquots of G-proteins for p-(f) and s-polarized light (OE). Solid curves correspond to hyperbolic fits to the data; binding constant values are given in Table I. D, data points obtained from plotting the shifts in the resonance position minimum of the PWR spectra obtained after several incremental additions of aliquots of G-proteins to the lipid bilayer with no receptor incorporated for p-(f) and s-polarized light (OE). Binding constants are given in Table I. E and F, PWR spectra obtained before (f) and after (Ⅺ) addition of GTP␥S (total concentration in the sample cell was 1 M) to the DPDPE⅐hDOR⅐G-protein complex for p-and s-polarized light, respectively. G, binding curves for the interaction of GTP␥S with the DPDPE⅐hDOR⅐G-protein complex for p-(f) and s-polarization (OE). Solid curves correspond to hyperbolic fits to the data; binding constants are given in Table I. H and I, time resolved experiments for the interaction of the G-protein with the agonist bound receptor and for the spectral shifts occurring upon GTP␥S treatment, for p-(f) and s-polarization (OE), respectively. Solid curves correspond to single exponential fits to the data. parameters: ␣ (or ), the spectral width, and the resonance depth. These depend on the refractive index (n), the extinction coefficient (k), and the thickness (t) of the plasmon-generating and emerging media, the latter including a thin film deposited on the silica surface (a proteolipid membrane in the present work) in contact with an aqueous solution. For non-spherical molecules oriented uniaxially on the resonator surface, which is the case in our studies, n and k will be different for s-and p-polarization, and this allows characterization of molecular orientation and conformational changes. Resonance spectra in this study were obtained using a Beta PWR instrument from Proterion Corp. (Piscataway, NJ) that records the relative reflectance versus the absolute angle with a resolution of 1 millidegree.

RESULTS
Binding of G-proteins to Various Liganded States of the hDOR-From PWR spectral measurements such as those illustrated in Fig. 2 (A and B), it can be seen that bilayer deposition, agonist-bound receptor incorporation, and addition of a G-protein solution to the aqueous compartment of the PWR cell lead to increases in the resonance angle minimum and changes in the resonance depth for both p-and s-polarized exciting light. These are related to increases of refractive index due to an increase in deposited mass on the resonator surface, as well as to increases in proteolipid film thickness (9,22). To quantify these changes, it is necessary to carry out a full theoretical analysis of these spectra. Such analyses are presently underway and will be reported separately.
In these experiments, we do not directly determine the concentrations of receptor and G-protein in the PWR cell. Affinities are determined based on the PWR spectral changes that occur due to mass increases in the proteolipid system upon incremental addition of G-protein to the cell. Only material that is deposited on the resonator surface affects the PWR signal, i.e. there is no interference from the material that is in the bulk solution. Thus, the spectral changes are proportional to the amount of G-protein bound to the receptor and plots of spectral shifts versus bulk G-protein concentration allow a direct determination of binding affinity. In other words, each concentration point in a saturation curve corresponds to the total G-protein added to the aqueous compartment versus the amount bound, and it is assumed that the bulk material is able to freely diffuse and equilibrate with the membrane.
In the case of G-protein binding, control experiments (Fig.  2D) demonstrate that much smaller spectral shifts (Ͻ10%) occur when the same concentrations of G-protein solution as in Fig. 2 (A and B) are added to a bilayer that has not had hDOR incorporated. Plots of the G-protein concentration (final concentration in the cell compartment) versus the resonance position angular shift (Fig. 2C) yield a hyperbolic curve that can be fit to obtain the K d value for G-protein binding to the hDOR (Table I). In the absence of receptor, the smaller spectral shifts observed do not follow a hyperbolic curve over the concentration range used and thus are non-saturating (Fig. 2D); presumably, these correspond to nonspecific G-protein binding to the lipid bilayer. The K d values obtained for DPDPE-liganded hDOR are in good agreement with the previously determined EC 50 for DPDPE (19.1 Ϯ 7.2 nM) obtained from [ 35 S]GTP␥S membrane assays (23). We should also point out that the total shifts in the PWR spectra in the presence of hDOR were larger for p-polarization than for s-polarization (ϳ50 millidegrees for p-polarization versus ϳ40 millidegrees for s-polarization), which is characteristic of an anisotropic structural change, as was also observed previously for agonist binding to the hDOR (13,15). This is consistent with the cylindrical shape of the receptor⅐G-protein complex. To our knowledge, the only other direct observations of G-protein binding to a GPCR have been carried out using surface plasmon resonance measurements of the rhodopsin/transducin system (17,21,24). Fig. 2 (E and F) shows the effect of adding GTP␥S (the non-hydrolyzable form of GTP) to the DPDPE⅐hDOR⅐G-protein complex. This caused a decrease in the incident angle of the resonance, which can be approximated by a lowering of the refractive index resulting from a decrease in mass in the proteolipid system (if the membrane thickness had changed, this should have produced a change in resonance amplitude as