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.

The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB 1 receptors, is still a matter of debate. In the present study, we report that CB 1 and adenosine A 2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB 1 receptor signaling was found to be completely dependent on A 2A receptor activation. Accordingly, blockade of A 2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB 1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A 2A and CB 1 receptors.

G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu5 receptors (mGlu5R) and dopamine D2 receptors (D2R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer (SRET) technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu5R, D2R and adenosine A2A receptor (A2AR). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu5R, D2R and A2AR in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu5R/D2R/A2AR oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

Adenosine A2A-dopamine D2 receptor interactions play a very important role in striatal function. A2A-D2 receptor interactions provide an example of the capabilities of information processing by just two different G protein-coupled receptors. Thus, there is evidence for the coexistence of two reciprocal antagonistic interactions between A2A and D2 receptors in the same neurons, the GABAergic enkephalinergic neurons. An antagonistic A2A-D2 intramembrane receptor interaction, which depends on A2A-D2 receptor heteromerization and Gq/11-PLC signaling, modulates neuronal excitability and neurotransmitter release. On the other hand, an antagonistic A2A-D2 receptor interaction at the adenylyl-cyclase level, which depends on Gs/olf-and Gi/o-type V adenylyl-cyclase signaling, modulates protein phosphorylation and gene expression. Finally, under conditions of upregulation of an activator of G protein signaling (AGS3), such as during chronic treatment with addictive drugs, a synergistic A2A-D2 receptor interaction can also be demonstrated. AGS3 facilitates a synergistic interaction between Gs/olf -and Gi/o-coupled receptors on the activation of types II/IV adenylyl cyclase, leading to a paradoxical increase in protein phosphorylation and gene expression upon co-activation of A2A and D2 receptors. The analysis of A2-D2 receptor interactions will have implications for the pathophysiology and treatment of basal ganglia disorders and drug addiction.Key Words: Adenosine A 2A Receptor, Dopamine D 2 Receptor, G Protein-Coupled Receptors, Receptor Heteromers, Striatum, Basal Ganglia Disorders, Drug Addiction. LOCALIZATION OF THE A 2A -D 2 RECEPTOR HETERO-MERApplying a broad definition of "neurotransmitter" [1], adenosine can be considered as an important neurotransmitter in the CNS, which acts through different subtypes of G protein-coupled receptors (GPCRs). From the four cloned adenosine receptors (adenosine A 1 , A 2A , A 2B and A 3 receptors ) , A 1 and A 2A receptors are the main targets for the physiological effects of adenosine in the brain [2]. A 1 receptor is widely distributed in the brain, including the striatum, while A 2A receptor is mostly concentrated in the striatum [2,3]. It is becoming increasingly obvious that the modulatory role of adenosine in the striatum is related to the ability of A 1 and A 2A receptors to heteromerize with themselves and with other GPCRs, such as dopamine, glutamate, cannabinoid and ATP receptors [4][5][6][7][8][9][10][11][12][13][14]. The present review focuses on the role of one particular adenosine receptor heteromer, the one constituted by the A 2A and the dopamine D 2 receptor, which is already having important implications for the treatment of neuropathologies involving the striatum (see below).Striatal medium spiny neurons are GABAergic efferent neurons which constitute more that 95% of the striatal neuronal population. They receive two main afferents, cortical-limbic-thalamic glutamatergic inputs and dopaminergic mesencephalic inputs, from the substantia nigra pars compac...

Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.

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.