Homodimerization of adenosine A2A receptors: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

J Neurochem. 2004 Feb;88(3):726-34. doi: 10.1046/j.1471-4159.2003.02200.x.

Abstract

The results presented in this paper show that adenosine A2A receptor (A2AR) form homodimers and that homodimers but not monomers are the functional species at the cell surface. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques have been used to demonstrate in transfected HEK293 cells homodimerization of A2AR, which are heptaspanning membrane receptors with enriched expression in striatum. The existence of homodimers at the cell surface was demonstrated by time-resolved FRET. Although agonist activation of the receptor leads to the formation of receptor clusters, it did not affect the degree of A2AR-A2AR dimerization. Both monomers and dimers were detected by immunoblotting in cell extracts. However, cell surface biotinylation of proteins has made evident that more than 90% of the cell surface receptor is in its dimeric form. Thus, it seems that homodimers are the functional form of the receptor present on the plasma membrane. A deletion mutant version of the A2A receptor, lacking its C-terminal domain, was also able to form both monomeric and dimeric species when cell extracts from transfected cells were analyzed by immunoblotting. This suggests that the C-terminal tail does not participate in the dimerization. This is relevant as the C-terminal tail of A2AR is involved in heteromers formed by A2AR and dopamine D2 receptors. BRET ratios corresponding to A2AR-A2AR homodimers were higher than those encountered for heterodimers formed by A2AR and dopamine D2 receptors. As A2AR and dopamine D2 receptors do indeed interact, these results indicate that A2AR homodimers are the functional species at the cell surface and that they coexist with A2AR/D2 receptor heterodimers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cyclodextrins / chemistry
  • Dimerization
  • Fluorescence Resonance Energy Transfer / methods*
  • HeLa Cells
  • Humans
  • Luminescent Measurements
  • Receptor, Adenosine A2A / chemistry*
  • Receptor, Adenosine A2A / metabolism*
  • beta-Cyclodextrins*

Substances

  • Cyclodextrins
  • Receptor, Adenosine A2A
  • beta-Cyclodextrins
  • betadex