Biochem. J. (2006) 396, 139–146 (Printed in Great Britain)
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doi:10.1042/BJ20051422
The allosteric enhancer PD81,723 increases chimaeric A1/A2A adenosine receptor coupling with Gs Samita BHATTACHARYA*1 , Rebecca L. YOUKEY*, Kobina GHARTEY*, Matthew LEONARD*, Joel LINDEN*†‡ and Amy L. TUCKER*†‡2 *Department of Internal Medicine, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A., †Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A., and ‡Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A.
PD81,723 {(2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)phenyl]methanone} is a selective allosteric enhancer of the Gi coupled A1 AR (adenosine receptor) that is without effect on Gs -coupled A2A ARs. PD81,723 elicits a decrease in the dissociation kinetics of A1 AR agonist radioligands and an increase in functional agonist potency. In the present study, we sought to determine whether enhancer sensitivity is dependent on coupling domains or G-protein specificity of the A1 AR. Using six chimaeric A1 /A2A ARs, we show that the allosteric effect of PD81,723 is maintained in a chimaera in which the predominant G-proteincoupling domain of the A1 receptor, the 3ICL (third intracellular loop), is replaced with A2A sequence. These chimaeric receptors are dually coupled with Gs and Gi , and PD81,723 increases the potency of N 6 -cyclopentyladenosine to augment cAMP accumu-
lation with or without pretreatment of cells with pertussis toxin. Thus PD81,723 has similar functional effects on chimaeric receptors with A1 transmembrane sequences that couple with Gi or Gs . This is the first demonstration that an allosteric regulator can function in the context of a switch in G-protein-coupling specificity. There is no enhancement by PD81,723 of Gi -coupled A2A chimaeric receptors with A1 sequence replacing A2A sequence in the 3ICL. The results suggest that the recognition site for PD81,723 is on the A1 receptor and that the enhancer acts to directly stabilize the receptor to a conformational state capable of coupling with Gi or Gs .
INTRODUCTION
allosteric modulator may vary with the orthosteric ligand and cell system [2]. The effects of adenosine are mediated through its interaction with four subtypes of GPCRs, A1 , A2A , A2B and A3 [6–8]. The A1 and A3 AR subtypes couple with Gi /Go [9–12], while A2A and A2B couple with Gs [13–15]. There is evidence that the A2B receptor can also couple with calcium mobilization, possibly via Gq [16]. Stimulation of the A1 receptor results in a reduction of cellular cAMP levels, while stimulation of the A2A receptors causes cAMP accumulation. We and others have previously characterized chimaeric ARs in which the 3ICL (third intracellular loop) of the A1 AR has been replaced by A2A sequence (A1 /A2A L) or vice versa (A2A /A1 L) [13,14]. These chimaeras demonstrate the importance of the 3ICL of the A2A AR in the activation of Gs . The chimaeric receptors are dually coupled with Gi and Gs and produce cAMP accumulation when activated with appropriate agonists [13,14]. A series of 2-amino-3-benzoylthiophene compounds, including PD81,723 {(2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)phenyl]methanone}, are allosteric regulators of the A1 AR, acting to increase agonist radioligand binding and agonist potency to activate the A1 AR in vivo and in cultured cells [17–22]. Among AR subtypes, PD81,723 appears to be A1 -selective, having no effect on ligand binding to recombinant rat [17] or canine [22]
Allosteric modulators alter receptor activity, either positively or negatively, through their interactions with binding sites distinct from the orthosteric site, and may offer some therapeutic advantages over traditional orthosteric ligands [1,2]. Allosteric modulators of ion channel-coupled receptors, including GABAA (γ -aminobutyric acid receptor, subtype A) [3] and nicotinic acetylcholine [4] receptors, have been developed into successful therapeutics. Several GPCRs (G-protein-coupled receptors), among which are the α 1 -, α 2A -, α 2B -, α 2D - and β 1 -adrenoceptors, as well as multiple subtypes each of dopaminergic, muscarinic and serotoninergic receptors and ARs (adenosine receptors), have been shown to undergo allosteric modulation [1]. At least one group of these compounds, allosteric modulators for the calciumsensing GPCR for the treatment of hyperparathyroidism, is in clinical trials [5]. These compounds offer some theoretical advantages over orthosteric ligands, including saturability, and greater tissue and receptor subtype selectivity [1]. Allosteric modulators can positively or negatively alter the affinity of agonist or antagonist ligands, and their functional effects to activate G-proteins and effector systems may parallel, oppose or be independent of effects on agonist binding, underscoring the dissociability of agonist ligand binding and G-protein activation [2]. Effects of a given
Key words: adenosine receptor, allosteric enhancer, canine, G-protein, N 6 -cyclopentyladenosine (CPA), PD81,723.
