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Allosteric Interactions Between Agonists and Antagonists Within The Allosteric interactions between agonists and PNAS PLUS antagonists within the adenosine A2A receptor- dopamine D2 receptor heterotetramer Jordi Bonaventuraa,b, Gemma Navarroa, Verònica Casadó-Angueraa, Karima Azdadc, William Reab, Estefanía Morenoa, Marc Brugarolasa, Josefa Mallola, Enric I. Canelaa, Carme Lluísa, Antoni Cortésa, Nora D. Volkowd, Serge N. Schiffmannc, Sergi Ferréb,1, and Vicent Casadóa,1 aDepartment of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, and Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas and Institute of Biomedicine of the University of Barcelona, 08028 Barcelona, Spain; bIntegrative Neurobiology Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD 21224; cLaboratory of Neurophysiology, Universite Libre de Bruxelles-Neuroscience Institute, 1070 Brussels, Belgium; and dNational Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892 Edited by Susan G. Amara, National Institute of Mental Health, Bethesda, MD, and approved May 27, 2015 (received for review April 21, 2015) Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) het- of an A2AR agonist to abolish the decreased excitability of eromers are key modulators of striatal neuronal function. It has GABAergic striatopallidal neurons induced by high concen- been suggested that the psychostimulant effects of caffeine de- tration of a D2R agonist (9), which should overcome the de- pend on its ability to block an allosteric modulation within the crease in affinity. Furthermore, a differential effect of allosteric A2AR-D2R heteromer, by which adenosine decreases the affinity modulations of different agonist-mediated signaling responses and intrinsic efficacy of dopamine at the D2R. We describe novel (i.e., functional selectivity) can occur within GPCR heteromers unsuspected allosteric mechanisms within the heteromer by which (1, 2, 8). Again, the A2AR-D2R heteromer provides a valuable not only A2AR agonists, but also A2AR antagonists, decrease the example. A recent study has shown that different levels of in- 2+ affinity and intrinsic efficacy of D2R agonists and the affinity of tracellular Ca exert different modulations of A2AR-D2R D2R antagonists. Strikingly, these allosteric modulations disappear heteromer signaling (8). This depends on the ability of low and PHARMACOLOGY + on agonist and antagonist coadministration. This can be explained high Ca2 to promote a selective interaction of the heteromer 2+ by a model that considers A2AR-D2R heteromers as heterote- with different Ca -binding proteins, which differentially modulate tramers, constituted by A2ARandD2R homodimers, as demon- allosteric interactions in the heteromer (8). strated by experiments with bioluminescence resonance energy It has been hypothesized that the allosteric interactions be- transfer and bimolecular fluorescence and bioluminescence com- tween A2AR and D2R agonists within the A2AR-D2R heteromer plementation. As predicted by the model, high concentrations of provide a mechanism responsible not only for the depressant A R antagonists behaved as A R agonists and decreased D R 2A 2A 2 effects of A2AR agonists, but also for the psychostimulant effects function in the brain. of adenosine A2AR antagonists and the nonselective adenosine receptor antagonist caffeine (9, 11, 12), with implications for adenosine A2A receptor | dopamine D2 receptor | caffeine | several neuropsychiatric disorders (13). In fact, the same mech- GPCR heteromers anism has provided the rationale for the use of A2AR antagonists ost evidence indicates that G protein-coupled receptors Significance M(GPCRs) form homodimers and heteromers. Homodimers seem to be a predominant species, and oligomeric entities can be G protein-coupled receptors (GPCRs) constitute the largest viewed as multiples of dimers (1). It has been proposed that plasma membrane protein family involved in cell signaling. GPCR heteromers are constituted mainly by heteromers of GPCR homodimers are predominant species, and GPCR het- homodimers (1, 2). Allosteric mechanisms determine a multi- eromers likely are constituted by heteromers of homodimers. plicity of unique pharmacologic properties of GPCR homo- The adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) dimers and heteromers (1, 3). First, binding of a ligand to one of heteromer is a target for the nonselective adenosine receptor the receptors in the heteromer can modify the affinity of ligands antagonist caffeine. This study uncovers allosteric modulations for the other receptor (1, 3, 4). The most widely reproduced of A2AR antagonists that mimic those of A2AR agonists, chal- allosteric modulation of ligand-binding properties