Abstract
Agonists bind with higher affinity to G protein-coupled heptahelical receptors than to uncoupled receptors. Recombinant A1 and A3 adenosine receptors couple well to Gi/o, but recombinant human A2A adenosine receptors (hA2AAR) couple poorly to Gs and bind agonists with K i values in binding assays that are much higher than ED50 values for functional responses such as coronary dilation and inhibition of neutrophil oxidative burst. In this study, we produced hA2AAR-G protein complexes in membranes derived from Sf9 cells quadruply infected with receptors and heterotrimeric G protein subunits. The composition of Gβ markedly influences coupling such that A2AAR-αsβ1γ2 are 8 ± 2% coupled whereas equivalently expressed A2AAR-αsβ4γ2 are 40 ± 2% coupled. Hence, we were able for the first time to accurately measure high-affinity agonist binding to hA2AAR. The agonist 2-[2-(4-amino-3-[125I]iodophenyl)ethylamino]adenosine binds to coupled and uncoupled hA2AAR withK D values of 0.46 nM and 26 nM, respectively, a difference in affinity of 57-fold. The addition of GTPγS converts all receptors to the low-affinity state. A2AAR coupling does not influence binding of antagonists including,125I-4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (125I-ZM241385), K D = 0.5 nM. Based on a comparison of high-affinity binding sites,N 6-3-iodo-2-chlorobenzyladenosine-5′-N-methyluronamide is only 8-fold A3 selective (A2AK i, H = 18.3 ± 3.2 nM; A3 K i, H = 2.4 ± 0.3 nM) and 2-chloro-N 6-cyclopentyladenosine is only 33-fold A1 selective (A2AK i, H = 11.0 ± 1.9; A1 K i, H = 0.3 ± 0.1). We conclude that recombinant hA2AAR can form a high-affinity receptor-G protein complex with αsβ4γ2 that is useful for determining receptor selectivity.
A2AARs are one of four subtypes (A1, A2A, A2B, and A3) of GPCRs that respond to the purine adenosine, which is released from tissues in response to metabolic stress or ischemia. The A2AAR is an important pharmacological target because of the generally anti-inflammatory effects elicited when it is activated (Sullivan and Linden, 1998;Linden, 2001). Like other GPCRs, the A2AAR population is composed of receptors in two conformational states: those coupled to a heterotrimeric G protein, forming an R-G complex, and those that are uncoupled. GPCRs can be converted to uncoupled receptors upon binding of guanine nucleotides such as GTPγS to the G protein. Coupled GPCRs have a higher affinity for agonist molecules than do their uncoupled counterparts.
We have shown previously that the radiolabeled agonist [125I]APE binds to two affinity states of rat striatal A2A adenosine receptors (K D = 1.3 and 19 nM) and < 20% of striatal receptors are found in the high-affinity conformation (Luthin et al., 1995). [3H]CGS21680 also binds to two affinity states of rat striatal membranes (K D = 3.9 and 51 nM) (Luthin et al., 1995) and to two affinity states in human brain preparations (Wennmalm, 1988). The high-affinity state of the recombinant human A2AAR has not been easily observable because recombinant A2AARs do not seem to form R-G complexes to a significant degree. Poor A2Acoupling was noted in COS-7 cells assayed with [125I]APE (Luthin et al., 1995) and in Chinese hamster ovary cells assayed with [3H]NECA (Klotz et al., 1998). Similarly, little GTPγS-sensitive [3H]CGS21680 binding is detected to A2AARs transfected into COS-7 cells or human embryonic kidney 293 cells, suggesting that few receptors are coupled to G proteins (Piersen et al., 1994; Rosin et al., 1996). The inability to detect the high-affinity agonist binding conformation of the hA2AAR may have resulted in an underestimation of the relative affinity of agonists for hA2AAR compared with hA1ARs and hA3ARs (Sullivan et al., 2001).