Abbreviations used: 3ICL, third intracellular loop; ADA, adenosine deaminase; AR, adenosine receptor; CPA, N 6 -cyclopentyladenosine; CPX, 1,3-dipropyl-8-cyclopentylxanthine; GPCR, G-protein-coupled receptor; GTP[S], guanosine 5 -[γ-thio]triphosphate; HEK-293 cell, human embryonic kidney 293 cell; 125 I-ABA, 125 I-N 6 -4-amino-3-iodo-benzyladenosine; 125 I-APE, 125 I-2-[2-(4-amino-3-iodo-phenyl)ethylamino]adenosine; NECA, 5 -Nethylcarboxamidoadenosine; R–G complex, receptor–G-protein complex; TM, transmembrane domain. 1 Present address: Department of Microbiology, Columbia University, New York, NY 10032, U.S.A. 2 To whom correspondence should be addressed, at Box 801394, MR5 Room G219, University of Virginia Health System, Charlottesville, VA 22908, U.S.A. (email
[email protected]). c 2006 Biochemical Society
140 Table 1
S. Bhattacharya and others Ligands used and their pharmacological properties
CPT, 8-cyclopentyltheophylline; 125 I-HPIA, 125 I-hydroxyphenylisopropyladenosine.
A1 A2A Non-selective
Agonist
Antagonist
125
[3 H]CPX
I-ABA, 125 I-HPIA, CPA I-APE, CGS 21680 NECA
125
CPT, BW-A1433
A2A AR or to canine A3 AR [22]. Allosteric modulators structurally distinct from PD81,723 have been described for other AR subtypes, including the A3 [23–25] and A2A [26] receptors. Amiloride analogues allosterically modulate binding to A1 , A2A and A3 receptors [24]. The allosteric binding site for PD81,723 has not been characterized, but T277A mutation of the A1 AR, which decreases agonist binding, also inhibits the effects of PD81,723 [27]. In radioligand binding studies in CHO cells (Chinese-hamster ovary cells) stably expressing the human A1 AR, PD81,723 was found to increase the fraction of receptors found in the high-affinity agonist binding conformation state, suggesting that PD81,723 stabilizes A1 AR–G complexes [28]. Given the lipophilic nature of PD81,723 and its action to stabilize R–G (receptor–G-protein) complexes, it is possible that it interacts with transmembrane or intracellular domains of the A1 AR and that it might interact with sites on receptors and G-proteins. Although PD81,723 is unlikely to bind exclusively to Gi since it has no allosteric effect on M2 -muscarinic, α 2 -adrenergic or δopioid receptors [17], the possibility of an interaction between PD81,723 and elements of Gi in R–G complexes has not been ruled out. Regardless of the binding site for PD81,723, it is also possible that its allosteric effects on the A1 AR depend on selective receptor coupling with Gi . In the present study, using chimaeric A1 /A2A ARs that couple with Gs , we test whether the effects of PD81,723 on binding and functional enhancement of the A1 AR are dependent on G-protein specificity and/or on the sequence of the intracellular coupling domain of the A1 AR. We show that PD81,723 stabilizes the interaction between chimaeric A1 /A2A receptors and Gs . This is the first demonstration that an allosteric modulator of a GPCR can enhance function following a switch in G-protein coupling. The results imply that PD81,723 acts directly to stabilize agonist binding to A1 AR ligand recognition domains and only indirectly to stabilize R–G complexes, resulting in parallel enhancement of binding and function.
EXPERIMENTAL Materials
The ligands used in the present study are listed in Table 1. Tris, Hepes, MgCl2 , CPA (N 6 -cyclopentyladenosine) and forskolin were purchased from Sigma (St. Louis, MO, U.S.A.); R-N 6 (2-phenylisopropyl)adenosine, 8-cyclopentyltheophylline, CGS 21680 {2-[4-(2-carboxyethyl)phenethylamino]-5 -N-ethylcarboxamidoadenosine; an A2A AR-selective agonist} and CPX (1,3-dipropyl-8-cyclopentylxanthine) were from Research Biochemicals International (Natick, MA, U.S.A.); ADA (adenosine deaminase) was from Boehringer Mannheim; DMSO was from Fisher Scientific (Fair Lawn, NJ, U.S.A.) and [3 H]CPX from NEN (Boston, MA, U.S.A.). Canine A1 and A2A receptor cDNAs were a gift from Dr G. Vassart (Universit´e Libre de Bruxelles, Brussels, Belgium). Pertussis toxin was a gift from Dr Erik Hewlett (University of Virginia). Tissue culture sup c 2006 Biochemical Society
Figure 1 Design of the canine A1 /A2A AR chimaeras characterized in the present study (A) Diagrams of TM V–TM VII of the canine A1 (left-hand panel) and A2A (right-hand panel) ARs. Residues represented by black circles denote those into which silent restriction sites were introduced. The 3ICL sequence is between TM V and TM VI and the C-terminal sequence follows TM VII. (B) Schematic representation of the receptors used in the present study. A1 sequence is shown as a solid line and A2A as striped.
plies were from Gibco BRL. 125 I-ABA (125 I-N 6 -4-amino-3iodobenzyladenosine) and 125 I-APE {125 I-2-[2-(4-amino-3-iodophenyl)ethylamino]adenosine} were synthesized as previously described [29,30]. Canine A1 /A2A AR chimaeras
Chimaeras were constructed by introducing restriction sites into canine A1 and A2A AR cDNAs expressed in pALTER using oligonucleotide-directed mutagenesis as previously described [14]. Briefly, silent restriction sites were introduced into the A1 and A2A cDNAs in regions to be used as chimaeric receptor splice boundaries. SpeI sites were introduced at positions corresponding to Val189 and Val186 of the canine A1 and A2A receptors respectively and StuI sites at Phe241 of the A1 and Gly239 of A2A receptor (Figure 1). These sites flank the 3ICL. NsiI sites were introduced just proximal to the putative CT (C-terminus) at Ala289 (A1 ) and Ala288 (A2A ) (Figure 1). These sites were used to create six chimaeric receptors in which sequences for the 3ICL, CT, or both, had been switched from one subtype to the other. The chimaeric receptors were subcloned into the CLDN10B expression vector, sequenced and stably expressed in HEK-293 cells (human embryonic kidney 293 cells). The radioligand binding characteristics and G-proteincoupling characteristics of these chimaeric receptors have been investigated in detail [14]. Previously published K d (nM) values for these receptors are: for 125 I-ABA, K d = 6.7 + − 0.7 for A1 , K d = 10.5 + − 1.4 for A1 /A2A L, K d = 10.7 + − 2.5 for A1 /A2A T and
Allosteric enhancement of Gs -coupled adenosine receptors 125 K d = 7.2 + − 0.1 for A1 /A2A LT; for I-APE, K d = 13.9 + − 2.5 for + A2A , K d = 7.9 − 0.6 for A2A /A1 L, K d = 24.1 + − 3.5 for A2A /A1 T and K d = 12.2 + − 3.4 for A2A /A1 LT [14] (where A1 /A2A T is a receptor chimaera with A1 sequence except for A2A sequence in the CT, and A1 /A2A LT is a receptor chimaera with A1 sequence except for A2A sequence in the 3ICL and the CT). The affinity of chimaeric receptors for ligands is determined by the transmembrane sequences of the chimaera, while G-protein coupling is determined principally by the composition of the 3ICL.
cAMP assays
cAMP was measured in extracts of HEK-293 cells stably expressing recombinant ARs. Cells were dissociated from 150 mm2 culture dishes using 5 mM EDTA in PBS for 5–10 min. In some cases, to inactivate Gi/o , cells were pretreated with 100 ng/ml pertussis toxin for 18 h at 37 ◦C prior to removal from plates. Suspended cells were centrifuged at 500 g for 1 min and resuspended in Dulbecco’s modified Eagle’s medium buffered with 20 mM Hepes (pH 7.4) containing 10 units/ml ADA to degrade most endogenous adenosine and 20 µM Ro-20-1724 to block phosphodiesterase. Forskolin (5 µM), BW-A1433 (1,3dipropyl-8-( p-acrylic) phenylxanthine; 10 µM) and PD81,723 (20 µM) were added where indicated and cells were divided into aliquots (200 µl) in assay tubes. Various concentrations of ligand were added in 50 µl aliquots and cells were incubated for 10 min at 37 ◦C in a shaking water bath. Incubations were terminated with the addition of HCl to a final concentration of 0.1 M in a final volume of 750 µl and cellular debris was pelleted by centrifugation for 15 min at 2000 g. cAMP (500 µl) in the acid extract was acetylated by the addition of 22.5 µl of triethylamine/acetic anhydride (3.5:1, by vol.) and the cAMP was measured by RIA.
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Dissociation of 125 I-ABA or 125 I-APE from receptors with A1 AR or A2A AR transmembrane sequence
The allosteric effect of PD81,723 is characterized by a decrease in the dissociation kinetics of a prebound agonist radioligand. To measure radioligand dissociation kinetics, 20 µg of membrane protein derived from HEK-293 cells expressing recombinant wildtype or chimaeric receptors was incubated with 1 nM 125 I-ABA or 125 I-APE in HE buffer supplemented with 2.5 mM MgCl2 for 2 h at 25 ◦C in a volume of 100 µl. PD81,723 (20 or 100 µM) or vehicle (0.2–1 % DMSO) was added for 5 min. This time was found to be sufficiently long for PD81,723 to bind to an allosteric site, but short enough to minimally affect equilibrium radioligand binding. Dissociation was initiated by the addition of 100 µM NECA (5 -N-ethylcarboxamidoadenosine) or, in some cases, by a combination of 100 µM 8-cyclopentyltheophylline plus 50 µM GTP[S], and membranes were filtered after various times. In some cases, the effect of PD81,723 was evaluated at a single point in time after initiating radioligand dissociation in the presence of NECA, a modification of the method described by Figler et al. [32]. The optimal time to evaluate the effect of PD81,723 was determined in preliminary experiments to be 10 min for the canine A1 AR and 6 min for the canine A2A AR. Non-specific binding was determined by adding 100 µM NECA prior to the radioligand in the absence of PD81,723. Data analysis
Kinetic data were optimally fitted to a one- or two-site exponential equation based on the method of Motulsky and Ransnas [33]. Differences in residual binding measured at a single time point after the addition of NECA were evaluated using the paired Student’s t test. Significance of differences between means for two groups was determined using Student’s t test and between more than two groups using ANOVA.
