©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Fluorescent Labeling of Purified Adrenergic Receptor
EVIDENCE FOR LIGAND-SPECIFIC CONFORMATIONAL CHANGES (*)

(Received for publication, August 8, 1995; and in revised form, September 18, 1995)

Ulrik Gether (1) Sansan Lin (1) Brian K. Kobilka (1) (2)(§)

From the  (1)Howard Hughes Medical Institute and the (2)Division of Cardiovascular Medicine, Stanford University Medical School, Stanford, California 94305

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The purpose of the present study was to develop an approach to directly monitor structural changes in a G protein-coupled receptor in response to drug binding. Purified human beta(2) adrenergic receptor was covalently labeled with the cysteine-reactive, fluorescent probe N,N`-dimethyl-N-(iodoacetyl)-N` - (7 - nitrobenz -2 - oxa -1,3diazol-4-yl)ethylenediamine (IANBD). IANBD is characterized by a fluorescence which is highly sensitive to the polarity of its environment. We found that the full agonist, isoproterenol, elicited a stereoselective and dose-dependent decrease in fluorescence from IANBD-labeled beta(2) receptor. The change in fluorescence could be plotted against the concentration of isoproterenol as a simple hyperbolic binding isotherm demonstrating interaction with a single binding site in the receptor. The ability of several adrenergic antagonists to reverse the response confirmed that this binding site is identical to the well described binding site in the beta(2) receptor. Comparison of the response to isoproterenol with a series of adrenergic agonists, having different biological efficacies, revealed a linear correlation between biological efficacy and the change in fluorescence. This suggests that the agonist-mediated decrease in fluorescence from IANBD-labeled beta(2) receptor is due to the same conformational change as involved in receptor activation and G protein coupling. In contrast to agonists, negative antagonists induced a small but significant increase in base-line fluorescence. Despite the small amplitude of this response, it supports the notion that antagonists by themselves may alter receptor structure. In conclusion, our data provide the first direct evidence for ligand-specific conformational changes occurring in a G protein-coupled receptor. Furthermore, the data demonstrate the potential of fluorescence spectroscopy as a tool for further delineating the molecular mechanisms of drug action at G protein-coupled receptors.


INTRODUCTION

The beta(2) adrenergic receptor is a prototype member of the G protein-coupled receptor family(1) . The receptor family constitutes the largest group of plasma membrane receptors, which are characterized by a remarkable diversity in the chemical structure of their endogenous ligands(1, 2, 3) . The receptors are all believed to share a common topology with seven alpha-helical, transmembrane segments; however, the helical arrangement and actual three-dimensional structure of the receptors remain unknown(1, 2, 3) . Mutagenesis studies in the beta(2) adrenergic receptor as well as in many other G protein-coupled receptors have been able to assign distinct receptor functions, such as ligand binding and G protein coupling, to specific receptor domains(1, 2, 3) . Several molecular models of these receptors have also been generated based on the structure of bacteriorhodopsin and rhodopsin for which more detailed structural information is available(3, 4, 5) . Nevertheless, very little is known about the molecular events and structural changes in the receptor that provide the important link between ligand binding and transmission of the signal across the membrane.

Drugs acting at G protein-coupled receptors are traditionally classified in biological assays as either full agonists, which elicit the maximal response, as partial agonists, which only elicit a fractional response, or as antagonists, which block the response induced by agonists(6, 7) . In addition, recent data have suggested that antagonists should be subclassified into at least two categories: neutral antagonists, which have no effect on basal receptor activity, and negative antagonists (also referred to as inverse agonists), which inhibit basal receptor activity occurring in the absence of agonist (7, 8, 9, 10, 11, 12, 13) . Thus, in contrast to the conventional view, it has now become evident even in vivo(13) that many antagonists actively inhibit receptor function rather than just passively block access of the agonist to its binding site(8, 9, 10, 11, 12, 13) . However, the structural basis for the biological classification of drug action at G protein-coupled receptors is not yet known. Any direct evidence for the existence of discrete ligand-specific conformational states of G protein-coupled receptors has not been obtained. To date, the conformational state of G protein-coupled receptors has been assessed only by indirect methods, such as the effect of receptor conformation on G protein GTPase activity or on the activity of the effector enzymes.

In the present study we have developed a fluorescence spectroscopy approach to directly monitor ligand-induced conformational changes in a G protein-coupled receptor. Our approach takes advantage of the sensitivity of many fluorescent molecules to the polarity of their molecular environment(14, 15, 16, 17) . Fluorescent labels incorporated into proteins can therefore often be used as sensitive indicators of conformational changes and of protein-protein interactions that cause changes in polarity of the environment surrounding the probe(14, 15, 16, 17) . To accomplish these experiments, we expressed the human beta(2) adrenergic receptor in SF-9 insect cells and established a purification procedure, which allowed us to obtain the required amount of pure protein for the spectroscopy analysis. The purified receptor was labeled with the cysteine-specific and environmentally sensitive fluorescent probe N,N`-dimethyl-N-(iodoacetyl)-N`-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD). (^1)We found that binding of full agonists and strong partial agonists to the fluorescently labeled beta(2) adrenergic receptor produced a stereospecific, dose-dependent, and reversible decrease in fluorescence emission, which is not observed in response to weaker partial agonists or in response to neutral or negative antagonists. The magnitude of the response correlated with agonist efficacy, suggesting that this change in fluorescence emission is due to the same conformational change as involved in agonist activation of the receptor.