The composition of G protein β-subunits influences the potency of βγ to stimulate guanine nucleotide exchange in assays with A2AAR-G protein complexes such that Gβ4 is more potent than Gβ1 (McIntire et al., 2001). This prompted us investigate the influence of G protein β-subunit composition on the degree of coupling to recombinant A2A receptors. Recombinant baculoviruses encoding the hA2AAR and three heterotrimeric G protein subunits were overexpressed in Sf9 cells. Expression of αsβ4γ2with hA2AAR results in well-coupled receptors. We have used membranes from these Sf9 cells to investigate the affinities of various agonists for the high-affinity conformational state of hA2AARs.
Experimental Procedures
Materials.
ZM241385 (Poucher et al., 1995) was a gift from Simon Poucher (Astra-Zeneca Pharmaceuticals, Cheshire, UK). Carrier-free 125I-ZM241385 and [125I]APE were synthesized and purified using high-performance liquid chromatography as described previously (Linden et al., 1984; Sullivan et al., 1999). ATL 146e was prepared as described previously (Rieger et al., 2001). MRS 1220 (Jacobson, 1998) was a gift from Kenneth Jacobson (National Institutes of Health; Bethesda, MD). CGS21680, NECA, XAC, CPA, and IB-MECA were purchased from Sigma/RBI (Natick, MA).
CCPA was purchased from SRI (Menlow Park, CA). ABA was a gift from Susan Daluge (GlaxoSmithKline, Research Triangle Park, NC). Adenosine deaminase was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA). The following reagents were purchased from Sigma Chemical Co. (St. Louis, MO): GTPγS, GDP, PMSF, leupeptin, pepstatin, aprotinin, and theophylline. Recombinant baculoviruses encoding the G protein subunits αs, β1, β4, and γ2 were kindly provided by James C. Garrison at the University of Virginia. The baculovirus encoding the hA2AAR was constructed as described previously (Robeva et al., 1996).
Cell Culture and Membrane Preparation.
Sf9 cells were cultured in Grace's medium supplemented with 10% fetal bovine serum, 2.5 μg/ml amphotericin B, and 50 μg/ml gentamycin in an atmosphere of 50% N2/50% O2. Viral infection was performed at a density of 2.5 × 106 cells/ml with a multiplicity of infection of two for each virus used. Infected cells were harvested 3 days postinfection and washed twice in insect PBS, pH 6.3. Cells were then resuspended in lysis buffer [20 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM MgCl2, 1 mM β-mercaptoethanol, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 1 μg/ml aprotinin, and 0.1 mM PMSF] and snap-frozen for storage at −80°C. Cells were thawed on ice, brought to 30 ml of total volume in lysis buffer, and burst by N2 cavitation (600 psi for 20 min). A low-speed centrifugation was performed to remove any unlysed cells (1000g for 10 min), followed by a high-speed centrifugation (17,000g for 30 min). The pellet from the final centrifugation was homogenized in buffer containing 20 mM HEPES, pH 8, 100 mM NaCl, 1% glycerol, 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 2 μg/ml aprotinin, 0.1 mM PMSF, and 10 μM GDP using a small glass homogenizer followed by passage through a 26-gauge needle. Membranes were aliquoted, snap frozen in liquid N2, and stored at −80°C. Membranes from cells stably expressing the human A1 AR (Chinese hamster ovary K1 cells) or A3 AR (human embryonic kidney 293 cells) were prepared as described previously (Robeva et al., 1996).
Western Blotting.