well (22), which was not observed). The angular shifts also followed a hyperbolic binding curve (Fig. 2G) with K d values as given in Table I. It is known that the receptor-activated exchange of GDP by GTP in the ␣ subunit of the G-protein causes release of this subunit from the receptor and from the ␤␥ subunit. Thus, if the ␣-subunit leaves the membrane entirely in the present system, the decrease in the membrane-bound mass in the proteolipid system could correspond to this process. However, it is known that the G-protein subunits are palmitoylated, myristoylated, and prenylated, and thus the ␣-subunit may remain membrane-bound even after activation. A second possibility is  with no ligand (B). Binding curves were obtained by plotting the shifts in the resonance position minimum of the PWR spectra obtained after several incremental additions of aliquots of G-proteins for p-(f) and s-polarized light (OE). Solid curves correspond to hyperbolic fits to the data; binding constant values are given in Table I. All spectra were measured with 543-nm exciting light.

TABLE I
Binding constants for the interaction between G-proteins and the hDOR either unbound or prebound to agonist or antagonist K d values were obtained from plotting the resonance minimum position for the PWR spectra as a function of G-protein concentration and fitting to the following hyperbolic function that describes the binding of a ligand to a receptor: Y ϭ (B max ϫ X)/(K d ϩ X). B max represents the maximum concentration bound and K d is the concentration of ligand required to reach half-maximal binding. a change in the structure of the receptor complex that results in displacement of lipid from the bilayer into the Gibbs border that anchors the bilayer to the Teflon spacer separating the resonator from the aqueous compartment, as we have previously observed for agonist binding to the hDOR (13,15). Further studies are required to clarify this point. GTP binding to transducin was also observed in the earlier surface plasmon resonance studies of rhodopsin (17,21,24). Because our previous work (13, 15) 2 has shown that high affinity ligand binding to the hDOR occurs under the conditions of the present experiments, the fact that we can now also bind G-proteins with high affinity shows that the receptor incorporates into the lipid bilayer in a bidirectional manner, i.e. some of the molecules insert with their ligand binding site facing the external medium and others with their G-protein binding site facing in that direction. In Fig. 2 (H and I) we show the time courses for G-protein binding to the receptor (pre-bound with agonist) and for the spectral shifts, possibly due to ␣-subunit dissociation, following GTP␥S addition. By fitting the data to an exponential function (solid lines), we have obtained the following rate constants: for G-protein binding to the receptor, 0.29 min Ϫ1 for p-polarized light and 0.23 min Ϫ1 for s-polarized light; for the putative dissociation process, 0.19 min Ϫ1 for p-polarized light, and 0.17 min Ϫ1 for s-polarized light.
Applying the same strategy, we have studied the binding of the G-proteins to hDOR pre-bound with the antagonist naltrin-dole (NTI) and have found that the binding to the receptor still occurred but with a 50-fold lower affinity (Table I). As in the case of the agonist-bound state, we found the process to be anisotropic with shifts in the p-polarization resonance of ϳ40 and ϳ30 millidegrees for the s-polarization resonance and to follow a hyperbolic saturation curve (Fig. 3A). The difference in G-protein binding affinity between agonist-bound and antagonist-bound receptor states is in agreement with studies using fluorescence spectroscopy, which support the existence of conformational heterogeneity of G-protein-coupled receptors, depending on the nature of the ligand being bound (25)(26)(27), as well as with recent studies of GPCRs using PWR, which have directly shown that an antagonist places the receptor into a different conformation from that produced by an agonist or inverse agonist (13,15). 2,3 It is also well known from pharmacological studies that antagonist binding produces conformations that are not favorable for G-protein binding and activation. Consistent with these observations is the fact that GTP␥S addition to the antagonist-liganded hDOR⅐G-protein complex produced no additional PWR spectral shifts (Fig. 4, A and B). It is known that point mutations in receptors (for example, mutations of Phe-303 in the ␣ 1B -adrenoreceptor) can preserve high affinity for G-proteins but eliminate the ability of receptor agonists to produce G-protein activation (28). This suggests that the receptor conformations for binding to G-protein and for its activation may be distinct. It is also worth noting that the FIG. 4. G-protein and GTP␥S interactions with the hDOR pre-bound with the antagonist NTI and inverse agonist TMT-L-Tic. PWR spectra were obtained using 543-nm excitation. A and B, PWR spectra for p-and s-polarization, respectively, for the NTI⅐hDOR⅐G-protein complex before (Ⅺ) and after (f) addition of GTP␥S (final concentration 1 M). The hDOR concentration was 4 nM and the G-protein concentration was 800 nM, the latter corresponding to the amount needed to reach saturation. C and D, PWR spectra for p-and s-polarization, respectively, obtained with the TMT-L-Tic⅐hDOR complex before (q) and after (f) addition of G-proteins (the receptor concentration in the sample cell was 4 nM and the G-protein concentration was 800 nM). existence of antagonist⅐receptor⅐G-protein complexes has been observed for the -opioid receptor using the GTP␥S assay (29).