EXPERIMENTAL PROCEDURES

Expression and Purification of the beta(2) Adrenergic Receptor

DNA sequences encoding the human beta(2) adrenergic receptor, epitope-tagged at the amino terminus with the cleavable influenza-hemagglutinin signal-sequence followed by the ``FLAG''-epitope (IBI, New Haven, CT), and tagged at the carboxyl terminus with six histidines, were cloned into the baculovirus expression vector pVL1392 (Invitrogen, San Diego, CA) and expressed in SF-9 insect cells according to previously described methods(18) . For purification, the cells were grown in 1000-ml cultures in SF 900 II medium (Life Technologies, Inc.) containing 5% fetal calf serum (Gemini, Calabasas, CA) and 0.1 mg/ml gentamicin (Life Technologies, Inc.). Cells were routinely infected with a 1:30-40 dilution of a high titer virus stock at a density of 6-8 times 10^6 cells/ml and harvested after 48 h. Cell pellets were kept at -70 °C until used for purification. The receptor was purified to homogeneity using a three-step purification procedure as described elsewhere(19) . Briefly, lysed cell membranes were solubilized in 0.8% n-dodecyl beta-D-maltoside (DbetaM) (CalBiochem, La Jolla, CA) followed by Ni-column chromatography using chelating Sepharose (Pharmacia, Uppsala, Sweden). The eluate from the Ni-column was further purified on an M1 anti-FLAG antibody column (IBI) and finally by alprenolol-affinity chromatography. This procedure ensured that only noncleaved and functional protein was purified. Approximately 5 nmol of pure protein generally could be obtained from a 1000-1500-ml culture.

Fluorescent Labeling of Purified beta(2) Receptor

Purified beta(2) receptor (1-1.5 nmol) was labeled with 10-15-fold molar excess of IANBD (Molecular Probes, Eugene, OR) (150 µM) in a total volume of 100 µl of buffer (20 mM Tris-buffer, pH 7.4, containing 100 mM NaCl and 0.05% DbetaM). The reaction was allowed to proceed for 1 h at room temperature in the dark and was quenched by addition of 1 mM cysteine. Cysteine-reacted dye was removed by desalting on a Sephadex G50 gel filtration column (0.5 cm times 9 cm) followed by concentrating the resulting sample to 100 µl using a Centricon-30 concentrator (Amicon, Beverly, MA). The labeled protein was diluted approximately 100-fold in buffer for the fluorescence measurements. Fluorescence in control samples without receptor or with receptor incubated with cysteine-reacted IANBD was negligible (Fig. 1b). The stoichiometry of the labeling was determined by measuring absorption at 481 nm and using an extinction coefficient of 21,000 M cm for IANBD and a molecular mass of 50,000 Da for the beta(2) receptor. Protein concentration was determined using the Bio-Rad DC protein assay kit (Bio-Rad). The labeling procedure resulted in incorporation of 1.2 ± 0.16 mol of IANBD per mol of receptor (mean ± S.E., n = 7).


Figure 1: Fluorescence properties of IANBD and IANBD-labeled beta(2) receptor. a, emission spectra of cysteine-reacted IANBD (0.3 µM) in solvents of different polarity. Excitation was set at 481 nm. b, emission spectrum of IANBD-labeled beta(2) receptor (0.15 µM receptor, 1.2 mol IANBD per mol receptor). Control is emission spectrum of 0.15 µM beta(2) receptor ``labeled'' with IANBD prebound to free cysteine instead of free IANBD to asses possible nonspecific attachment of the probe to the receptor during labeling. Insert, 10% SDS-polyacrylamide gel electrophoresis of IANBD-labeled beta(2) receptor. Lane 1, 150 pmol of IANBD-labeled beta(2) receptor; lanes 2 and 3, 150 pmol of beta(2) receptor preincubated before exposure to IANBD with iodoacetamide (lane 2) and N-ethylmaleimide (lane 3). Inset: left panel, Coomassie Blue staining of gel; right panel, gel photographed under UV light. The weak band with an apparent molecular mass of 32.5 kDa is a degradation product of the receptor.



