Ligand-dependent oligomerization of dopamine D(2) and adenosine A(2A) receptors in living neuronal cells.
Adenosine A(2A) and dopamine D(2) receptors (A(2A) and D(2)) 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 A(2A) and D(2) oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A(2A)/A(2A), D(2)/D(2), and A(2A)/D(2) BiFC pairs. Robust fluorescence complementation reflecting A(2A)/D(2) 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 A(2A)/dopamine D(1) BiFC pairs. Multicolor BiFC was used to simultaneously visualize A(2A) and D(2) homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D(2) stimulation with quinpirole lead to the internalization of D(2)/D(2) and A(2A)/D(2) oligomers and resulted in decreased A(2A)/D(2) relative to A(2A)/A(2A) oligomer formation. Opposing effects were observed in cells treated with D(2) antagonists or with the A(2A) 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 A(2A)/D(2) receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.
Dept. of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA.
Pierre-Alexandre
PA
Vidi
Benjamin R
BR
Chemel
Chang-Deng
CD
Hu
Val J
VJ
Watts
D002470:Cell Survival; D002478:Cells, Cultured; D005453:Fluorescence; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D008024:Ligands; D043705:Receptor, Adenosine A2A; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins