The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that

The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic glutamate receptors in ␥-aminobutyric acid (GABA)ergic striatal neurons is intriguing. Here we provide in vitro and in vivo evidence for a synergism between adenosine and glutamate based on subtype 5 metabotropic glutamate (mGluR5) and adenosine A2A ( A denosine is a neuromodulator that plays a very important role in basal ganglia function (1). Its actions are mediated by specific G protein-coupled receptors, which are currently classified in A1, A2A, A2B, and A3 subtypes (2). Compared with the other adenosine receptor subtypes, A2A receptors (A2ARs) are concentrated in the striatum (1, 3), where they are expressed mostly by ␥-aminobutyric acid (GABA)ergic striatopallidal neurons (4). The recent ultrastructural analysis performed by Hettinger et al. (5) has demonstrated that, in the rat, A2ARs are localized mostly postsynaptically in the dendrites and dendritic spines of striatal GABAergic neurons. A2AR immunoreactivity was observed primarily at glutamatergic (asymmetric) synapses (5). Therefore, it was suggested that A2AR plays a prominent role in modulating glutamatergic input to striatal GABAergic neurons (5).Glutamate acts on both ionotropic and metabotropic G protein-coupled receptors (mGluRs). Molecular and pharmacological characterization studies have currently divided the mGluR family into three groups (I-III) (6). Group I mGluR includes mGluR1 and mGluR5, with the latter being highly expressed in the striatum, particularly in the striatal GABAergic efferent neurons (7). In the striatopallidal complex in primates, mGluR5 showed a localization very similar to that described for A2AR in rats. Thus, mGluR5 immunoreactivity was commonly found postsynaptically and perisynaptically to asymmetric synapses (8). These studies provide a morphological basis for the possible existence of functional interactions between striatal A2AR and mGluR5. In fact, in recent in vivo microdialysis experiments we found functional evidence for the possible existence of synergistic A2AR͞mGlluR5 interactions modulating the function of the GABAergic striatopallidal neurons originating in the nucleus accumbens (9). In the present study we provide evidence for the existence of A2AR͞mGluR5 heteromeric complexes in membrane preparations from human embryonic kidney (HEK)-293 cells transiently cotransfected with both receptors and from rat striatum. Furthermore, the same kind of functional A2AR͞mGluR5 synergistic interaction (induction of the immediate-early gene c-fos) could be demonstrated both in cotransfected cells and the rat striatum. These results suggest that A2AR͞mGluR5 synergistic interactions can have important implications for striatal neuronal function and dysfunction.

These findings contribute to evidence identifying the σ(1) receptor as a modulator of activity-induced spinal sensitization and pain hypersensitivity, and suggest σ(1) receptor antagonists as potential novel treatments for neuropathic pain.

Previous results from FRET and BRET experiments and computational analysis (docking simulations) have suggested that a portion of the third intracellular loop (I3) of the human dopamine D2 receptor (D2R) and the C-tail from the human adenosine A2A receptor (A2AR) are involved in A2AR-D2R heteromerization. The results of the present studies, using pull-down and mass spectrometry experiments, suggest that A2AR-D2R heteromerization depends on an electrostatic interaction between an Arg-rich epitope from the I3 of the D2R (217RRRRKR222) and two adjacent Asp residues (DD401-402) or a phosphorylated Ser (S374) residue in the C-tail of the A2AR. A GST-fusion protein containing the C-terminal domain of the A2AR (GST-A2ACT) was able to pull down the whole D2R solubilized from D2R-tranfected HEK-293 cells. Second, a peptide corresponding to the Arg-rich I3 region of the D2R (215VLRRRRKRVN224) and bound to Sepharose was able to pull down both GST-A2ACT and the whole A2AR solubilized from A2AR-tranfected HEK-293 cells. Finally, mass spectometry and pull-down data showed that the Arg-rich D2R epitope binds to two different epitopes from the C-terminal part of the A2AR, containing the two adjacent Asp residues or the phosphorylated Ser residue (388HELKGVCPEPPGLDDPLAQDGAVGS412 and 370SAQEpSQGNT378). The present results are the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization, a new, expanding area of protein-protein interactions.