Membrane preparation
HEK-293 cells expressing recombinant ARs were scraped from 150 mm2 culture dishes in 25 ml of ice-cold Buffer A (10 mM Hepes, 10 mM EDTA and 0.1 mM benzamidine) at pH 7.4. Cells were homogenized using a Brinkmann polytron on setting 5 for 30 s. The homogenates were centrifuged at 20 000 g for 30 min. Pellets were resuspended in 25 ml of ice-cold HE (10 mM Hepes, 1 mM EDTA and 0.1 mM benzamidine, pH 7.4) and washed twice by centrifugation. The final pellets were suspended in HE supplemented with 10 % (w/v) sucrose at a protein concentration [31] of 1 mg/ml and divided into aliquots and frozen at − 20 ◦C. Radioligand binding
For equilibrium binding studies, 10 µg of membrane protein was incubated in 100 µl with 2 units/ml ADA and with radioligand in HE buffer containing MgCl2 at a final concentration of 4.9 mM in the presence or absence of competing compounds. Binding reactions were incubated at 25 ◦C for 2–3 h and terminated by rapid filtration through Whatman glass fibre (GF/C) filters using a Brandel cell harvester. 125 I-ABA served as an agonist radioligand and 1 µM CPX was used to determine non-specific binding in experiments on receptors with A1 transmembrane sequence. 125 I-APE was the radioligand and 10 µM CGS 21680 was used to determine non-specific binding for those receptors with A2A transmembrane sequence. The G-protein-coupled fraction of receptors is defined as the fraction of GTP[S] (guanosine 5 [γ -thio]triphosphate) (50 µM)-sensitive specific agonist radioligand binding per total specific binding.
RESULTS
PD81,723 allosterically decreases the dissociation kinetics of agonists from the A1 AR and increases coupling with Gi/o proteins [28]. PD81,723 has no allosteric effect on antagonist binding to the A1 ARs, e.g. the kinetics of [3 H]CPX dissociation are not affected, but at high concentrations (> 20 µM) PD81,723 acts as a weak competitive antagonist in equilibrium binding assays. It is not known if the specificity of PD81,723 for A1 versus A2A or A2B receptors is due to differences in the ligand recognition TMs (transmembrane domains) of receptors or due to differences in G-protein coupling. To address this question, chimaeric A1 /A2A ARs were used in which the 3ICL, C-terminal tail or both were switched from A1 to A2A sequence and vice versa. We have demonstrated previously that these chimaeric receptors retain the ability to bind radioligands and couple with G-proteins [14]. The structure–activity profile of chimaeric receptors is conferred by the identity of the transmembrane helices, while coupling specificity (Gi versus Gs ) is determined primarily by the sequence of the 3ICL, with a minor contribution by the CT region [14]. PD81,723 does not enhance the effects of CPA on cAMP accumulation in untransfected HEK-293 cells
cAMP accumulation was measured in untransfected cells to identify non-A1 AR-mediated effects of CPA and PD81,723. At submicromolar doses, CPA has no effect on cAMP accumulation in untransfected HEK-293 cells, but at concentrations above 10 µM, CPA stimulates an increase in cAMP by activating endogenous A2B receptors on these cells (EC50 = 46 µM, 95 % c 2006 Biochemical Society
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S. Bhattacharya and others G-protein coupling of chimaeric ARs
Figure 2 Effects of PD81,723 and BW-A1433 on the dose dependence of CPA to increase cAMP accumulation in untransfected HEK-293 cells Cells were washed and incubated with vehicle (0.2 % DMSO), PD81,723 (20 µM) or BWA1433 (10 µM), and 10 units/ml ADA, Ro-20-1724 (20 µM) and various concentrations of CPA at 37 ◦C for 10 min. Each point is the mean + − S.E.M. for triplicate determinations. The experiment was repeated twice with similar results. Basal cAMP levels are not significantly different: 6 + + 15.7 + − 1.5 pmol/10 cells with CPA, 16.8 − 0.05 with CPA + PD81,723 and 18.2 − 0.10 with CPA + BW-A1433. Maximal cAMP levels were different among the three groups: 6 6 + 110.5 + − 5.5 pmol/106 cells with CPA, 91.1 − 5.9 pmol/10 cells with CPA + PD81,723 and cells with CPA + BW-A1433 ( P < 0.05, ANOVA). 25.9 + 0.1 pmol/10 −
confidence internal = 34–64 µM) with a maximal 7-fold increase in cAMP (Figure 2). In untransfected cells, the addition of 20 µM PD81,723 with CPA fails to stimulate cAMP above levels seen with CPA; in fact there is a slight, but significant, reduction that can be attributed either to the competitive antagonist activity of PD81,723 or to its direct inhibition of adenylate cyclase [34]. cAMP accumulation in HEK-293 cells is likely mediated by an AR subtype other than A1 , because the non-selective AR antagonist BW-A1433 blocks CPA-induced increases in cAMP, but has no effect on basal cAMP levels. There is no significant binding of 1 nM 125 I-ABA or 125 I-APE to untransfected HEK-293 cells (results not shown), consistent with the known low affinity of these radioligands for A2B ARs and the absence of A1 or A2A ARs in these cells. Taken together, these results indicate that high doses of CPA can activate A2 ARs, resulting in stimulation, rather than inhibition, of cAMP. The effects of PD81,723, if any, are to inhibit cAMP, possibly through an AR-independent mechanism.