The addition of G-proteins to the inverse agonist (TMT-L-Tic)-bound receptor was found to give even more pronounced differences than those obtained with antagonist. Thus, the G-protein does not recognize the receptor when in the inverse agonist-bound state (Fig. 4, C and D), as evidenced by the fact that the small shifts in the PWR spectra obtained here were indistinguishable from the nonspecific binding of the G-protein to the lipid bilayer that was observed in our control experiments (see above). This is consistent with the well known elimination of the basal activity of GPCRs (i.e. G-protein activation in the absence of agonist binding) by inverse agonists.
Again, as expected, addition of GTP␥S produced no spectral changes (not shown).
We also have studied G-protein binding to a receptor in the unliganded state and have found that binding indeed occurs, although with a lower affinity than that observed for the agonist-bound state (Table I). Again, the process is anisotropic but to a lesser extent than for the agonist or antagonist-bound state, and the total shifts also were smaller (ϳ30 millidegrees for p-polarization and ϳ26 millidegrees for s-polarization, as seen in Fig. 3B), suggesting structural differences in the states produced. Constitutive activity has become a well described characteristic of many GPCRs and has helped to redefine the concept of how they function. Such ligand-independent activity FIG. 5. Interactions of G␣ i2 plus ␤␥ and GTP␥S with the hDOR pre-bound with agonist DPDPE. PWR spectra were obtained using 543-nm excitation. A and B, PWR spectra for p-and s-polarization, respectively, obtained with the DPDPE⅐hDOR complex before (q) and after (f) addition of G␣ i2 plus ␤␥ (the receptor concentration in the sample cell was 4 nM, and the G-protein concentration was 125 nM). C, data points obtained from plotting the shifts in the resonance position minimum of the PWR spectra obtained after several incremental additions of aliquots of G␣ i2 and ␤␥ subunits to the lipid bilayer with agonist bound receptor incorporated for p-(f) and s-polarized light (OE). Solid curves correspond to hyperbolic fits to the data; binding constants are given in Table II. D, binding curves for the interaction of GTP␥S with the DPDPE⅐hDOR⅐G␣ i2 ϩ ␤␥ complex for p-(f) and s-polarization (OE). Solid curves correspond to hyperbolic fits to the data; binding constants are given in Table II.   TABLE II Binding affinities between the individual G-protein subtypes and the hDOR either unliganded or DPDPE-bound and between GTP␥ S and the receptor⅐G-protein complex K d values were determined as in Table I. has been described for the opioid receptors, either in their wild-type form or in mutated forms (30). Even though it has long been thought that the inactivated receptor should be able to interact with G-proteins, and this state has been proposed by Kenakin as a component of the cubic ternary complex model of GPCR function (31,32), up until now there has been no direct evidence of the existence of that state. The present studies directly demonstrate that the unliganded receptor corresponds to a distinct state from that of agonist, antagonist, and inverse agonist-bound species, because it binds to the G-protein with a different affinity and produces a different structure. Previous studies with the ␤ 2 -adrenergic receptor point to this same conclusion based on thermal denaturation and proteolysis experiments (33,34). It is important to point out, however, that, as in the antagonist and inverse agonist cases, we have observed no changes in the PWR spectra upon addition of GTP␥S to the receptor⅐G-protein complex, suggesting that the unliganded receptor may not allow activation. This interesting observation requires further study.