Binding Assay

Binding assays on solubilized and purified beta(2) receptor were performed using [^3H]dihydroalprenolol as radioligand (Amersham Corp.). To routinely assess total amount of receptor, solubilized and purified beta(2) receptor was incubated with 10 nM [^3H]dihydroalprenolol in a total volume of 100 µl of buffer (20 mM Tris-buffer, pH 7.4, containing 100 mM NaCl and 0.05% DbetaM) for 1 h. In competition binding assays, unlabeled or IANBD-labeled purified receptor was incubated with 1.2 nM [^3H]dihydroalprenolol in a total volume of 100 µl of buffer and indicated concentrations of alprenolol or isoproterenol for 1 h. The binding assay was stopped and free [^3H]dihydroalprenolol separated from bound by desalting on Sephadex G50 columns (4 cm times 0.5 cm) using ice-cold buffer. Nonspecific binding was determined in presence of 10 µM alprenolol. IC values were determined by nonlinear regression analysis using Inplot 4.0 from GraphPad Software, San Diego, CA.

SDS-Polyacrylamide Gel Electrophoresis

Purified beta(2) receptor labeled with IANBD was analyzed by 10% SDS-polyacrylamide gel electrophoresis according to Laemmli(20) . Incorporated fluorophore was visualized by photographing the gel under UV light. The protein was visualized by standard Coomassie Blue staining.

Fluorescence Spectroscopy

Fluorescence spectroscopy was performed at room temperature on a SPEX Fluoromax spectrofluorometer with photon counting mode using an excitation and emission bandpass of 4.2 nm. In emission scan experiments, 30-60 pmol of IANBD-labeled beta(2) receptor in 400 µl of buffer (20 mM Tris-buffer, pH 7.4, containing 100 mM NaCl and 0.05% DbetaM) were used. Excitation was 481 nm, and emission measured from 490 to 625 nm with an integration time at 0.3 s/nm. Time course experiments were performed with 30-60 pmol of IANBD-labeled beta(2) receptor in 500 µl of buffer under constant stirring. Excitation was 481 nm, and emission was 523 nm. The volume of the ligands (or water) was always 1% of total volume, and fluorescence was corrected for this dilution in all experiments shown. Fluorescence quenching experiments were performed by sequential addition to the cuvette of potassium iodide (KI) from a freshly made 1 M stock containing 10 mM of Na(2)S(2)O(3). The experiments were done with 50-60 pmol of receptor in an initial volume of 400 µl. After each addition of KI, the sample was thoroughly mixed, and fluorescence was recorded at 523 nm (excitation at 481 nm). To correct for dilution/ionic strength effects on fluorescence changes, 1 M KCl was added to control samples as described above for KI. The corrected data were plotted according to Stern-Volmer equation(21) , F(0)/F = 1 + K[KI], where F(0)/F is the ratio of fluorescence intensity in the absence and presence of KI, and K is the Stern-Volmer quenching constant. All of the compounds tested had an absorbance of less than 0.01 at 481 and 523 nm in the concentrations used excluding any ``inner filter'' effect in the fluorescence experiments (data not shown).

Membrane Preparation from SF-9 Insect Cells

Membranes were prepared from SF-9 cells (30-ml cultures at a density of 3 times 10^6 cells/ml) infected with beta(2) receptor baculovirus for 24 h to obtain low receptor density (approximately 1.2 pmol/mg of protein) and 48 h to obtain high receptor density (approximately 7 pmol/mg of protein). Cells were washed with phosphate-buffered saline and lysed using 25 strokes with a Dounce homogenizer in 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml benzamidin. The lysates were centrifuged for 5 min at 500 times g, and the resulting supernatant was centrifuged at 40,000 times g for 30 min. The membrane pellet was resuspended in binding buffer (75 mM Tris-HCl, pH 7.4, with 12.5 mM MgCl(2) and 1 mM EDTA). Protein was determined using the Bio-Rad DC protein assay kit (Bio-Rad). Adenylate cyclase in membranes was measured as described elsewhere(22) .


RESULTS

Fluorescent Properties of IANBD and Purified beta(2) Receptor Labeled with IANBD

IANBD is a highly fluorescent, cysteine-selective reagent(16, 17) . The fluorescence of IANBD increased as the polarity of the solvent decreased and was more than 10-fold stronger in 1-butanol and n-hexane than in aqueous buffer (Fig. 1a). There was a parallel blue-shift in the emission maximum from 540 nm in aqueous buffer to 530 nm in 1-butanol and 510 nm in n-hexane (Fig. 1a). Labeling of the beta(2) adrenergic receptor, purified from SF-9 insect cells, with IANBD at a stoichiometry of about 1.2 mol of IANBD per mol of receptor revealed a strong fluorescence signal with an emission maximum at 523 nm (Fig. 1b). The blue-shift in emission maximum, as compared to cysteine-reacted IANBD in aqueous buffer, indicated that the modified cysteine(s) are located in an environment that, on the average, is of lower polarity than 1-butanol but higher than n-hexane (Fig. 1b). This would likely involve labeling of one or more of the five cysteine residues that are located in the transmembrane, hydrophobic core of the receptor. This was also supported by the observation that denaturation of the labeled receptor with guanidinium chloride dramatically reduced emission around 80% and caused a 2-3-nm red-shift in the emission maximum (data not shown). The covalent modification of the receptor was confirmed by SDS-polyacrylamide gel electrophoresis of the labeled receptor, and the specificity of the labeling was verified by blocking the incorporation of IANBD with the cysteine-specific, nonfluorescent reagents, iodoacetamide and N-ethylmaleimide (Fig. 1b, inset). The fluorescent labeling did not perturb the pharmacological properties of the receptor as illustrated in Fig. 2. Thus, the agonist, isoproterenol, and the antagonist, alprenolol, inhibited [^3H]dihydroalprenolol binding to IANBD-labeled beta(2) receptor with IC values of 1.3 ± 0.5 µM (n = 3, mean ± S.E.) and 2.8 ± 0.6 nM (n = 3, mean ± S.E.), respectively, as compared to 0.8 ± 0.2 µM (n = 3, mean ± S.E.) and 2.9 ± 0.9 nM (n = 3, mean ± S.E.) for the unlabeled receptor. These data are in agreement with previous binding data on purified beta(2) receptor(23) . Labeling of the beta(2) receptor with IANBD did not affect the total number of binding sites as assessed from binding assays using a saturating concentration of [^3H]dihydroalprenolol (10 nM) (data not shown).


