On the Existence of a Possible A2A–D2–β-Arrestin2 Complex: A2A Agonist Modulation of D2 Agonist-Induced β-Arrestin2 Recruitment

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Abstract

Given that coactivation of adenosine A2A (A2AR) and dopamine D2 (D2R) receptors results in the coaggregation, cointernalization, and codesensitization of the A2AR and D2R and the role of scaffolding protein β-arrestin2 in the desensitization, internalization, and signaling of G-protein-coupled receptors, in this study we explored the ability of the A2AR agonist CGS21680 in A2AR–D2R-coexpressing cells to modulate the D2R agonist-induced recruitment of β-arrestin2 to the D2R by means of proximity-based bioluminescence resonance energy transfer (BRET2) and co-trafficking analysis. We found evidence that CGS21680 can increase the maximal BRET2 signal between β-arrestin2RLuc and D2LRGFP2 upon D2R activation, by increasing the potency of the D2R agonist to exert this action. In addition, this change was associated with an increased formation of cytoplasmic clusters containing β-arrestin2GFP2 and D2LRYFP as seen from the co-trafficking analysis. Furthermore, the A2AR agonist advanced the time for the increase in Akt phosphorylation obtained with the D2R agonist. Finally, using a novel bioinformatics approach to predict the protein–protein interface, we have also found that amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the -induced β-arrestin2 recruitment by A2AR–D2R heteromers. Taken together, the results indicate that the antagonistic A2AR–D2R allosteric receptor–receptor interaction in A2AR–D2R heteromers favors β-arrestin2 recruitment to the D2LR protomer with subsequent cointernalization associated with a reduced time onset of Akt phosphorylation followed by a rapid dephosphorylation. Thus, β-arrestin2 action becomes more rapid and short-lasting and, in this way, mimics G-protein-mediated signaling.

Research Highlights

► A2A agonist can increase the maximal BRET2 signal between β-arrestin2 and the D2R. ► BRET2 change is associated with an increased formation of cytoplasmic clusters. ► A2A agonist advances the time for the increase of Akt phosphorylation upon D2R activation. ► A set of amino acid pro-triplets may be crucial for β-arrestin2 recruitment.

Introduction

Adenosine A2A receptor (A2AR)–dopamine D2 receptor (D2R) heteromerization has been demonstrated by means of biochemical and biophysical methods [co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), and fluorescence resonance energy transfer analyses] to occur upon transient cotransfection of the two receptors in cell lines including HEK293 T cells.1, 2, 3 Antagonistic allosteric A2AR–D2R receptor–receptor interactions have been shown to exist in A2AR–D2R heteromers, reducing the affinity of the D2R agonist binding sites, Gi/o coupling, and signaling via adenylate cyclase, phospholipase C, and mitogen-activated protein kinases.2, 4, 5, 6

In D2R-cotransfected neuroblastoma cells, coactivation of A2AR and D2R results in the coaggregation, cointernalization, and codesensitization of the A2AR and D2R.7 However, it is unknown how the scaffolding protein β-arrestin2, being recognized to participate in the desensitization, internalization, and signaling of G-protein-coupled receptors, is involved in these events.8 The sequence amino acid 212-IYIV-215 in the N-terminal part of the third intracellular loop (IC-3) of the D2R appears critical for β-arrestin2 binding.9, 10 Furthermore, Lys149 in the second intracellular loop (IC-2) in the D2R gives preferential β-arrestin2 binding to D2R IC-2 versus D3R IC-2, as shown in studies with glutathione S-transferase fusion proteins of D2R and D3R IC-2.10 β-Arrestin2 is also involved in mediating the Akt/GSK3 signaling of D2Rs.11, 12

In this study, we explored the ability of the A2AR agonist CGS21680 in A2AR–D2R-cotransfected cells to modulate the D2R-like agonist-induced recruitment of β-arrestin2 to the D2R by means of BRET2 and co-trafficking analyses. In parallel, the A2AR agonist modulation of the D2R-induced changes in the temporal dynamics of protein kinase B (Akt) phosphorylation was studied to link changes in signaling to the BRET2 and co-trafficking analyses. Therefore, according to the experimental results and using a novel bioinformatics approach to predict the protein–protein interface, the amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the agonist-induced β-arrestin2 recruitment by A2AR–D2R heteromers.

Section snippets

D2R agonist-mediated internalization is increased upon combined A2AR and D2R agonist treatment

A2AR–D2R heteromers are known to be formed after transient coexpression of the two receptors in HEK cells,2, 3 resulting in co-trafficking of the D2R and A2AR.13 We investigated whether A2AR affects the D2R agonist-mediated D2R internalization in transiently cotransfected HEK293T cells using a cell surface receptor expression assay.

HEK293T cells coexpressing 3xHA-D2LR and A2AR were incubated in the presence of the D2LR agonist quinpirole (0.25 μM) with or without the A2AR agonist CGS21680

Discussion

Previous works have shown that transient cotransfection of the A2AR and D2R in HEK cells results in the formation of A2AR–D2R heteromers.2, 3 Therefore, the observation that the A2AR agonist CGS21680 can increase the maximal BRET2 signal between β-arrestin2RLuc and D2LRGFP2 upon D2R activation with quinpirole by increasing the potency of the D2R agonist to exert this action is of substantial interest. It indicates that the A2AR agonist-induced activation of the A2AR in the heteromer can favor

Plasmid constructs

β-Arrestin2Rluc and β-arrestin2GFP2 were generated by PCR amplification of the coding sequences of human β-arrestin2 (kindly provided by M. Pérez-Alea, Covalab, France) without their stop codons and subcloned in pGFP2-N3 (Perkin-Elmer, Madrid, Spain) and pRluc-N3 (Packard Bioscience, Spain). On the other hand, the cDNA encoding the D2LR without its stop codon was subcloned in pGFP2-N1 (Perkin-Elmer, Spain) and pEYFP-N1 (Clontech, Germany).

Cell culture and transfection

HEK293T cells (American Type Culture Collection, USA)

Acknowledgements

This work was supported by grants from the Swedish Research Council (04X-715), the Torsten and Ragnar Söderberg Foundation, the Hjärnfonden and Marianne and Marcus Wallenberg Foundation (to K.F.), and Ministerio de Ciencia e Innovación (SAF2008-01462 and Consolider-Ingenio CSD2R008-00005 to F.C.). A.O.T. did not receive any support for this work.

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