G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html.

The adenosine A_2A receptor (A_2AR), dopamine D₂ receptor (D₂R) and metabotropic glutamate receptor type 5 (mGluR₅) form A_2AR-D₂R-mGluR₅ heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A_2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D₂R-mGluR₅ heteromeric component of the A_2AR-D₂R-mGluR₅ complex in vitro and in vivo. The A_2AR and mGluR₅ protomers interact and modulate D₂R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A_2AR in HEK293T cells co-expressing D₂R and mGluR₅ resulted in a significant and marked increase in the formation of the D₂R-mGluR₅ heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D₂R (D2R_{Ki High)} values was found upon cotreatment with the mGluR₅ and A_2AR agonists in the cells expressing A_2AR, D₂R and mGluR₅ with a significant effect observed also with the mGluR₅ agonist alone compared to cells expressing only D₂R and mGluR₅. In cells co-expressing A_2AR, D₂R and mGluR₅, stimulation of the cells with an mGluR₅ agonist like or D₂R antagonist fully counteracted the D₂R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR₅ and D₂R. In agreement, the mGluR₅-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A_2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR₅ and A_2AR in enhancing D₂R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.

Cocaine was previously shown to act at the Sigma1R which is a target for counteracting cocaine actions. It therefore becomes of interest to test if the monoamine stabilizer (–) OSU-6162 (OSU-6162) with a nanomolar affinity for the Sigma1R can acutely modulate in low doses the effects of cocaine self-administration. In behavioral studies, OSU-6162 (5 mg/kg, s.c.) did not significantly change the number of active lever pressing and cocaine infusions. However, a trend to reduce cocaine readouts was found after 3 days of treatment. In contrast, in maintenance of cocaine self-administration, the proximity ligation assay performed on brains from rats pretreated with OSU-6162 showed highly significant increases in the density of the D2R-Sigma1R heteroreceptor complexes in the shell of the nucleus accumbens versus OSU-6162 induced increases in this region of yoked saline rats. In cocaine self-administration, highly significant increases were also induced by OSU-6162 in the A2AR-D2R heteroreceptor complexes in the nucleus accumbens shell versus vehicle-treated rats. Furthermore, ex vivo, the A2AR agonist CGS21680 (100 nM) produced a marked and significant increase of the D2R Ki high values in the OSU-6162-treated versus vehicle-treated rats under maintenance of cocaine self-administration. These results indicate a substantial increase in the inhibitory allosteric A2AR-D2R interactions following cocaine self-administration upon activation by the A2AR agonist ex vivo. The current results indicate that OSU-6162 via its high affinity for the Sigma1R may increase the number of accumbal shell D2R-Sigma1R and A2AR-D2R heteroreceptor complexes associated with further increases in the antagonistic A2AR-D2R interactions in cocaine self-administration.

It was previously demonstrated that rat adenosine A2AR transmembrane V peptide administration into the nucleus accumbens enhances cocaine self-administration through disruption of the A2AR-dopamine (D2R) heteroreceptor complex of this region. Unlike human A2AR transmembrane 4 (TM4) and 5 (TM5), A2AR TM2 did not interfere with the formation of the A2AR-D2R heteroreceptor complex in cellular models using BRET¹ assay. A2AR TM2 was proposed to be part of the of the receptor interface of the A2AR homomer instead and was therefore tested in the current article for effects on rat cocaine self-administration using rat A2AR synthetic TM2 peptide bilaterally injected into the nucleus accumbens. The injected A2AR TM2 peptide failed to significantly counteract the inhibitory action of the A2AR agonist CGS 21680 (0.1 mg/Kg) on cocaine self-administration. In line with these results, the microinjected A2AR TM2 peptide did not reduce the number of proximity ligation assay blobs identifying A2AR-D2R heteroreceptor complexes in the nucleus accumbens. In contrast, the A2AR TM2 peptide significantly reduced the number of A2AR-A2AR homoreceptor complexes in the nucleus accumbens. As to effects on the receptor–receptor interactions in the A2AR-D2R heteroreceptor complexes, the A2AR TM2 peptide did not alter the significant increase in the D2R Ki, high values produced by the A2AR agonist CGS 21680 ex vivo in the ventral striatum. The results indicate that the accumbal A2AR-A2AR homomeric complexes are not involved in mediating the A2AR agonist-induced inhibition of cocaine self-administration.

Background: Antagonistic adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) receptor–receptor interactions have previously been demonstrated in A2AR–D2R heteroreceptor complexes in the rat dorsal striatum. They mainly involve a reduction of affinity in the high-affinity component of the D2R agonist binding site upon activation in vivo of the A2AR by an A2AR agonist. Upon cocaine self-administration, this antagonistic A2AR–D2R interaction disappeared in the dorsal striatum.Methods: In the current experiments, it was tested whether such modifications in the antagonistic A2AR–D2R receptor–receptor interactions can develop also after an acute systemic injection of a low cocaine dose (1 mg/kg; sc).Results: Microdialysis experiments indicated that acute cocaine did not significantly alter the extracellular dopamine levels in the dorsal striatum of the awake Wistar rats. Competition dopamine receptor binding experiments demonstrated that in the acute cocaine group, the A2AR agonist CGS-21680 produced significantly larger increases in the D2RK_{i, High}values (reduction of high-affinity) versus the saline-injected (i.e. control) group. Furthermore, in the dorsal striatum membrane preparation from acute cocaine-injected rats, CGS-21680 also produced significant increases in the D2RK_{i, Low}values (reduction of low-affinity) and in the proportion of D2Rs in the high-affinity state (RH). Such significant effects were not observed with CGS-21680 in the control group.Conclusions: The molecular mechanism involved in the acute cocaine-induced increase in the antagonistic allosteric A2AR–D2R receptor–receptor interactions may be an increased formation of higher-order complexes A2AR–D2R-sigma1R in which cocaine by binding to the sigma1R protomer also allosterically enhances the inhibitory A2AR–D2R interaction in this receptor complex.