{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T23:07:51Z","timestamp":1790636871626,"version":"4.1.0"},"reference-count":36,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2019,7,23]],"date-time":"2019-07-23T00:00:00Z","timestamp":1563840000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"FEDER\/Ministerio de Ciencia, Innovaci\u00f3n y Universidades\u2013Agencia Estatal de Investigaci\u00f3n","award":["SAF2017-87349-R"],"award-info":[{"award-number":["SAF2017-87349-R"]}]},{"DOI":"10.13039\/501100004587","name":"ISCIII","doi-asserted-by":"publisher","award":["PIE14\/00034"],"award-info":[{"award-number":["PIE14\/00034"]}],"id":[{"id":"10.13039\/501100004587","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008666","name":"Fundaci\u00f3 la Marat\u00f3 de TV3","doi-asserted-by":"publisher","award":["Grant 20152031"],"award-info":[{"award-number":["Grant 20152031"]}],"id":[{"id":"10.13039\/100008666","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003130","name":"FWO","doi-asserted-by":"publisher","award":["SBO-140028"],"award-info":[{"award-number":["SBO-140028"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Catalan government","award":["2017 SGR 1604"],"award-info":[{"award-number":["2017 SGR 1604"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Background: Several biophysical techniques have been successfully implemented to detect G protein-coupled receptors (GPCRs) heteromerization. Although these approaches have made it possible to ascertain the presence of GPCR heteromers in animal models of disease, no success has been accomplished in pathological human post-mortem brains. The AlphaScreen technology has been consistently used to quantify small analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range and sensitivity exhibited by this technology support that it may be suitable to detect GPCR heteromers even under non-optimal conditions. Methods: Here, we describe the development of a new AlphaScreen assay to detect GPCR oligomers in human post-mortem brain. Results: Adenosine A2A-dopamine D2 receptor (A2AR\/D2R) heteromer formation was monitored in caudate from healthy and Parkinson\u2019s disease (PD) subjects. The approach was first validated using striatal membranes from wild type and A2AR deficient mice. Secondly, we took advantage of the 6-hydroxydopamine hemiparkinsonian rat model to validate previous results. In addition, finally, A2AR\/D2R heteromer formation was assessed in caudate membranes from human post-mortem brains. Importantly, our preliminary results revealed an increase in A2AR\/D2R heteromer formation in PD brains. Conclusions: The new AlphaScreen assay allowed assessing GPCR heteromers in human post-mortem brains with high sensitivity.<\/jats:p>","DOI":"10.3390\/ijms20143600","type":"journal-article","created":{"date-parts":[[2019,7,23]],"date-time":"2019-07-23T10:44:51Z","timestamp":1563878691000},"page":"3600","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":42,"title":["Revealing Adenosine A2A-Dopamine D2 Receptor Heteromers in Parkinson\u2019s Disease Post-Mortem Brain through a New AlphaScreen-Based Assay"],"prefix":"10.3390","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7834-2965","authenticated-orcid":false,"given":"V\u00edctor","family":"Fern\u00e1ndez-Due\u00f1as","sequence":"first","affiliation":[{"name":"Unitat de Farmacologia, Departament de Patologia i Terap\u00e8utica Experimental, Facultat de Medicina i Ci\u00e8ncies de la Salut, IDIBELL, Universitat de Barcelona, 08907 L\u2019Hospitalet de Llobregat, Spain"},{"name":"Institut de Neuroci\u00e8ncies, Universitat de Barcelona, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4412-8108","authenticated-orcid":false,"given":"Maricel","family":"G\u00f3mez-Soler","sequence":"additional","affiliation":[{"name":"Unitat de Farmacologia, Departament de Patologia i Terap\u00e8utica Experimental, Facultat de Medicina i Ci\u00e8ncies de la Salut, IDIBELL, Universitat de Barcelona, 08907 L\u2019Hospitalet de Llobregat, Spain"},{"name":"Institut de Neuroci\u00e8ncies, Universitat de Barcelona, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marta","family":"Valle-Le\u00f3n","sequence":"additional","affiliation":[{"name":"Unitat de Farmacologia, Departament de Patologia i Terap\u00e8utica Experimental, Facultat de Medicina i Ci\u00e8ncies de la Salut, IDIBELL, Universitat de Barcelona, 08907 L\u2019Hospitalet de Llobregat, Spain"},{"name":"Institut de Neuroci\u00e8ncies, Universitat de Barcelona, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masahiko","family":"Watanabe","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Hokkaido University School of Medicine, 060-0818 Sapporo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9888-8754","authenticated-orcid":false,"given":"Isidre","family":"Ferrer","sequence":"additional","affiliation":[{"name":"Institut de Neuroci\u00e8ncies, Universitat de Barcelona, 08035 Barcelona, Spain"},{"name":"Institut de Neuropatologia, Servei d\u2019Anatomia Patol\u00f2gica, IDIBELL-Hospital Universitari de Bellvitge, 08907 L\u2019Hospitalet de Llobregat, Spain"},{"name":"CIBERNED, Instituto Carlos III, 28035 Madrid, Spain"},{"name":"Unitat de Anatomia Patol\u00f2gica, Departament de Patologia i Terap\u00e8utica Experimental, Facultat de Medicina i Ci\u00e8ncies de la Salut, IDIBELL, Universitat de Barcelona, 08907 L\u2019Hospitalet de Llobregat, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco","family":"Ciruela","sequence":"additional","affiliation":[{"name":"Unitat de Farmacologia, Departament de Patologia i Terap\u00e8utica Experimental, Facultat de Medicina i Ci\u00e8ncies de la Salut, IDIBELL, Universitat de Barcelona, 08907 L\u2019Hospitalet de Llobregat, Spain"},{"name":"Institut de Neuroci\u00e8ncies, Universitat de Barcelona, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,7,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"322","DOI":"10.3109\/10799893.2010.508166","article-title":"G protein-coupled receptor oligomerization for what?","volume":"30","author":"Ciruela","year":"2010","journal-title":"J. Recept. Signal Transduct. Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1124\/jpet.106.104463","article-title":"Functional selectivity and classical concepts of quantitative pharmacology","volume":"320","author":"Urban","year":"2007","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_3","first-page":"2455","article-title":"Receptor heterodimerization leads to a switch in signaling: beta-arrestin2-mediated ERK activation by mu-delta opioid receptor heterodimers","volume":"21","author":"Rozenfeld","year":"2007","journal-title":"Faseb J. Off. Publ. Fed. Am. Soc. Exp. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1124\/mol.114.093096","article-title":"Functional Selectivity of Allosteric Interactions within G Protein-Coupled Receptor Oligomers: The Dopamine D1-D3 Receptor Heterotetramer","volume":"86","author":"Guitart","year":"2014","journal-title":"Mol. Pharmacol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4952","DOI":"10.1007\/s12035-017-0696-y","article-title":"Antipsychotic-Like Efficacy of Dopamine D2 Receptor-Biased Ligands is Dependent on Adenosine A2A Receptor Expression","volume":"55","author":"Sahlholm","year":"2018","journal-title":"Mol. Neurobiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1016\/S1474-4422(06)70471-9","article-title":"Epidemiology of Parkinson\u2019s disease","volume":"5","author":"Breteler","year":"2006","journal-title":"Lancet. Neurol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1257","DOI":"10.1016\/S1474-4422(16)30230-7","article-title":"The epidemiology of Parkinson\u2019s disease: risk factors and prevention","volume":"15","author":"Ascherio","year":"2016","journal-title":"Lancet Neurol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e18","DOI":"10.1111\/j.1755-5949.2009.00126.x","article-title":"Adenosine-dopamine interactions in the pathophysiology and treatment of CNS disorders","volume":"16","author":"Fuxe","year":"2010","journal-title":"CNS Neurosci. Ther."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"111","DOI":"10.2144\/000113719","article-title":"Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum","volume":"51","author":"Trifilieff","year":"2011","journal-title":"Biotechniques"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.neuropharm.2013.10.036","article-title":"l-DOPA-treatment in primates disrupts the expression of A2A adenosine-CB1 cannabinoid-D2 dopamine receptor heteromers in the caudate nucleus","volume":"79","author":"Bonaventura","year":"2014","journal-title":"Neuropharmacology"},{"key":"ref_11","first-page":"57","article-title":"Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats","volume":"8","author":"Taura","year":"2015","journal-title":"Dis. Models Mech."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Longhena, F., Faustini, G., Missale, C., Pizzi, M., and Bellucci, A. (2018). Dopamine transporter\/\u03b1-synuclein complexes are altered in the post mortem caudate putamen of Parkinson\u2019s disease: An in situ proximity ligation assay study. Int. J. Mol. Sc., 19.","DOI":"10.3390\/ijms19061611"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5426","DOI":"10.1073\/pnas.91.12.5426","article-title":"Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence","volume":"91","author":"Ullman","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1518","DOI":"10.1093\/clinchem\/42.9.1518","article-title":"Luminescent oxygen channeling assay (LOCI): sensitive, broadly applicable homogeneous immunoassay method","volume":"42","author":"Ullman","year":"1996","journal-title":"Clin. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2","DOI":"10.2174\/1875397300801010002","article-title":"The Use of AlphaScreen Technology in HTS: Current Status","volume":"1","author":"Eglen","year":"2008","journal-title":"Curr. Chem. Genom."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1007\/978-1-4939-3673-1_5","article-title":"AlphaScreen-Based Assays: Ultra-High-Throughput Screening for Small-Molecule Inhibitors of Challenging Enzymes and Protein-Protein Interactions","volume":"1439","author":"Yasgar","year":"2016","journal-title":"Methods Mol. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1177\/108705719900400206","article-title":"A simple statistical parameter for use in evaluation and validation of high throughput screening assays","volume":"4","author":"Zhang","year":"1999","journal-title":"J. Biomol. Screen."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"7038","DOI":"10.1007\/s12035-018-0887-1","article-title":"Disruption of A2AR-D2R Heteroreceptor Complexes After A2AR Transmembrane 5 Peptide Administration Enhances Cocaine Self-Administration in Rats","volume":"55","author":"Wydra","year":"2018","journal-title":"Mol. Neurobiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e12432","DOI":"10.1111\/gbb.12432","article-title":"Behavioral control by striatal adenosine A2A-dopamine D2 receptor heteromers","volume":"17","author":"Taura","year":"2018","journal-title":"Genes Brain Behav."