Figure 3
Specific binding was measured in the presence and absence of the uncoupling reagent GTP[S] in order to compare coupling efficiency of native and chimaeric receptors. 125 I-ABA (1 nM) binding to recombinant wild-type A1 receptors and chimaeric A1 /A2A receptors with A1 transmembrane sequence is shown in Figure 3(A). The radioligand binds predominantly to G-proteincoupled A1 receptors (93 %) as defined by the reduction of specific radioligand binding seen upon the addition of GTP[S]. In chimaeric receptors in which A2A sequence replaced A1 sequence in the 3ICL (A1 /A2A L), most receptors remain coupled with G-proteins. The insensitivity of A1 AR–G protein complexes to GTP[S] reported in some native membranes is not observed in our recombinant system [35]. We have shown previously that the A1 /A2A L chimaera is dually coupled with Gi and Gs , and that activation of this receptor causes an increase in cellular cAMP in response to CPA [14]. Unlike A1 receptors, for which binding to 125 I-ABA measures primarily coupled receptors, 125 IAPE binds primarily uncoupled A2A receptors as measured by poor sensitivity to GTP[S]. Only 4.1 % of wild-type A2A receptors are coupled with Gs in the stably transfected HEK-293 cells we used, consistent with prior published observations [36]. However, chimaeric A2A /A1 L receptors are well coupled at 54.0 % (Figure 3B). A1 /A2A and A2A /A1 receptor chimaeras are able to couple with G-proteins, with coupling fractions that are intermediate between those of the wild-type receptors. PD81,723 slows agonist dissociation from chimaeric ARs with A1 transmembrane sequence
Radioligand dissociation from wild-type and chimaeric receptors was measured to determine whether the sensitivity of the A1 receptor to PD81,723-mediated enhancement of radioligand binding was dependent on the presence of A1 receptor domains that determine G-protein specificity. Figure 4 summarizes the effects of PD81,723 on one-point dissociation assays of radioligands from wild-type and chimaeric ARs. At predetermined time points, PD81,723 increases the fraction of 125 I-ABA bound to wildtype A1 receptor and all chimaeric A1 /A2A receptors containing A1 transmembrane segments. In contrast, PD81,723 fails to significantly retard the dissociation of 125 I-APE from wild-type A2A receptors or any chimaeric A2A /A1 receptors that contain A2A sequence in the transmembrane regions and/or CT region. We conclude from these results that PD81,723 does not interact directly with the 3ICL or CT region of the A1 AR. The failure of PD81,723 to slow 125 I-APE dissociation from the A2A /A1 L chimaera is particularly notable, given that this receptor differs from the A2A AR in that it is well-coupled. Hence the failure of
Effects of GTP[S] (GTPγ S) on radioligand binding to wild-type and chimaeric A1 (A) and A2A (B) ARs
Equilibrium binding was measured as described in the Experimental section. Each bar represents the composite means + − S.E.M. for three independent experiments each performed in triplicate. c 2006 Biochemical Society
Allosteric enhancement of Gs -coupled adenosine receptors
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Figure 5 Effect of PD81,723 on the kinetics of dissociation of 125 I-ABA from membranes derived from HEK-293 cells expressing recombinant canine A1 AR (A) or A1 /A2A L AR (B) chimaeras Dissociation was initiated by the addition of 100 µM 8-cyclopentyltheophylline (CPT) and 50 µM GTP[S] (GTPγ S) and kinetics were measured and evaluated as described in the Experimental section. The data are fitted to a two-site exponential decay model, with the exception of the A1 /A2A L + PD81,723 data, which fit better to a one-site model. The graphs are composites representing data from two or three independent experiments consisting of one data point/time point. Dissociation from the low-affinity site was too rapid to allow characterization of t 1/2 (half-life). The t 1/2 for ligand dissociation from the high-affinity A1 AR site increased from 1.3 min in the absence of PD81,723 to 4.0 min in the presence of PD81,723 (P = 0.005, t test). The t 1/2 for the A1 /A2A L chimaera increased from 0.99 to 1.9 min with the addition of PD81,723 (P = 0.04, t test).
