Human A2A Adenosine Receptors: High-Affinity Agonist Binding to Receptor-G Protein Complexes Containing G

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 Ki 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 G markedly influences coupling such that A2AARs 1 2 are 8 2% coupled whereas equivalently expressed A2AARs 4 2 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-[I]iodophenyl)ethylamino]adenosine binds to coupled and uncoupled hA2AAR with KD values of 0.46 nM and 26 nM, respectively, a difference in affinity of 57-fold. The addition of GTP S converts all receptors to the low-affinity state. A2AAR coupling does not influence binding of antagonists including, I-4-(2-[7amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (I-ZM241385), KD 0.5 nM. Based on a comparison of high-affinity binding sites, N-3-iodo-2-chlorobenzyladenosine5 -N-methyluronamide is only 8-fold A3 selective (A2A Ki, H 18.3 3.2 nM; A3 Ki, H 2.4 0.3 nM) and 2-chloro-N cyclopentyladenosine is only 33-fold A1 selective (A2A Ki, H 11.0 1.9; A1 Ki, H 0.3 0.1). We conclude that recombinant hA2AAR can form a high-affinity receptor-G protein complex with s 4 2 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 GTP S 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 [I]APE binds to two affinity states of rat striatal A2A adenosine receptors (KD 1.3 and 19 nM) and 20% of striatal receptors are found in the high-affinity conformation (Luthin et al., 1995). [H]CGS21680 also binds to two affinity states of rat striatal membranes (KD 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 A2A coupling was noted in COS-7 cells assayed with [I]APE (Luthin et al., 1995) and in Chinese hamster ovary cells assayed with [H]NECA (Klotz et al., 1998). Similarly, little GTP S-sensitive [H]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 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; [ I]APE, 2-[2-(4-amino-3-[I]iodophenyl)ethylamino]adenosine; CGS21680, 2-[4-(2-carboxyethyl)phenethylamino]-5 -N-ethylcarboxamidoadenosine; GTP S, 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-3,4dihydroxy-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)aminocarbonylmethyloxy)phenyl)-1–3-dipropylxanthine; CPA, N-cyclopentyladenosine; IB-MECA, N-3-iodobenzyladenosine-5 -N-methyluronamide, PMSF, phenylmethylsulfonyl fluoride; TBST, Tris-buffered saline/Tween 20; HE, HEPES/EDTA; [I]ABA, N-(4-amino-3-[I]iodo-benzyl)adenosineCCPA, 2-chloro-N-cyclopentyladenosine; Cl-IB-MECA, N-3-iodo-2-chlorobenzyladenosine-5 -N-methyluronamide. 0026-895X/02/6102-455–462$3.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 2 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1238/965671 Mol Pharmacol 61:455–462, 2002 Printed in U.S.A. 455 at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from affinity of agonists for hA2AAR compared with hA1ARs and hA3ARs (Sullivan et al., 2001). The composition of G protein -subunits influences the potency of to stimulate guanine nucleotide exchange in assays with A2AAR-G protein complexes such that G 4 is more potent than G 1 (McIntire et al., 2001). This prompted us investigate the influence of G protein -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 s 4 2 with hA2AAR results in wellcoupled 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). Carrierfree I-ZM241385 and [I]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): GTP S, GDP, PMSF, leupeptin, pepstatin, aprotinin, and theophylline. Recombinant baculoviruses encoding the G protein subunits s, 1, 4, and 2 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 g/ml amphotericin B, and 50 g/ml gentamycin in an atmosphere of 50% N2/50% O2. Viral infection was performed at a density of 2.5 10 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 -mercaptoethanol, 5 g/ml leupeptin, 5 g/ml pepstatin A, 1 g/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 g/ml leupeptin, 2 g/ml pepstatin A, 2 g/ml aprotinin, 0.1 mM PMSF, and 10 M 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 g 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 -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 common and NEI800 for common) 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 [I]APE (Luthin et al., 1995) or the radiolabeled antagonist I-ZM241385. To detect the highaffinity, GTP S-sensitive state of A1 and A3 AR, we used the agonist [I]ABA (Linden et al., 1985, 1993). Binding experiments were performed in triplicate with 5 g (A2A) or 25 g (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 M GTP S. 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 150l 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 M NECA. For binding isotherms, nonspecific binding and free radioligand were fit by leastsquares 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 [I]APE were carried out using isotope dilution (100 nM unlabeled I-APE and 5 nM [I]APE before serial dilutions) to create a range of radioligand concentrations useful for detecting both highand low-affinity binding sites. Saturation binding assays using I-ZM241385 did not require isotope dilution. Bmax and KD 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 equation Y B ( B 4AC) / 2A, where Y [L]bound/[L]free, A KD1 KD2, B X(KD1 KD2) Bmax1 KD2 Bmax2, C X (X Bmax1 Bmax2), and X specific binding. Optimal parameter values were determined by nonlinear least-squares interpo-