Binding of Individual G-protein Subtypes to Unliganded and Agonist-bound hDOR-We have also investigated the interaction of the individual G-protein subtypes with the unliganded and DPDPE-bound states of the receptor. As an illustration of the results obtained in these experiments, Fig. 5 (A and B) presents the PWR spectra obtained for the interaction of the G␣ i2 plus ␤␥ subunits (1:1 ratio) with the agonist-bound receptor. It should be pointed out that the total shift obtained upon G-protein binding to the receptor was comparable to the shifts obtained with the G-protein mixture, as expected because approximately the same amount of receptor was present in both cases. As can be seen in Fig. 5 (B and C) and Table II, the K d values obtained for the binding of the G␣ i2 plus ␤␥ subunits to the agonist-bound receptor and the GTP␥S affinity for the receptor⅐G-protein complex are comparable to those obtained for the G-protein mixture. Table II also presents a summary of the results obtained with the other G-protein subtypes. It is very interesting to note the high level of specificity in the binding of the G-protein subunits to the receptor, both in the unliganded and the agonist-bound states. The agonist-bound receptor has the highest affinity for the G␣ i2 and the G␣ o subunits (ϳ7 and 10 nM, respectively; slightly higher than the G-protein mixture), intermediate affinity for the G␣ i3 subunit, and lowest affinity for the G␣ i1 subunit. The unliganded receptor also has the highest affinity for the G␣ o subtype (ϳ20 nM), whereas it has intermediate affinity for the G␣ i1 (ϳ80 nM, higher than the agonistbound state) and the G␣ i3 subtypes, and, in contrast to the agonist-bound state, it has the lowest affinity for the G␣ i2 subtype. Previous coimmunoprecipitation studies of the mouse ␦-opioid receptor⅐G-protein complexes performed with antisera directed against different G␣ and G␤ subunits noted changes in the G-protein subtype association upon agonist stimulation (35). Thus, agonist binding causes an increase in association with G␣ i and a decrease in association with G␣ o , dissociation of G␣ i1 and association with G␣ i2 , and no change in association with G␣ i3 and G ␤ . These results correlate with ours.
As seen in Table II, the affinities of GTP␥S toward the receptor⅐G-protein complex are also very much dependent on the G-protein subtype that is bound, as well as on the receptor state. The highest affinity was found for the receptor⅐G␣ i1 complex (ϳ4 nM) and the lowest for the receptor⅐G␣ o complex (ϳ400 nM). In contrast to the agonist-bound state of the receptor, no PWR spectral shifts were observed upon addition of GTP␥S (concentrations up to 5 M), except for the receptor⅐G␣ o complex. It is also interesting to note that the GTP␥S affinities do not track the G-protein affinities, adding another level of selectivity to these interactions. Previous GTPase activity studies done with the hDOR also observed that the G␣ i1 -bound receptor promoted greater GTP exchange than the G␣ o subtype, activating about three times more efficiently (36). Also of interest, in a recent study the dopamine receptor was expressed with four different G-protein subtypes (the same as used in the present work), and the coupling between receptor and G-protein was investigated upon agonist treatment (37). A high level of selectivity in the receptor⅐G-protein interaction was also found, as well as differential agonist activation of the four G-protein subtypes. DISCUSSION The present work has demonstrated that PWR spectroscopy can provide important new insights into membrane signaling by GPCRs. The results are consistent with the formation of distinct conformational states of the hDOR by binding of various types of ligand that interact differently with G-proteins and that correlate well with the known pharmacological activities of these different ligand classes. We are presently carrying out a more detailed analysis of the data described above, including theoretical fitting of the PWR spectra to evaluate refractive indices and thickness parameters. This will provide greater insights into the structural conformations of the various states produced in these experiments and will be reported separately.
The results obtained with the individual G-protein subunits reveal a high degree of diversity and selectivity that may be important in controlling the specific interactions of these receptors with multiple cellular effector systems. We are currently applying the same strategy to study receptor⅐G-protein interactions upon different agonist treatments. This should provide new insights into the basis for differential physiological and/or pharmacological effects of drug activity. It is important to point out that PWR spectroscopy allows one to pursue a great variety of studies without having to rely on labeling protocols, as well as avoiding more complicated and time-consuming techniques. We also note that it should also be possible to design experiments to elucidate events occurring further downstream in the signal transduction process, such as receptor down-regulation. As a consequence, the methods described here will allow new light to be shed on the pathways of signal transduction and should be extremely useful in drug discovery protocols.