Figure 4 Effect of PD81,723 on radioligand dissociation from recombinant wild-type or chimaeric receptors with A1 AR transmembrane sequence Dissociation was measured at a single time point as described in the Experimental section. (A) Method for one-point dissociation experiments. AE represents allosteric enhancer. (B) Dissociation of 125 I-ABA from receptors containing A1 transmembrane sequence. Each bar is a composite of the means + − S.E.M. for data from four to seven independent experiments, each performed in triplicate. In all cases, vehicle and PD81,723 groups differed significantly (A1 AR, P = 0.04; A1 /A2A L, P = 0.01; A1 /A2A T, P = 0.003 and A1 /A2A LT, P = 0.003, paired t test) based on paired analysis of experiments performed on different days. (C) Dissociation of 125 I-APE from receptors containing A2A transmembrane sequence. Each bar is a composite of the means + − S.E.M. for data from four or five independent experiments each performed in triplicate. In all cases, vehicle and PD81,723 groups were not significantly different (P > 0.05, paired t test).
PD81,723 to enhance agonist binding to the wild-type A2A AR cannot be attributed to the fact that it is poorly coupled with G-proteins. We also examined the effect of PD81,723 on the kinetics of agonist radioligand dissociation from the wild-type A1 AR and chimaeric A1 /A2A L receptors. Dissociation was accelerated by the addition of GTP[S]. The effect of PD81,723 on GTP[S]accelerated dissociation kinetics of 125 I-ABA from the wild-type A1 AR and the A1 /A2A chimaeric receptor is shown in Figure 5. PD81,723 reduces the fraction of A1 ARs that rapidly convert into a low-affinity conformational state upon the addition of GTP[S].
PD81,723 slows radioligand dissociation from the wild-type A1 AR by 3-fold, and from the A1 /A2A L AR by 2-fold. As has been previously described in the literature [22], PD81,723 does not increase coupling or dissociation rates when added to membranes expressing the wild-type canine A2A receptor (results not shown). Our results suggest that domains of the A1 AR primarily involved in G-protein coupling are not necessary for enhancement of radioligand binding by PD81,723. Effects of PD81,723 and BW-A1433 on cAMP responses in HEK-293 cells expressing chimaeric receptors
In order to measure the CPA-dependent effects of PD81,723 on cAMP accumulation in cells expressing recombinant A1 and A1 /A2A L ARs, we wanted to minimize differences in baseline cAMP for each receptor observed with the addition of PD81,723 alone. Transfection of HEK-293 cells with wild-type ARs changes basal cAMP levels, particularly in receptors coupled with Gs . Moreover, the addition of PD81,723 in the absence of AR agonists produces changes in cellular cAMP consistent with the compound acting as an enhancer of the effects of low levels of endogenous adenosine, as a potentiator of constitutive receptor activity, or as a direct inhibitor of adenyate cyclase. PD81,723 has been observed to demonstrate each of these effects in a c 2006 Biochemical Society
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Figure 6 Effect of PD81,723 on the dose dependence of BW-A1433 to modify forskolin-stimulated cAMP accumulation in HEK-293 cells expressing (A) canine A1 AR and (B) A1 /A2A L AR chimaeras Cells were washed and incubated with vehicle (DMSO), or PD81,723 (20 µM), 10 units/ml ADA, Ro-20-1724 (20 µM), forskolin (10 µM) and various concentrations of BW-A1433 for 10 min. Each graph is a composite of the means + − S.E.M. for two to four experiments each assayed in triplicate.
system-specific fashion [22,34,37]. Of these, one would expect that each except the direct inhibition of adenylate cyclase by PD81,723 would be blocked by an AR antagonist, which could then be used in subsequent experiments to equalize basal cAMP levels in the absence of CPA. Figure 6 shows the effects of PD81,723 and the non-selective AR antagonist BW-A1433 on forskolin-stimulated cAMP levels in HEK-293 cells expressing wild-type A1 or A1 /A2A L ARs in the absence of an agonist ligand. The A1 AR is functionally coupled with adenylate cyclase inhibition, while the A1 /A2A L AR is functionally coupled with stimulation. In the absence of antagonist, 20 µM PD81,723 causes a 30 % inhibition of cAMP levels in cells expressing the A1 6 AR (198 + − 6 versus 257 + − 7 pmol/10 cells, P = 0.02) and a 61 % increase in cells expressing the A1 /A2A L AR (1068 + − 115 versus 6 1719 + 64 pmol/10 cells, P = 0.05). Increasing concentrations of − antagonist block these effects of PD81,723 as well as constitutive effects of A1 /A2A AR expression. BW-A1433 reduces cAMP levels in forskolin-stimulated cells expressing the A1 /A2A L AR to levels similar to those seen in cells expressing the wild-type A1 AR. BW-A1433 was added to subsequent functional experiments at a concentration of 10 µM in order to block the effects of endogenous adenosine and/or constitutive receptor activity and clearly assess functional allosteric effects mediated by PD81,723. PD81,723 decreases the EC50 for cAMP stimulation observed on addition of CPA to the A1 /A2A L AR
Assuming that the effects of PD81,723 to enhance functional responses to A1 AR agonist binding are independent of the c 2006 Biochemical Society
Figure 7 Effect of PD81,723 on the dose dependence of CPA to modify cAMP accumulation in HEK-293 cells expressing canine A1 AR (A) and A1 /A2A L AR (B) chimaeras Cells were washed and incubated with vehicle (DMSO), or PD81,723 (20 µM), 10 units/ml ADA, Ro-20-1724 (20 µM) and various concentrations of CPA for 10 min. (A) HEK-293-A1 cells were stimulated with forskolin (10 µM) during the incubation. HEK-293-A1 /A2A L cells were treated without forskolin (B) or with (C) pretreatment with pertussis toxin (PTX). Each point depicts the mean + − S.E.M. for triplicate data points from a representative experiment that was repeated three times.
