Heterobivalent Ligand for the Adenosine A2A–Dopamine D2 Receptor Heteromer

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’s 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.

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Evidence for the heterotetrameric structure of the adenosine A2A–dopamine D2 receptor complex

Biochemical Society Transactions ◽

10.1042/bst20150276 ◽

2016 ◽

Vol 44 (2) ◽

pp. 595-600 ◽

Cited By ~ 20

Author(s):

Verònica Casadó-Anguera ◽

Jordi Bonaventura ◽

Estefanía Moreno ◽

Gemma Navarro ◽

Antoni Cortés ◽

...

Keyword(s):

Structural Model ◽

Dopamine D2 Receptor ◽

Ex Vivo ◽

D2 Receptor ◽

Drug Dosage ◽

D1 Receptor ◽

Structural Basis ◽

Dopamine D2 ◽

Allosteric Mechanisms ◽

Adenosine A2a

Heteromers of G-protein-coupled receptors (GPCRs) have emerged as potential novel targets for drug development. Accumulating evidence indicates that GPCRs can form homodimers and heteromers, with homodimers being the predominant species and oligomeric receptors being formed as multiples of dimers. Recently, heterotetrameric structures have been proposed for dopamine D1 receptor (D1R)–dopamine D3 receptor (D3R) and adenosine A2A receptor (A2AR)–dopamine D2 receptor (D2R) heteromers. The structural model proposed for these complexes is a heteromer constituted by two receptor homodimers. The existence of GPCR homodimers and heteromers provides a structural basis for inter-protomer allosteric mechanisms that might account for a multiplicity of unique pharmacological properties. In this review, we focus on the A2AR–D2R heterotetramer as an example of an oligomeric structure that is key in the modulation of striatal neuronal function. We also review the interfaces involved in this and other recently reported heteromers of GPCRs. Furthermore, we discuss several published studies showing the ex vivo expression of A2AR–D2R heteromers. The ability of A2AR agonists to decrease the affinity of D2R agonists has been reported and, on the basis of this interaction, A2AR antagonists have been proposed as potential drugs for the treatment of Parkinson's disease. The heterotetrameric structure of the A2AR–D2R complex offers a novel model that can provide new clues about how to adjust the drug dosage to the expected levels of endogenous adenosine.

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