Figure 2: Competition binding profiles of unlabeled and IANBD-labeled, purified beta(2) receptor. a and b, competition binding of [^3H]dihydroalprenolol (1.2 nM) with isoproterenol (a) and alprenolol (b) to unlabeled, purified beta(2) receptor. c and d, competition binding of [^3H]dihydroalprenolol (1.2 nM) with isoproterenol (a) and alprenolol (b) to IANBD-labeled, purified beta(2) receptor. Data are expressed as percent of maximum bound [^3H]dihydroalprenolol (mean ± S.E., n = 3).



Stereospecificity, Dose Dependence, and Reversibility of Isoproterenol-mediated Decrease in Fluorescence from IANBD-labeled beta(2) Receptor

Binding of the full agonist, isoproterenol, to IANBD-labeled beta(2) receptor caused a decrease in fluorescence intensity without detectable change in the wavelength at which maximal emission occurred (Fig. 3). The decrease was stereospecific as illustrated in Fig. 3by comparing the effect of 30 µM of the (-)-isomer of isoproterenol with 30 µM of the less active (+)-isomer. In addition, no response to isoproterenol was observed with IANBD-labeled receptor denatured in guanidinium chloride (data not shown). To investigate the kinetics of this agonist-mediated change, the fluorescence intensity was measured as a function of time ( Fig. 4and Fig. 5). Prior to adding ligand we observed a slight but constant decline in base-line fluorescence ( Fig. 4and Fig. 5). This loss of fluorescence over time is likely due to factors such as bleaching and hydrolysis of the probe during the experiments. It is unlikely that this decline in fluorescence is due to denaturation of the protein, since a similar loss of fluorescence also was observed with labeled receptor that was intentionally denatured in guanidinium chloride (see Fig. 7d). It should also be noted that the decrease over time was unaffected by addition of 0.1% bovine serum albumin, 10% glycerol, or phospholipids to the cuvette (data not shown).


Figure 3: Stereospecificity of isoproterenol induced decrease in fluorescence from IANBD-labeled beta(2) receptor. a, emission spectra of IANBD-labeled beta(2) receptor obtained immediately (t = 0) and after 15 min (t = 15) following addition of 30 µM of the less active (+)-isomer of the agonist, isoproterenol ((+)ISO). b, Emission spectra of IANBD-labeled beta(2) receptor obtained immediately (t = 0) and after 15 min (t = 15) following addition of 30 µM of the active (-)-isomer of the agonist, isoproterenol ((-)ISO). The experiments shown is representative of three identical experiments. Fluorescence measurements were done as described under ``Experimental Procedures'' with excitation set at 481 nm.




Figure 4: Time course and dose-dependence of isoproterenol induced decrease in fluorescence from IANBD-labeled beta(2) receptor. a, emission from IANBD-labeled beta(2) receptor measured over time following stimulation with indicated concentrations of isoproterenol. Excitation was 481 nm and emission measured at 523 nm. Isoproterenol (ISO) was added at the time indicated by the arrow. Fluorescence in the individual traces was normalized to the fluorescence observed immediately after addition of ligand. The experiment shown is representative of four identical experiments. b, the percent change in fluorescence at t = 15 min following addition of isoproterenol plotted against the isoproterenol concentration and fitted to a simple hyperbolic function (F = F(0)L/K + L; F, change in fluorescence at the ligand concentration L; F(0), maximum change in fluorescence; K, affinity constant). c, the relative change in fluorescence at t = 15 min following addition of isoproterenol plotted against the logarithm of the isoproterenol concentration and fitted to a one-site sigmoid curve. The percent change in fluorescence was calculated as the change in fluorescence relative to the extrapolated base line at t = 15 min after addition of isoproterenol. Percent change is given as mean ± S.E. of the following number of experiments, 1000 µM, n = 5; 300 µM, n = 4; 100 µM, n = 4; 30 µM, n = 4; 10 µM, n = 4; 3 µM, n = 4; 1 µM, n = 2; 0.1 µM, n = 2, H(2)O, n = 5. Curve fittings were performed using Inplot 4.0 from GraphPad Software, San Diego, CA.