For each membrane preparation, 100 μg of membrane protein was added to 2× electrophoresis buffer (20% glycerol, 150 mM Tris, 0.05 mg/ml bromphenol blue, 4% SDS) plus 1 mM β-mercaptoethanol, loaded onto 10% Tris-Glycine Gradigels and electrophoresed at a constant voltage of 150 V for 90 min. Samples were transferred onto Westran polyvinylidene difluoride membranes (Schleicher and Schuell) using a constant current of 150 mA for 90 min. Nonspecific sites were blocked by incubating blots overnight at 4°C in a solution of TBST (50 mM Tris, 150 mM NaCl, and 0.5% Tween 20) containing 5% milk at pH 8. Blots were rinsed 4 × 5 min in TBST and then incubated with the primary antibody (NEN808 for βcommon and NEI800 for αcommon) in 2% milk in TBST. Blots were again rinsed 4 × 5 min with TBST before incubating for 90 min with donkey anti-rabbit IgG-horseradish peroxidase-linked F(ab′)2 at a dilution of 1:3000. Blots were rinsed 3 × 5 min in TBST, exposed to enhanced chemiluminescence reagents for 1 min, and placed on Kodak X-ray film for 15 s.
Radioligand Binding Assays.
Radioligand binding to recombinant human A2A receptors in Sf9 cell membranes was performed using either the radiolabeled agonist [125I]APE (Luthin et al., 1995) or the radiolabeled antagonist 125I-ZM241385. To detect the high-affinity, GTPγS-sensitive state of A1and A3 AR, we used the agonist [125I]ABA (Linden et al., 1985, 1993). Binding experiments were performed in triplicate with 5 μg (A2A) or 25 μg (A1 and A3) membrane protein in a total volume of 0.1 ml HE buffer (20 mM HEPES and 1 mM EDTA) with1 U/ml adenosine deaminase and 5 mM MgCl2 with or without 50 μM GTPγS. Membranes were incubated with radioligands at room temperature for 3 h (for agonists) or 2 h (for antagonists) in Millipore Multiscreen 96-well GF/C filter plates and assays were terminated by rapid filtration on a cell harvester (Brandel, Gaithersburg, MD) followed by four 150-μl washes over 30 s with ice-cold 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2. Nonspecific binding was measured in the presence of 50 μM NECA. For binding isotherms, nonspecific binding and free radioligand were fit by least-squares regression to a straight line. The extrapolated fit value of nonspecific binding for each free concentration of radioligand was subtracted from total binding to calculate specific binding. Equilibrium binding assays using [125I]APE were carried out using isotope dilution (100 nM unlabeled I-APE and 5 nM [125I]APE before serial dilutions) to create a range of radioligand concentrations useful for detecting both high- and low-affinity binding sites. Saturation binding assays using125I-ZM241385 did not require isotope dilution.B
max andK
D values were fit using nonlinear least-squares interpolation (Marquardt, 1963) for single or two-site binding models. For curvilinear 2-site Scatchard analyses (plots of [L]bound versus [L]bound/[L]free), [L]bound/[L]free was calculated from specific binding using the quadratic equationY = −B + (
Competition binding assays were performed as described previously (Robeva et al., 1996) using 0.5 to 1 nM [125I]APE, 125I-ZM241385, or [125I]ABA. We found that it was sometimes important to change pipette tips after each serial dilution to prevent transfer on tips of potent hydrophobic compounds. TheK
i values for competing compound binding to a single site were derived from IC50values with correction for radioligand and competing compound depletion as described previously (Linden, 1982). For determining twoK
i values for agonists in competition for an antagonist radioligand binding with the same affinity to both sites, we used nonlinear least-squares fitting to solve three simultaneous equations:
To determine the rate of association of [125I]APE to A2AAR-αsβ4γ2, 50 μl of membrane protein solution (0.03 mg/ml membrane protein in HE buffer containing 2 U/ml adenosine deaminase) was dispensed onto filter plates. At various time points, 50 μl of radioligand solution (0.68 nM [125I]APE in HE buffer containing 10 mM MgCl2) was added to the membrane solution yielding final concentrations of 0.34 nm [125I]APE and 5 mM MgCl2. The experiment was terminated by rapid filtration as described above. For determination of dissociation kinetics, radioligand, and membrane solution were dispensed into filter plates and allowed to incubate for 3 h. Dissociation was started by addition of 50 μl of HE buffer containing 5 mM MgCl2 and 50 μM ZM241385 at various time points followed by rapid filtration.
