QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: mol.108.047472v1 74/3/544    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Vidi, P.-A. Articles by Watts, V. J. PubMed PubMed Citation Articles by Vidi, P.-A. Articles by Watts, V. J.

Accelerated Communication

Ligand-Dependent Oligomerization of Dopamine D₂ and Adenosine A_2A Receptors in Living Neuronal Cells

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana

Adenosine A_2A and dopamine D₂ receptors (A_2A 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 A_2A 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 A_2A/A_2A, D₂/D₂, and A_2A/D₂ BiFC pairs. Robust fluorescence complementation reflecting A_2A/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 A_2A/dopamine D₁ BiFC pairs. Multicolor BiFC was used to simultaneously visualize A_2A 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 A_2A/D₂ oligomers and resulted in decreased A_2A/D₂ relative to A_2A/A_2A oligomer formation. Opposing effects were observed in cells treated with D₂ 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/D₂ receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.

Address correspondence to: Val J. Watts, Dept. of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907. E-mail: wattsv{at}purdue.edu.