Figure 5: Reversibility of isoproterenol-induced decrease in fluorescence from IANBD-labeled beta(2) receptor. a, control addition of water (H(2)O). b and c, reversal of the response to isoproterenol (ISO) by the active(-)-isomer of the antagonist propranolol,(-)PROP (b), but not by the less active (+)-isomer, (+)PROP (c). Dotted lines indicate extrapolated base line. Excitation was 481 nm and emission measured at 523 nm. Fluorescence in all the individual traces shown was normalized to the fluorescence observed immediately after addition of ligand. All traces shown are representative of at least three identical experiments.




Figure 7: Effect of antagonists on fluorescence from IANBD-labeled beta(2) receptor. a, emission from IANBD-labeled beta(2) receptor measured over time following stimulation with 1 µM(-)-propranolol ((-)PROP), 1 µM (+)-propranolol ((+)PROP) and water (H(2)O). b, emission from IANBD-labeled beta(2) receptor measured over time following stimulation with 10 µM ICI 118,551 (ICI), 10 µM pindolol (PIND), and water (H(2)O). c, emission from IANBD-labeled beta(2) receptor measured over time following exposure to 10 µM dichloroisoproterenol (DCI), alprenolol (ALP) and water (H(2)O). d, emission from guanidinium chloride denatured IANBD-labeled beta(2) receptor measured over time following exposure to 10 µM(-)-propranolol ((-)-PROP) and 10 µM ICI 118,551 (ICI). The compounds were added at the time indicated by the arrows. Excitation was 481 nm, and emission was measured at 523 nm. Fluorescence in the individual traces was normalized to the fluorescence observed immediately after addition of ligand. The experiment shown is representative of at least three identical experiments.



The time course analysis showed that the response to isoproterenol was dose-dependent and reached a maximum amplitude below the extrapolated base line after 10-15 min (Fig. 4a). The relative change in fluorescence at t = 15 min following addition of isoproterenol could be plotted against the isoproterenol concentration and fitted to a simple hyperbolic function, describing a single binding site with a K(d) of 29 µM (Fig. 4b). Similarly, the fluorescent change could be plotted against the logarithm of the isoproterenol concentration showing the best fit to a one-site sigmoid curve with an EC of 19 µM (Fig. 4c). The K(d) value of 29 µM and the EC value of 19 µM for isoproterenol are higher than the binding constants observed using conventional radioligand binding techniques as shown in Fig. 2(1 µM). One explanation for this apparent discrepancy could be technical differences in the methods by which the binding constants were obtained (the fluorescence studies were done with more than 100-fold higher receptor concentrations (100 nM receptor) for 15 min). Although apparent maximal change in fluorescence was observed already after 15 min, full equilibrium may not have been reached. Unfortunately, the fluorescent change upon ligand binding cannot be reliably determined after 1 h of incubation due to magnification of base-line differences over a longer period of time. Another potential explanation for the difference in apparent K(D) values could be the incomplete labeling of the cysteine in the beta(2) receptor that is responsible for the agonist-induced response. The fraction of labeled receptors may exhibit a lowered agonist affinity in contrast to the fraction of receptors labeled at other sites. However, this explanation is inconsistent with our radioligand binding data, which detect only one agonist affinity site (Fig. 2). Furthermore, we do not observe a change in the total number of binding sites following labeling with IANBD.

We also investigated whether the response seen following stimulation with isoproterenol could be reversed by addition of antagonist. As shown in Fig. 5, the response to isoproterenol could be readily reversed by the active(-)-isomer of the antagonist propranolol but not by the less active (+)-isomer. The response to isoproterenol was similarly reversed by several other antagonists, including alprenolol, ICI 118,551, pindolol, and dichloroisoproterenol (data not shown).

Comparison of the Isoproterenol-mediated Response with Other Adrenergic Agonists

A series of adrenergic agonists were tested for their ability to affect fluorescence of IANBD-labeled beta(2) receptor. The efficacy of these compounds was determined by measuring adenylate cyclase activity in membranes from SF-9 cells expressing the beta(2) adrenergic receptor (Fig. 6b) and was found to be in agreement with previous studies(6, 11, 24) . The full agonists, epinephrine and isoproterenol, which produced the largest increase in adenylate cyclase activity (Fig. 6b), also caused the largest decrease in fluorescence (5-6%) (Fig. 6a); whereas the relatively strong partial agonist salbutamol decreased fluorescence about 2.6% in agreement with a lower intrinsic activity in the adenylyl cyclase assay (Fig. 6b)(24) . The weaker partial agonists, dobutamine and ephedrine, did not cause a decrease in fluorescence relative to the extrapolated base line but rather produced a slight increase (Fig. 6b). Interestingly, plotting percent change in base-line fluorescence induced by this series of adrenergic agonists versus percent change in basal adenylate cyclase activity revealed a linear correlation (squared correlation coefficient, r^2 = 0.98) (Fig. 6c).


Figure 6: a, Effect of different adrenergic agonists on fluorescence from IANBD-labeled beta(2) receptor. The percent change (mean ± S.E.) in fluorescence was calculated as the change in fluorescence relative to the extrapolated base line at t = 15 min after addition of ligand. The ligands and concentrations used were (number of experiments in parentheses); H(2)O, water (n = 5); EPH, 10M ephedrine (n = 3); DOB, 10M dobutamine (n = 4); SAL, 10M salbutamol (n = 3); ISO, 10M isoproterenol (n = 5); and EPI, 10M epinephrine (n = 3). Responses significantly different from water are indicated by *p < 0.000005 (unpaired t test). The ligand concentrations used were chosen to ensure full saturation of the receptor with all ligands. This was confirmed by the ability of all compounds (at the concentration used) to fully displace [^3H]dihydroalprenolol from the receptor in a binding assay (data not shown). b, effect of adrenergic agonists on adenylate cyclase activity in membranes from SF-9 cells. Data are maximal response to indicated ligands (see above) expressed as percent of basal activity (mean ± S.E., n = 2) in membranes from cells infected with beta(2) receptor baculovirus at a density of 1.2 pmol/mg of protein. The dotted bar shows basal activity in an equivalent amount of membranes from non-infected cells in percent of basal activity in membranes from the infected cells. c, plot of percent change in fluorescence at t = 15 min against percent change in adenylate cyclase activity. The squared correlation coefficient (r^2) was 0.98.



Effect of Antagonists on Fluorescence from IANBD-labeled beta(2) Receptor

We also examined the effect of a series of adrenergic antagonists on fluorescence intensity from IANBD-labeled receptor over time. Propranolol and ICI 118,551, which both have been described as negative antagonists(10, 11, 13) , caused a relative increase in fluorescence intensity from IANBD-labeled beta(2) receptor followed by apparent stabilization of the rate at which fluorescence decreased over time (Fig. 7, a and b, and 8a). We did not observe any parallel change in the wavelength at which maximal emission occurred (data not shown). The response to propranolol was stereoselective since it was not elicited by the less active (+)-propranolol, indicating that the change must involve binding of propranolol to the receptor (Fig. 7a). This was also supported by the absence of response to propranolol and ICI 118,551 when they were exposed to denatured receptor (Fig. 7d). Alprenolol, pindolol, and dichloroproterenol, which in biological assays have been shown to exhibit either weak partial agonism, neutral antagonism or sometimes negative antagonism(6, 10, 11, 24) , induced a smaller but nevertheless reproducible increase in fluorescence (Fig. 7, b and c, and 8a).

The fluorescent changes were compared with the ability of the antagonists to affect basal adenylate cyclase activity in SF-9 cell membranes. The effect of the antagonists on basal adenylate cyclase activity was evaluated in SF-9 cells expressing a high level of beta(2) receptor (approximately 7 pmol/mg of protein) which leads to an elevated receptor-mediated basal level of adenylate cyclase activity (Fig. 8b). In contrast, partial agonists were evaluated at a lower receptor density (approximately 1.2 pmol/mg of protein) (Fig. 6b), as partial agonists are difficult to differentiate from full agonists in the presence of a large receptor reserve(25) . Alprenolol, pindolol, propranolol, dichloroisoproterenol, or ICI 118,551 did not affect basal adenylate cyclase activity at the low receptor density (data not shown). However, ICI 118,551 and propranolol, which induced the largest increase in fluorescence, potently decreased basal adenylate cyclase activity 40-60% at the high receptor density (Fig. 8b). Alprenolol and pindolol induced an approximately 30% decrease in basal activity, whereas dichloroisoproterenol revealed weak agonist activity at this high receptor density by increasing activity about 30%.


Figure 8: a, effect of different adrenergic antagonists on fluorescence from IANBD-labeled beta(2) receptor. The percent change (mean ± S.E.) in fluorescence was calculated as the change in fluorescence relative to the extrapolated base line at t = 15 min after addition of ligand. The ligands and concentrations used were (number of experiments in parentheses); H(2)O, water (n = 5); PIND, 10M pindolol (n = 4); ALP, 10M(-)alprenolol (n = 3); DCI, 10M dichloroisoproterenol (n = 3); (-)PROP, 10M(-)-propranolol (n = 3); and ICI, 10M ICI 118,551 (n = 4). Responses significantly different from water are indicated by *p < 0.0005 (unpaired t test). The ligand concentrations used were chosen to ensure full saturation of the receptor with all ligands. This was confirmed by the ability of all compounds (at the concentration used) to fully displace [^3H]dihydroalprenolol from the receptor in a binding assay (data not shown). b, effect of adrenergic antagonists on adenylate cyclase activity in membranes from SF-9 cells. Data are maximal response to indicated ligands (see above) expressed as percent of basal activity (mean ± S.E., n = 2) in membranes from cells infected with beta(2) receptor baculovirus at a density of 7 pmol/mg protein. The dotted bar shows basal activity in an equivalent amount of membranes from noninfected cells in percent of basal activity in membranes from the infected cells.



Quenching of Fluorescence from IANBD-labeled Receptor

The agonist-induced decrease in fluorescence of IANBD-labeled beta(2) receptor could be due to changes in the receptor structure that increase the exposure of one or more labeled cysteines to the aqueous solvent. Therefore, we investigated whether the fluorescent change was accompanied by greater accessibility of the aqueous quencher potassium iodide to the incorporated probe. Importantly, KI did not affect ligand binding to the receptor in a concentration up to 250 mM (data not shown). A strong quenching of fluorescence from IANBD-labeled beta(2) receptor was observed following addition of increasing concentrations of KI (Fig. 9). Stern-Volmer plots showed that the negative antagonists ICI 118,551 did not affect this quenching with the quenching constant K = 4.477 ± 0.074 (mean ± S.E., n = 3) in absence of any ligand and K = 4.436 ± 0.073 (mean ± S.E., n = 3) in presence of ICI 118,551 (Fig. 9). However, the presence of isoproterenol unexpectedly caused slightly less quenching as compared to buffer and ICI 118,551 (K = 4.209 ± 0.036, mean ± S.E., n = 3). The difference was significant with p < 0.05 both when comparing isoproterenol with buffer and isoproterenol with ICI 118,551 (unpaired t test).


Figure 9: Stern-Volmer plots of quenching of IANBD-labeled beta(2) receptor. Increasing concentrations of KI were added sequentially to labeled receptor (open square), labeled receptor treated with 10 µM ICI 118,551 (open circle) and 100 µM(-)-isoproterenol (closed circle). Fluorescence was measured and plotted as described under ``Experimental Procedures.'' The quenching constant K was 4.477 ± 0.074 (mean ± S.E., n = 3) in absence of any ligand, 4.436 ± 0.073 (mean ± S.E., n = 3) in presence of ICI 118,551 and 4.209 ± 0.036 (mean ± S.E., n = 3) in presence of isoproterenol. The difference between isoproterenol and buffer and between isoproterenol and ICI 118,551 was significant (p < 0.05) (unpaired t test).




DISCUSSION

In the present study we have been able to directly monitor conformational changes in a G protein-coupled receptor. As a molecular reporter we have used the cysteine-selective and environmentally sensitive, fluorescent probe, IANBD, which can be covalently incorporated into the purified, human beta(2) adrenergic receptor without perturbing the pharmacological properties of the receptor. Using the IANBD-labeled protein we were able to detect an agonist specific and reversible decrease in fluorescence emission from the labeled receptor protein. The response to the full agonist, isoproterenol, was shown to be dose-dependent and could be plotted as a simple, hyperbolic binding isotherm demonstrating interaction with a single binding site in the receptor. The ability of several adrenergic antagonists to reverse the isoproterenol-induced response confirmed that this binding site must be identical to the well described ligand binding site in the beta(2) receptor. These data provide structural evidence in a pure system for the existence of ligand-specific conformational states of a G protein-coupled receptor.

The agonist induced change most likely represents changes in the polarity of the environment surrounding one of more labeled cysteines in the hydrophobic core of the protein. Two possible mechanisms that could account for these changes are outlined in Fig. 10. According to the first model, a change in the environment around the indicated fluorophore (F) could be the result of a ligand induced movement of a transmembrane segment perpendicular to the plane of the lipid bilayer (Fig. 10a). This could result in the exposure of the fluorophore to the solvent causing quenching of the fluorescence and thus a decrease in the net fluorescence from the labeled receptor. Alternatively, it could be imagined that the agonist could induce a rotation of the membrane spanning domain resulting in movement of the fluorophore into the core of the protein, which is predicted to be more polar (Fig. 10b)(3, 4, 5) . According to the first model, the fluorophore should be more accessible to an aqueous quencher like potassium iodide following agonist binding. However, we observed that the agonist isoproterenol caused a slight decrease in quenching of the fluorescence from the IANBD-labeled receptor (Fig. 9). This would favor the latter model in which the fluorophore is moved to a more hydrophilic pocket in the core of the protein following agonist binding. In this model aqueous quenchers might be expected to have a more limited access to the fluorophore. It should be noted that at this point we can only speculate on the molecular mechanism behind the ligand-induced changes in fluorescence. With a stoichiometry of labeling at 1.2 mol of IANBD per mol of receptor at least two sites are being labeled in the receptor. Thus, the isoproterenol-mediated decrease in fluorescence may involve a different labeled cysteine than that responsible for the isoproterenol-induced effect on KI quenching. The observed changes may therefore be due to a more complex mechanism than that illustrated in Fig. 10.


Figure 10: Illustration of two possible mechanisms that may cause changes in polarity of the environment around the IANBD fluorophore. a, transmembrane segments of the receptor viewed from within the lipid bilayer. A change in the environment around the indicated fluorophore could be the result of a ligand induced movement of a transmembrane segment perpendicular to the plane of the lipid bilayer. b, transmembrane segments viewed from the extracellular face of the membrane. In this model, ligands could induce a rotation of the membrane spanning domain resulting in either movement of the fluorophore into the core of the protein, which is predicted to be more polar, or into the more hydrophobic, membrane-embedded shell of the protein.