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1038\/npp.2013.242","article-title":"Moonlighting proteins and protein-protein interactions as neurotherapeutic targets in the G protein-coupled receptor field","volume":"39","author":"Fuxe","year":"2014","journal-title":"Neuropsychopharmacology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"S19","DOI":"10.1212\/01.WNL.0000095206.44418.5C","article-title":"Receptor heteromerization in adenosine A2A receptor signaling: relevance for striatal function and Parkinson\u2019s disease","volume":"61","author":"Fuxe","year":"2003","journal-title":"Neurology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"49","DOI":"10.4172\/jaa.1000035","article-title":"A novel high-throughput screening assay to identify inhibitors of HIV-1 gp120 protein interaction with DC-SIGN","volume":"3","author":"Tran","year":"2011","journal-title":"J. Antivir. Antiretrovir."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Cacabelos, R. (2017). Parkinson\u2019s disease: From pathogenesis to pharmacogenomics. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18030551"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Bohush, A., Niewiadomska, G., and Filipek, A. (2018). Role of Mitogen Activated Protein Kinase Signaling in Parkinson\u2019s Disease. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19102973"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Comi, C., Ferrari, M., Marino, F., Magistrelli, L., Cantello, R., Riboldazzi, G., Bianchi, M.L.E., Bono, G., and Cosentino, M. (2017). Polymorphisms of dopamine receptor genes and risk of l-dopa\u2013induced dyskinesia in parkinson\u2019s disease. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18020242"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1449","DOI":"10.1093\/brain\/122.8.1449","article-title":"Striatal dopaminergic markers in dementia with Lewy bodies, Alzheimer\u2019s and Parkinson\u2019s diseases: rostrocaudal distribution","volume":"122","author":"Piggott","year":"1999","journal-title":"Brain"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1002\/ana.410300210","article-title":"Reduced D2 dopamine and muscarinic cholinergic receptor densities in caudate specimens from fluctuating parkinsonian patients","volume":"30","author":"Ahlskog","year":"1991","journal-title":"Ann. Neurol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/0304-3940(94)90358-1","article-title":"A detailed anatomical analysis of neurotransmitter receptors in the putamen and caudate in Parkinson\u2019s disease and Alzheimer\u2019s disease","volume":"169","author":"Griffiths","year":"1994","journal-title":"Neurosci. Lett."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1096\/fj.09-141044","article-title":"A2A adenosine receptor overexpression and functionality, as well as TNF-alpha levels, correlate with motor symptoms in Parkinson\u2019s disease","volume":"24","author":"Varani","year":"2010","journal-title":"FASEB J."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1007\/s004010100372","article-title":"Choline acetytransferase activity and striatal dopamine receptors in Parkinson\u2019s disease in relation to cognitive impairment","volume":"102","author":"Mattila","year":"2001","journal-title":"Acta Neuropathol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1159\/000346370","article-title":"Postmortem brain levels of urate and precursors in Parkinson\u2019s disease and related disorders","volume":"12","author":"McFarland","year":"2013","journal-title":"Neurodegener. Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1016\/j.nbd.2014.05.030","article-title":"Increased striatal adenosine A2A receptor levels is an early event in Parkinson\u2019s disease-related pathology and it is potentially regulated by miR-34b","volume":"69","author":"Porta","year":"2014","journal-title":"Neurobiol. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/S0197-4580(02)00065-9","article-title":"Staging of brain pathology related to sporadic Parkinson\u2019s disease","volume":"24","author":"Braak","year":"2003","journal-title":"Neurobiol. Aging"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"674","DOI":"10.1038\/41771","article-title":"Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor","volume":"388","author":"Ledent","year":"1997","journal-title":"Nature"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1093\/ilar.38.1.41","article-title":"Special report: the 1996 guide for the care and use of laboratory animals","volume":"38","author":"Clark","year":"1997","journal-title":"ILAR J.Natl. Res. Counc. Inst. Lab. Anim. Resour."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1111\/nan.12386","article-title":"Locus coeruleus at asymptomatic early and middle Braak stages of neurofibrillary tangle pathology","volume":"43","author":"Carmona","year":"2017","journal-title":"Neuropathol. Appl. Neurobiol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/20\/14\/3600\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:08:44Z","timestamp":1760188124000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/20\/14\/3600"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,7,23]]},"references-count":36,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2019,7]]}},"alternative-id":["ijms20143600"],"URL":"https:\/\/doi.org\/10.3390\/ijms20143600","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,7,23]]}}}