Most evidence indicates that, as for family C G protein–coupled receptors (GPCRs), family A GPCRs form homo- and heteromers. Homodimers seem to be a predominant species, with potential dynamic formation of higher-order oligomers, particularly tetramers. Although monomeric GPCRs can activate G proteins, the pentameric structure constituted by one GPCR homodimer and one heterotrimeric G protein may provide a main functional unit, and oligomeric entities can be viewed as multiples of dimers. It still needs to be resolved if GPCR heteromers are preferentially heterodimers or if they are mostly constituted by heteromers of homodimers. Allosteric mechanisms determine a multiplicity of possible unique pharmacological properties of GPCR homomers and heteromers. Some general mechanisms seem to apply, particularly at the level of ligand-binding properties. In the frame of the dimer-cooperativity model, the two-state dimer model provides the most practical method to analyze ligand–GPCR interactions when considering receptor homomers. In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to different intrinsic efficacy of ligands for different signaling pathways (functional selectivity). This gives a rationale for the use of GPCR oligomers, and particularly heteromers, as novel targets for drug development. Herein, we review the functional and pharmacological properties of GPCR oligomers and provide some guidelines for the application of discrete direct screening and high-throughput screening approaches to the discovery of receptor-heteromer selective compounds.

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A₁receptors (A₁Rs) and A_2Areceptors (A_2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A₁R–A_2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A₁R and A_2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A₁R–A_2AR heteromer is the ability of A_2AR activation to reduce the affinity of the A₁R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A₁R–A_2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A₁R–A_2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

Antagonistic and reciprocal interactions are known to exist between adenosine and dopamine receptors in the striatum. In the present study, double immunofluorescence experiments with confocal laser microscopy showed a high degree of colocalization of adenosine A_2A receptors (A_2AR) and dopamine D₂ receptors (D₂R) in cell membranes of SH-SY5Y human neuroblastoma cells stably transfected with human D₂R and in cultured striatal cells. A_2AR/D₂R heteromeric complexes were demonstrated in coimmunoprecipitation experiments in membrane preparations from D₂R-transfected SH-SY5Y cells and from mouse fibroblast Ltk⁻ cells stably transfected with human D₂R (long form) and transiently cotransfected with the A_2AR double-tagged with hemagglutinin. Long term exposure to A_2AR and D₂R agonists in D₂R-cotransfected SH-SY5Y cells resulted in coaggregation, cointernalization and codesensitization of A_2AR and D₂R. These results give a molecular basis for adenosine-dopamine antagonism at the membrane level and have implications for treatment of Parkinson's disease and schizophrenia, in which D₂R are involved.

There is evidence for strong functional antagonistic interactions between adenosine A_2A receptors (A_2ARs) and dopamine D₂ receptors (D₂Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A_2AR-D₂R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A_2AR-D₂R interactions in co-transfected cells. The degree of A_2AR-D₂R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D₂R-D₁R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D₂R were replaced by those of the dopamine D₁ receptor (D₁R). Although the wild type D₂R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D₂R holds the site(s) for interaction with A_2AR. Modeling of A_2AR and D₂R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D₂R and A_2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D₂R approached helix 4 and the C-terminal portion of the C-tail from the A_2AR, respectively.