in a GPCR lenging the traditional view of antagonists as inactive ligands. heteromer is the ability of adenosine A2A receptor (A2AR) ag- These allosteric modulations disappear when agonist and an- onists to decrease the affinity of dopamine D2 receptor (D2R) tagonist are coadministered, however. A model is proposed agonists in the A2AR-D2R heteromer (5). A2AR-D2R hetero- that considers A2AR-D2R heteromers as heterotetramers, con- mers have been revealed both in transfected cells (6, 7), striatal stituted by A2AR and D2R homodimers. The model predicted neurons in culture (6, 8) and in situ, in mammalian striatum (9, that high concentrations of A R antagonists would behave as 10), where they play an important role in the modulation of 2A A2AR agonists and decrease D2R function in the brain. GABAergic striatopallidal neuronal function (9, 11). In addition to ligand-binding properties, unique properties for Author contributions: J.B., G.N., C.L., N.D.V., S.N.S., S.F., and V.C. designed research; J.B., each GPCR oligomer emerge in relation to the varying intrinsic G.N., V.C.-A., K.A., W.R., E.M., M.B., A.C., and V.C. performed research; J.B., G.N., V.C.-A., efficacy of ligands for different signaling pathways (1–3). In- K.A., W.R., E.M., M.B., J.M., E.I.C., C.L., A.C., S.N.S., S.F., and V.C. analyzed data; and J.B., G.N., C.L., S.N.S., S.F., and V.C. wrote the paper. trinsic efficacy refers to the power of the agonist to induce a The authors declare no conflict of interest. functional response, independent of its affinity for the receptor. This article is a PNAS Direct Submission. Thus, allosteric modulation of an agonist can potentially in- 1To whom correspondence may be addressed. Email: [email protected] or vcasado@ volve changes in affinity and/or intrinsic efficacy (1, 3). This ub.edu. principle can be observed in the A2AR-D2R heteromer, where a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. decrease in D2R agonist affinity cannot alone explain the ability 1073/pnas.1507704112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1507704112 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 in patients with Parkinson’s disease (13, 14). The initial aim of protein. The decrease in [3H]quinpirole binding by CGS 21680 the present study was to study in detail the ability of caffeine to and caffeine was related to a noncompetitive inhibition, with de- counteract allosteric modulations between A2AR and D2R ago- creasing affinity (i.e., increase in KD1 values), as shown in compe- 3 nists (affinity and intrinsic efficacy) within the A2AR-D2R het- tition experiments of [ H]quinpirole vs. quinpirole (Table 1). eromer. Unexpectedly, when performing control radioligand- Previous studies have shown that in the A2AR-D2R heteromer, binding experiments, not only an A2AR agonist, but also caffeine, a strong electrostatic interaction occurs between an arginine-rich significantly decreased D2R agonist binding. However, when coad- epitope localized in the N-terminal part of the third intracellular ministered, the A2AR agonist and caffeine co-counteracted their loop of the D2R and a phosphorylated residue, serine-374, lo- ability to modulate D2R agonist binding. By exploring the molecular calized in the distal part of the C terminus of the A2AR (15, 16). mechanisms behind these apparent inconsistencies, the present Bioluminescence energy transfer experiments demonstrated that A374 study provides new insight into the quaternary structure and func- transfection with mutant A2AR(A2A R) or D2R lacking these tion of A2AR-D2R heteromers. key interacting residues leads to pronounced modification of the quaternary structure of the heteromer (15, 17, 18). In transfected A374 Results cells, A2A R showed a very similar expression (Bmax for 3 Caffeine Modulates D2R Agonist Binding: A New Biochemical Property [ H]ZM 241385 binding of 2.0 pmol/mg protein) and the same of the A2AR-D2R Heteromer. As expected, the A2AR agonist CGS affinity for caffeine or CGS 21680 compared with the wild-type 3 3 21680 significantly decreased D2R agonist [ H]quinpirole bind- A2AR. Identical competition curves of [ H]ZM 241385 vs. CGS ing in membrane preparations from both sheep striatum (Fig. 1A, 21680 or caffeine were obtained from cells transfected with A374 black bars) and Chinese hamster ovary (CHO) cells transiently D2R and either A2A Rorwild-typeA2AR(Fig. S1B). The 3 transfectedwithA2ARandD2R (Fig. 1A, red bars). Unexpectedly, ability of CGS 21680 and caffeine at modulating [ H]quinpirole caffeine also produced the same effect (Fig. 1B), and the effective binding was significantly reduced in the CHO cells transfected A374 concentrations of CGS 21680 and caffeine were in the same
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