Results
To produce membranes containing hA2AAR that are partially coupled to G proteins, Sf9 cells were simultaneously infected with a baculovirus encoding the hA2AAR or four baculoviruses encoding the receptor and the heterotrimeric G protein subunits αs, β2or β4, and γ2. The amount of receptor expression was determined from saturation binding isotherms using both the agonist [125I]APE and the antagonist 125I-ZM241385 as radioligands. The data from antagonist saturation experiments (found in Table1) shows that the neither the presence of G proteins nor addition of GTPγS significantly changed theB max for125I-ZM241385.
The data from agonist saturation binding summarized in Table 1 shows that the A2A receptor, when expressed alone, has a relatively low affinity for [125I]APE (K D = 27 nM). Scatchard analysis indicates that all agonist binding is optimally fit to a single affinity site. When G proteins are coexpressed, the receptor displays two different affinities for the agonist. Table 1 shows that Gβ1- and Gβ4-containing membranes have similar high-affinity (K H) binding dissociation constants for [125I]APE of 0.32 nM and 0.46 nM, respectively. The low-affinity (K L) dissociation constants also are similar (K L = 10.5 nM andK L = 26 nM). NeitherK H norK L are significantly different between membranes expressing Gβ1 or Gβ4.
The total number of receptors measured varied with the choice of ligand. The B max using125I-ZM241385 is significantly greater than that using [125I]APE. This is probably caused by partial dissociation of the agonist ligand from the low-affinity site during washing of glass fiber filters. This phenomenon would cause the low-affinity B max values to appear artificially depressed without affecting the observedK D. Thus, to determine the fraction of receptors found in the high-affinity state, we divided the high-affinity B max by the total number of receptors as determined by saturation binding of125I-ZM241385 (B max, ZM). This analysis shows that the Gβ subunit influences the efficiency of R-G coupling in this system. The fraction of receptors found in the high-affinity state in the presence of Gβ4 (40%) was significantly higher than that seen when β1 was present (8%) (Table 1). This difference cannot be attributed to a difference in the amount of expressed α- or β-subunits, because the amount of protein detected in Sf9 membranes by Western blotting with antibodies that detect α or β subunits was similar (Fig. 1). The anti-β antibody detects β1 and β4 with the same affinity because it recognizes a common epitope. Because the Gβ4-containing membranes had a greater fraction of coupled receptors, we used them to characterize agonist high-affinity binding sites in subsequent experiments.
The equilibrium binding of [125I]APE to membranes derived from Sf9 cells quadruply infected with hA2AAR-αsβ4γ2complexes with or without GTPγS is shown in Fig.2A. Coinfection with G protein subunits substantially increased the amount of specific binding at a given concentration without changing nonspecific binding. In the absence of GTPγS, specific binding to R-G complexes is fit significantly better to a two-site binding model (Fig. 2A, —▪—) than to a single site equation (Fig. 2A, - - ▪ - -). The two [125I]APE affinity states of hA2AAR-αsβ4γ2complexes are more clearly evident in the Scatchard plot shown in Fig.2B. Treatment of membranes expressing these receptor-G protein complexes with GTPγS (50 μM) eliminates the high-affinity site completely, resulting in equilibrium binding that is optimally fit to a single-site equation (K D = 32 nM) and characterized by a linear Scatchard plot. This affinity is similar to the low-affinity population of receptors detected in the absence of GTPγS (26 nM) or the single low-affinity site detected when the receptor is expressed alone (27 nM). This GTPγS sensitivity of binding confirms that the high-affinity site is due to the interaction of the receptor with G proteins.
We next conducted kinetic experiments to determinek 1 andk −1 for [125I]APE binding hA2AAR-αsβ4γ2complexes. The kinetics of [125I]APE (0.34 nM) association is illustrated in Fig. 3A. At this concentration, [125I]APE binds with ak obs of 0.0673 ± 0.0025 min−1. The binding is well fit by a single exponential equation, which is confirmed by the linearity of the transformed data shown in the Fig. 3, inset. The slope of this line defines a pseudo–first-order rate constant (k 1 × [[125I]APE] +k −1) and is calculated based on the assumption that free radioligand does not change significantly over time. During this experiment, only 15% of the radioligand was bound at equilibrium. The dissociation of [125I]APE is shown in Fig. 3B. The data is well fit by a single exponential equation, which is consistent with the proposition that a large fraction of the total binding is to the high-affinity site. We calculated k −1 to be 0.0291 ± 0.002 min−1 corresponding to at 1/2 of 24 min. A kinetic analysis of [125I]APE dissociation from the low-affinity site could not be accurately performed because of rapid dissociation of the radioligand.
From k obs andk −1 of the high-affinity site, and using (k obs =k 1 × [[125I]APE]+k −1) we calculatedk 1 as 1.12 × 108 ± 0.09 × 108min/M. The K D for [125I]APE was calculated (K D =k −1/k 1) to be 0.37 ± 0.02 nM, which is similar to theK D determined by equilibrium binding (0.46 nM). Thus, there is good agreement between equilibrium and kinetic binding parameters. By a similar analysis, we determined the kinetic binding parameters for hA2AAR-αsβ1γ2complexes: k 1 = 1.40 × 108 ± 0.12 × 108min/M, k −1 = 0.0295 ± 0.003 min−1, and K D = 0.21 ± 0.06 nM. These values are not significantly different from those for hA2AAR-αsβ4γ2.
We also observed two affinity sites of the receptor based on competition binding assays with agonists. Figure4 demonstrates that when125I-ZM241385 binds to membranes expressing the hA2AAR and G proteins, competition by the agonist, ATL 146e, for binding sites is biphasic. To accurately determine both K i values from these biphasic curves, we used a curve-fitting algorithm that directly calculated both K i values based on theK D of the radioligand and correcting for the depletion of both the radioligand and the competing compound during binding (see Experimental Procedures). When fit to a two-site model, the competition curve yields twoK i values for the potent A2A ligand ATL 146e: 0.18 and 58 nM. A comparison of [125I]APE and ATL 146e reveals a potency difference of 2.6- and 2.1-fold, respectively, for the high- and low-affinity hA2AAR binding sites. Addition of GTPγS in competition assays completely eliminates the high-affinityK i, leaving a low-affinityK i (58 nM), which is consistent with the K i for ATL 146e when competing for125I-ZM241385 binding sites in membranes expressing the receptor alone (68 nM, Table2). The high-affinityK i of ATL 146e in competition for125I-ZM241385 (0.18 nM) is not significantly different from the K i calculated from competition for [125I]APE (0.20 nM).
To determine the affinities of other adenosine receptor agonists at G protein-coupled A2A receptors, we performed competition experiments using low concentrations (0.3–0.5 nM) of [125I]APE as the radioligand. At the concentrations used, the radioligand was bound predominantly (>95%) to the high-affinity site. We chose several agonists that are widely used experimentally because they have been reported to be highly selective for binding to one of the four adenosine receptor subtypes: A2A-selective, ATL146e and CGS21680; A3 selective, IB-MECA and Cl-IBMECA; and A1 selective, CPA and CCPA. We also examined the nonselective agonist, NECA. As expected, these agonists have a much higher affinity for the G protein coupled hA2AAR than for the A2A receptor expressed without any G proteins. A typical experiment is shown in Fig.5 and the results of 60 experiments are summarized in Table 2.
We also examined four antagonists in competition assays: theophylline, MRS1220, XAC, and ZM241385. The results are summarized in Table3. TheK i values of antagonists do not vary significantly between the coupled and uncoupled receptors. These results are consistent with the expectation that antagonists do not differ substantially in their affinities for coupled and uncoupled receptors.
To examine the selectivity of selected compounds for various adenosine receptor subtypes, we performed competition radioligand binding experiments to membranes expressing the human A1AR or A3AR using the agonist [125I]ABA. At the [125I]ABA concentrations used, >80% of radioligand binding in these systems is GTPγS sensitive and therefore predominantly represents the high-affinity binding sites of these receptors. Resultant competition curves were fit with both one- and two-site models. In all cases, the one-site model better fit the data as determined by F tests (Motulsky and Ransnas, 1987).K i values were determined as described under Experimental Procedures. The high-affinityK i values from these experiments are summarized in Table 4. We also calculated the selectivity ratio of each agonist at high-affinity A1 and A3 receptors relative to both their low and high A2Aaffinities. The results indicate that agonists that are considered to be selective for A1 or A3receptors are less selective over A2A receptors than previously noted.
Discussion
We have developed a system in which the G protein-coupled state of the A2AAR can be accurately detected using a quadruple infection of Sf9 cells with baculoviruses encoding the hA2AAR and three G protein subunits. Previous studies using transfected COS-7 cells (Piersen et al., 1994) or HEK293 cells (Rieger et al., 2001; Sullivan et al., 2001) display little or no GTPγS-sensitive agonist binding, possibly because of the low levels of Gs expressed in these cell lines relative to the highly expressed receptor or to other factors that limit coupling of receptors to Gs.
In this study, we show that the identity of the β subunit can influence the coupling efficiency of the A2AAR. Overexpressing αsβ1γ2with the A2AAR in Sf9 cells resulted in only partially coupled A2AAR (8%), but substantially greater coupling was observed (40%) if β1 was replaced by β4. This difference was not due to variations in protein expression levels as determined by Western blots. The results are consistent with the recent report showing in reconstitution experiments that β4 is significantly more potent than β1 in stimulating agonist-induced guanine nucleotide exchange in Sf9 membranes expressing hA2AAR (McIntire et al., 2001). Our data imply that the difference in guanine nucleotide exchange is a consequence of changes in the stability of the agonist-receptor-G protein complex, which is dependent on the composition of the Gβ subunit. Gβ composition also influences the coupling of M2 muscarinic receptors such that guanine nucleotide exchange on αoβ4γ2is greater than on αoβ1γ2(Hou et al., 2001). The influence of G protein composition on A2A receptor coupling has not been as extensively studied as the A1 receptor. A1 receptors couple preferentially to G proteins containing γ2 or γ3over subunits that contain γ1 (Figler et al., 1997). Additional experimentation will be required to determine whether, like the A1 receptor, the coupling of A2A receptors is influenced by the composition of Gγ.
We used αsβ4γ2to carefully examine the high-affinity binding site of hA2AARs. Several lines of evidence support the conclusion that the high-affinity binding site observed in this study results from G protein coupling: 1) the agonist used ([125I]APE) was previously shown to bind to both coupled and uncoupled A2AAR receptors on rat striatal membranes (Luthin et al., 1995); 2) two affinity states were detected in both saturation and competition studies; 3) high-affinity binding to quadruply infected Sf9 membranes was completely inhibited by the addition of 50 μM GTPγS to binding assays; 4) the high-affinity site was absent in membranes expressing the receptor alone; and 5) agonists, but not antagonists, bound differentially to coupled and uncoupled receptors.