G-protein to which the receptor couples and of the receptor domains involved in coupling, then PD81,723 should increase cAMP accumulation in response to CPA for the Gs -coupled A1 /A2A L chimaera. In the presence of 10 µM BW-A1433, PD81,723 did not alter basal cAMP levels of either forskolinstimulated wild-type A1 AR (Figure 7A) or the A1 /A2A L AR chimaera (Figure 7B). PD81,723 amplified both CPA-induced inhibition of cAMP levels in cells expressing wild-type A1 AR (Figure 7A) and CPA-induced stimulation in cells expressing A1 /A2A L AR (Figure 7B). As shown in Figure 7(C), pertussis intoxication of HEK-293 cells expressing the A1 /A2A L AR in the presence of 10 µM BW-A1433 increased the average
Allosteric enhancement of Gs -coupled adenosine receptors
maximal cAMP levels observed with 10−4 M CPA from 226 + − 13 6 of PD81,723, and to 944 + − 80 pmol/10 cells in the absence 6 from 291 + − 12 to 1167 + − 138 pmol/10 cells in the presence of PD81,723. Figure 7(C) shows that pertussis intoxication primarily increases the magnitude of CPA-induced cAMP accumulation in the A1 /A2A L AR. The EC50 for cAMP accumulation in response to CPA in the presence of PD81,723 is 3.17 + − 0.37 µM with pertussis toxin versus 4.36 + − 1.28 µM without pertussis toxin. Thus the PD81,723-induced shift in the potency of CPA is independent of pertussis intoxication (compare Figures 7B and 7C). This implies that PD81,723 has a similar effect on receptors coupled with Gi or Gs . EC50 values could not reliably be calculated from curves in the absence of both PD81,723 and pertussis toxin because cAMP did not reach maximal levels at the highest concentrations of CPA used; however, in the presence of pertussis toxin, PD81,723 resulted in a decrease in EC50 from 176 + − 12 µM to 3.17 + − 0.37 µM (Figure 7C; P = 0.0002). The results suggest that the recognition site for PD81,723 is on domains of the A1 AR other than the 3ICL and that the enhancer acts to directly stabilize the receptor in a conformational state capable of coupling with either Gi or Gs . DISCUSSION
PD81,723 enhances the effects of agonists on A1 , but not A2A , ARs. Although there is high homology between the A1 and A2A AR subtypes [6], the receptor subtype selectivity of PD81,723 could be due to minor differences in the transmembrane ligand recognition domains, or the actions of PD81,723 could be restricted to receptors that couple with Gi/o . The results of the present study using chimaeric A1 /A2A receptors favour the former interpretation. When expressed in HEK-293 cells, each of the three chimaeric receptors engineered with A1 transmembrane sequence, as well as the wild-type A1 AR, are enhanced by PD81,723 as demonstrated by slowed agonist dissociation in ligand binding experiments. This enhancement occurs regardless of whether the receptor is primarily functionally coupled with Gi or Gs . Further, the potency of CPA to stimulate cAMP formation via the A1 /A2A L AR, which functionally couples with Gs and Gi , is increased by the addition of PD81,723, and this effect persists even if the coupling to Gi/o G-proteins is uncoupled with pertussis toxin. Conversely, PD81,723 does not slow radioligand dissociation from the wild-type A2A AR or from chimaeric receptors with A2A AR transmembrane sequence. Taken together, these observations demonstrate that, although PD81,723 may act to increase the coupled fraction of GPCRs, it probably does not interact directly with the 3ICL or G-proteins. Rather, PD81,723 likely works by causing a conformational change in the receptor through interactions with the extracellular domains or TMs. Our results suggest that PD81,723-induced conformational changes in the receptor result in positive co-operativity for both agonist binding and functional activation of the A1 AR. The binding site for PD81,723 has not been characterized. Of the GPCRs, the allosteric binding site for the muscarinic receptors has been most extensively studied and the prevailing model suggests that it resides predominantly in the extracellular domains superficial to the transmembrane location of the orthosteric site [1]. Similarly, studies using site-directed mutagenesis and receptor modelling to delineate the allosteric binding sites for the A3 AR demonstrate an energetically favourable binding site located at the top of TM VI and TM VII, superficial to the proposed orthosteric site for Cl-IB-MECA [2-chloro-N 6 -(3-iodobenzyl)adenosine-5 -methylcarboxamide; an AR agonist with relative A3 AR-selectivity] [38]. In the very limited analysis of amino acids important for allosterism of PD81,723 at the A1 AR, Thr277 ,
