TY - JOUR T1 - Ligand-Dependent Oligomerization of Dopamine D<sub>2</sub> and Adenosine A<sub>2A</sub> Receptors in Living Neuronal Cells JF - Molecular Pharmacology JO - Mol Pharmacol SP - 544 LP - 551 DO - 10.1124/mol.108.047472 VL - 74 IS - 3 AU - Pierre-Alexandre Vidi AU - Benjamin R. Chemel AU - Chang-Deng Hu AU - Val J. Watts Y1 - 2008/09/01 UR - http://molpharm.aspetjournals.org/content/74/3/544.abstract N2 - Adenosine A2A and dopamine D2 receptors (A2A and D2) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A2A and D2 oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A2A/A2A, D2/D2, and A2A/D2 BiFC pairs. Robust fluorescence complementation reflecting A2A/D2 heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A2A/dopamine D1 BiFC pairs. Multicolor BiFC was used to simultaneously visualize A2A and D2 homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D2 stimulation with quinpirole lead to the internalization of D2/D2 and A2A/D2 oligomers and resulted in decreased A2A/D2 relative to A2A/A2A oligomer formation. Opposing effects were observed in cells treated with D2 antagonists or with the A2A agonist 5′-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A2A/D2 receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization. ER -