It is significant that agonist radioligands of the A2A receptor such as [125I]APE and the widely used compound [3H]CGS21680 bind with high enough affinity to detect both uncoupled and coupled receptors. Consequently, attempts to fit radioligand binding data to a single site may result in detection of a composite apparent binding site that is intermediate in its affinity for coupled and uncoupled receptors. This will result in discrepancies in agonist binding constants that depend on various factors, including the receptor density, the fraction of coupled receptors, the concentration of radioligand, and the time and temperature of filter washing. The absolute affinity of the radioligand for the low-affinity site will influence its detection because the lowest affinity ligands are most prone to washing off the receptor during the wash phase of the filtration process. In this regard, it is notable that the rank affinity of agonists radioligands for the low-affinity site is: [125I]APE (26 nM) < [3H]NECA (85 nM) < [3H]CGS21680 (944 nM). Collectively, this may explain the wide differences in reported agonist binding affinities for the A2A receptors between laboratories and the fact that the high-affinity K i values found in this study are significantly lower than those reported previously (Robeva et al., 1996; Klotz et al., 1998) because previous studies have not had the benefit of a well-coupled receptor system and thus could not explicitly detect the high-affinity state.
We measured both the low- and high-affinityK i values of several widely used adenosine receptor agonists. It is notable that the ratio of binding affinity for the high- and low-affinity sites was highly variable among the agonists we examined and ranged from 862-fold with IB-MECA to 42-fold with NECA (Table 2). This is a significant observation because it indicates that even the relative affinities and rank order potency of agonists vary depending on the coupling state of receptors. Stated another way, these data indicate that binding to uncoupled A2A receptors is not highly predictive of binding to coupled receptors. The reason for the variance in the ratio between high- and low-affinity K i values is interesting and is the subject of ongoing work in our laboratory.
For comparing the selectivity of agonists for the four adenosine receptor subtypes, it makes sense to compare coupled receptors to coupled receptors, or uncoupled receptors to uncoupled receptors. In contrast to recombinant hA2AARs, recombinant hA1ARs and hA3ARs do couple sufficiently well to G proteins to readily allow detection of the high-affinity agonist binding conformation characterized by agonist binding that is largely inhibited by GTPγS (Gao et al., 1999). This may be related to the fact that Gi/o is more abundant in mammalian cells than is Gs. In addition, the Gi/o proteins seem to couple very tightly to their cognate receptors (Munshi et al., 1991; Gao et al., 1999). Consequently, the selectivity of agonists has been assessed in many instances based on competition of radioligand binding to well-coupled A1 and A3receptors, but to poorly coupled A2A receptors. Based on a comparison of well-coupled A1, A2A, and A3 receptors, we have shown in this study that among agonists generally used as A2A-selective ligands (ATL146e and CGS21680), the limited apparent selectivity for human A2Areceptors is actually substantially higher than previously thought (Sullivan et al., 2001). Moreover, the high-affinityK i values for agonists in our experiments are more in line with their functional EC50 values in other studies (Walker et al., 1997; Sullivan et al., 2001) than the low-affinityK i values determined here and by others.
We also examined agonists reported be highly A1and A3 selective. We confirmed that the radioligand binding in these assays is to a single GTPγS-sensitive site. CCPA and CPA both have subnanomolar affinities for the coupled A1AR and have been described as highly selective agonists of the A1 receptor. Previous studies have cited selectivity ratios for A1 over A2A receptors of over 300-fold for CPA and over 2000-fold for CCPA (Klotz et al., 1998). However, in this study we have shown that both of these compounds have only a 30- to 50-fold selectivity for A1 over A2Aat their high-affinity binding sites. This finding would seem to explain the effect of CCPA to increase interleukin-10 release and decrease tumor necrosis factor-α concentrations in endotoxemic mice at high concentrations (Hasko et al., 1996), actions that have been shown to be A2A dependent in vitro (Bouma et al., 1994; Sullivan and Linden, 1998).