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located in TM VII, has been found to be important; however, it is also important for agonist binding and G-protein coupling and may not directly interact with PD81,723 [27]. Based on our present study and previous work on the allosteric binding sites for bioamines, the most probable location for the allosteric site of PD81,723 is superficial to the orthosteric site, as opposed to near the G-protein-coupling domains. One emerging concept from investigations of adrenergic, muscarinic and purinergic allosteric sites is that mutable allosteric binding domains may exist that depend on the modulator itself as well as on the liganded state of the receptor [2,38]. A1 AR-independent actions of PD81,723 to directly inhibit adenylate cyclase have been proposed [34]. This makes the A1 / A2A L chimaeric receptor expressed in HEK-293 cells, in which the receptor-dependent effects of PD81,723 are to stimulate adenylate cyclase, a particularly useful reagent for distinguishing between receptor-mediated and direct effects of PD81,723. Furthermore, in untransfected HEK-293 cells, basal cAMP levels in the absence of CPA are not different between PD81,723-treated and -untreated cells. These results are not consistent with direct actions by PD81,723 to inhibit adenylate cyclase in HEK-293 cells. HEK-293 cells do not express endogenous A1 ARs; thus untransfected cells do not show inhibition of cAMP levels in response to CPA. At doses of CPA > 10 µM, cAMP levels rise due to activation of A2B receptors on these cells [39]. This effect is blocked by the non-selective AR antagonist BW-A1433 and is not enhanced by PD81,723, confirming that the effect of CPA on cAMP is mediated by A2B ARs. In addition to blocking endogenous A2B receptors, 10 µM BW-A1433 was also used in the present study to block constitutive activity of recombinant receptors, possible effects of endogenous adenosine, and the functional effects of adding PD81,723 alone. This strategy of using a competitive antagonist to block cellular effects of PD81,723 alone has been used in previous studies [18,28]. Since competitive antagonists eliminate the effect of PD81,723 on cAMP metabolism observed in the absence of an added agonist, but do not block the allosteric effect of PD81,723, the strategy is useful for distinguishing allosteric from direct receptor-mediated effects of PD81,723. It is notable, however, that the use of BW-A1433 causes a rightward shift in the dose–response curve of CPA, which has to be added at concentrations that are sufficiently high to overcome the competitive blockade. We selected the non-selective AR antagonist (BW-A1433) because it blocks A2B AR activation by CPA at concentrations that were necessary to use to overcome competitive blockade of A1 ARs (100 nM). When A2A L sequence is substituted for A1 sequence, the resulting receptor couples functionally with Gs when expressed in the HEK-293 cell system. The percentage of coupled receptors, as defined by GTP[S]-sensitive binding, is > 70 %. This is in contrast with the recombinant A2A AR, which displays only 4 % GTP[S]sensitive binding, consistent with previous findings [14,36]. These findings indicate that only a small percentage of receptors need to be coupled with Gs in order to generate an ample increase in cAMP levels with stimulation. The reason that there is a large percentage of coupled A1 /A2A L AR compared with the wild-type A2A AR is likely due to the dual coupling of the chimaeric receptor with both Gs and Gi demonstrated previously [14]. Dual coupling was confirmed in the present study by experiments performed on membranes expressing the A1 /A2A L AR in the presence and absence of pertussis toxin, which demonstrated that cAMP levels achieved in response to CPA in the presence of pertussis toxin were 4-fold higher than those in the absence of pertussis toxin. Failure by PD81,723 to enhance coupling of the A2A /A1 L receptor is significant since it establishes that the lack of efficacy c 2006 Biochemical Society
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S. Bhattacharya and others
of PD81,723 to enhance the A2A AR is not in some way related to its poor coupling with Gs . Also, PD81,723 shifts the CPA dose– response curve in HEK-293-A1 /A2A L cells, suggesting that the enhancer has similar effects on receptors coupled with Gi or Gs . In summary, the results of the present study show that PD81,723, a selective allosteric enhancer of A1 ARs, probably acts by binding to and stabilizing the high-affinity conformation state of the A1 AR–G protein complex without directly interacting with G-proteins or domains of the receptor that interact with Gproteins. The novel finding that chimaeric receptors coupled with Gs can also be enhanced suggests that the enhancer binds to the transmembrane or possibly extracellular receptor region. Hence, it should be possible to identify additional compounds that can bind to and enhance the coupling of various GPCRs with Gs . This work was supported by the National Institutes of Health, grant numbers R01-HL56111 (to J. L.) and K08-HL03268 (to A. L. T.), and by the Virginia Affiliate of the American Heart Association grant number VA-97-GS8 (to A. L. T.). We are grateful to Heidi Figler for her invaluable technical assistance. We thank Dr G. Vassart for the canine A1 and A2A receptor cDNAs and Dr Erik Hewlett for pertussis toxin.
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