Our fluorescence data suggest that the beta(2) receptor can exist in at least two conformational states, an unliganded state and an agonist-bound state. It is therefore tempting to consider these data in the context of the prevailing two-state model for activation of G protein-coupled receptors. This model predicts that the receptors exist in a dynamic equilibrium between two states, an inactive (R) and active conformation (R*); and that the biological response to a given ligand is governed by its intrinsic ability to change the overall equilibrium between the two states(8, 9, 10, 13, 24, 26, 27) . According to this model the fluorescence properties of the unoccupied IANBD-labeled receptor would be expected to represent the average fluorescence properties of the population of beta(2) receptors in the inactive state (R) and the active state (R*). The fluorescent properties of the R state alone should be observed in the presence of a negative antagonist, while those of the R* state alone should be observed in the presence of a full agonist(8, 9, 10, 13, 24, 26, 27) . Using our fluorescence assay we compared a series of adrenergic agonists, which exhibited distinct efficacies in a biological assay (Fig. 5b). We found an apparent linear correlation between the functional efficacy of these compounds and their ability to promote a change in fluorescence from the labeled receptor (Fig. 5c). These data can be interpreted in agreement with the two-state model. Hence, the different functional efficacies may result from differences in the ability of the different agonists to pull the equilibrium toward R* and thus decrease the fluorescence signal. Alternatively, the data also could support a multistate model in which the biological efficacy of an agonist may be a consequence of the magnitude of conformational change that it induces in the receptor, rather than just affecting the equilibrium between only two states. We should note, however, that without analyzing the fluorescently labeled receptor in a reconstituted system together with the corresponding G protein, we cannot exclude the possibility that the agonist-mediated change in fluorescence does not describe the structural changes of direct importance for G protein activation. Nevertheless, the correlation between intrinsic activity of the agonists and change in fluorescence suggests that the change is actually describing the conformational change in the receptor that leads to G protein activation.

It has been suggested, but never structurally verified, that antagonists may stabilize a conformation of the receptor that is distinct from unliganded receptor, and thus from the R state in the two-state model(11, 28, 29, 30, 31, 32) . For example, this has been proposed to explain the unexpected observation that non-peptide antagonists of G protein-coupled peptide receptors can act as competitive antagonists for agonist peptides without sharing apparent binding sites in the receptor(28, 29, 30, 31) . In other words, agonists and antagonists may be able to mutually exclude each others binding to the receptor by stabilizing different receptor conformations(28, 29, 30, 31) . It was therefore intriguing to observe that a series of adrenergic antagonists produced small but very reproducible increases in base-line fluorescence from the IANBD-labeled beta(2) receptor ( Fig. 7and Fig. 8). Except in the case of dichloroisoproterenol, the changes showed a correlation with the negative intrinsic activity of the tested compounds. Thus, propranolol and ICI 188,551, which exhibited the strongest negative intrinsic activity, caused the largest increase in base-line fluorescence, whereas alprenolol and pindolol with a smaller negative intrinsic activity also caused a smaller increase in fluorescence. However, given the small size of responses observed following stimulation with this group of compounds, it is difficult to assess the molecular significance of the fluorescence changes at the present stage. Nevertheless, the data are still of interest as they suggest that antagonists by themselves may alter receptor structure. The findings are also consistent with previous proteolysis studies on membrane bound receptor which show that agonists and antagonists are equivalent in protecting the beta(2) receptor from proteolysis(33) . This would not be expected if antagonists only changed the conformation of the small percentage of the receptor population which, in the absence of ligand, would be predicted to be in R* according to the two-state model.

Summarized, our data demonstrate the sensitivity of using fluorescence techniques for studying ligand-receptor interactions and their potential for delineating ligand-induced structural changes in G protein-coupled receptors. Importantly, if site-specific fluorescent labeling of the beta(2) receptor can be achieved, the approach may be useful for mapping conformational changes in the receptor structure to specific subdomains. In this way it should be possible to more precisely define the molecular mechanism of transmembrane signal transduction in G protein-coupled receptors.


FOOTNOTES

*
The work was supported by National Institutes of Health Grant RO 1 NS28471. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Howard Hughes Medical Institute, B157 Beckman Center, Stanford University Medical School, Stanford, CA 94305. Tel.: 415-723-7069; Fax: 415-498-5092.

(^1)
The abbreviations used are: IANBD, N,N`-dimethyl-N-(iodoacetyl)-N`-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine; DbetaM, n-dodecyl beta-D-maltoside.


ACKNOWLEDGEMENTS

We thank Drs. Tae R. Ji, Jeff Jasper, Elaine Sanders-Bush, and Mervyn Maze for critical reading of the manuscript. Drs. Robert J. Lefkowitz, Susan Senogles, Greg Dewey, and Lubert Stryer are thanked for helpful suggestions. Dr. Irene Sun is thanked for help with preparation of the protein.


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