Similarly, examination of the high-affinityK i values for IB-MECA and Cl-IB-MECA shows that neither is as selective for A3 over A2A receptors as has been reported (Gallo-Rodriguez et al., 1994; Klotz et al., 1999). Both of these agonists have only a ∼5-fold selectivity for A3over A2A for binding to the high-affinity site. This finding has critical importance for work involving discrimination of the physiological roles of the A3 and A2AARs in mediating the protective effects of adenosine. Various groups have relied on IB-MECA to define the role that the A3AR receptor plays modulating inflammatory responses (Hasko et al., 1996, 1998; Sajjadi et al., 1996). This was reasonable based on the relative potencies of these compounds reported in the prior literature. However, because the A2A and A3AR work through opposing mechanisms (Gαs versus Gαi), it seems unlikely that both receptors could have the generally anti-inflammatory effects noted in the same cells. The potency of IB-MECA at the A2AAR confirms our recent observation that the anti-inflammatory effects of IB-MECA on tumor necrosis factor-α production in human monocytes can be potently blocked by the selective A2Aantagonist, ZM241385 (Sullivan and Linden, 1998).
Comparing K i values of compounds is an excellent method for determining selectivity of agonist binding at various receptors. However, this does not give a complete picture of agonist action because these values do not incorporate the efficacy of the various agonists at each receptor. A given agonist could have equal potencies at two given receptors, but if its efficacy at one is much greater than at the other, the agonist would seem to be more potent at one receptor in functional assays. Because no work has been published on the relative efficacies of these compounds, we do not yet have a complete understanding of these agonists' actions.
In summary, we report here on the establishment of a method for expressing G protein coupled hA2AARs by quadruple infection of Sf9 cells and sensitivity of this coupling to Gβ composition. We have confirmed the existence of this high-affinity state by Scatchard analysis, competition assays, and kinetic experiments. Using this system, we have determined the affinity of several commonly used adenosine receptor agonists at the coupled A2AAR. This work has demonstrated that IB-MECA and Cl-IB-MECA can no longer be assumed to be highly selective agonists of the hA3AR or CCPA, a highly selective agonist of the hA1AR. Consequently, findings based on the use of these agonists must be critically evaluated with respect to possible involvement of the A2AAR in responses that have been previously ascribed to A1 or A3 receptors. We anticipate that the use of methods to express well coupled adenosine receptors will be valuable for detecting novel potent and selective AR agonists.
Acknowledgments
We gratefully acknowledge Dr. James Garrison of the University of Virginia for his generous gift of baculoviruses for G protein subunits, Dr. Ken Jacobson of the National Institute of Diabetes and Digestive and Kidney Diseases for his gift of MRS1220 and Simon Poucher of Astra-Zeneca for his gift of ZM241385. We also thank Dr. William McIntire and Heidi Figler for helpful advice.
Footnotes
- Received July 26, 2001.
- Accepted October 10, 2001.
-
This work was supported in part by National Institutes of Health Grants R01-HL37942 (J.L.).
Abbreviations
- A2AAR
- A2A adenosine receptor
- GPCR
- G protein coupled receptor
- [125I]APE
- 2-[2-(4-amino-3-[125I]iodophenyl)ethylamino]adenosine
- CGS21680
- 2-[4-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- ZM241385
- 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol
- ATL146e
- 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester
- MRS 1220
- N-(9-chloro-2-furan-2-yl-[1,2,4]triazolo[1,5-c]quinazolin-5-yl)-2-phenylacetamide
- NECA
- 5′-N-ethylcarboxamidoadenosine
- XAC
- 8-(4-((2-a-minoethyl)aminocarbonyl-methyloxy)phenyl)-1–3-dipropylxanthine
- CPA
- N6-cyclopentyladenosine
- IB-MECA
- N6-3-iodobenzyladenosine-5′-N-methyluronamide, PMSF, phenylmethylsulfonyl fluoride
- TBST
- Tris-buffered saline/Tween 20
- HE
- HEPES/EDTA
- [125I]ABA
- N6-(4-amino-3-[125I]iodo-benzyl)adenosineCCPA, 2-chloro-N6-cyclopentyladenosine
- Cl-IB-MECA
- N6-3-iodo-2-chlorobenzyladenosine-5′-N-methyluronamide
- The American Society for Pharmacology and Experimental Therapeutics