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.

The results presented in this paper show that adenosine A 2A receptor (A 2A R) 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 A 2A R, 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 A 2A R-A 2A R 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 A 2A 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 A 2A R is involved in heteromers formed by A 2A R and dopamine D2 receptors. BRET ratios corresponding to A 2A R-A 2A R homodimers were higher than those encountered for heterodimers formed by A 2A R and dopamine D2 receptors. As A 2A R and dopamine D2 receptors do indeed interact, these results indicate that A 2A R homodimers are the functional species at the cell surface and that they coexist with A 2A R/D2 receptor heterodimers. Keywords: 7TM receptors, dopamine receptors, G proteincoupled receptors, heteromerization, homomerization, receptor-receptor interactions. Heptaspanning membrane receptors (HSMRs) also known as G protein-coupled receptors (GPCRs) were previously considered to be monomeric proteins which only interacted with G proteins. However it has become clear that HSMRs are oligomeric structures formed by receptor homodimers, heterodimers and multimers and a variety of proteins interacting at the horizontal and the vertical level (Agnati et al. 2002Bouvier, 2001;Milligan and White 2001;Rios et al. 2001;Franco et al. 2003

This work aimed to evaluate the potential role of the 5-HT(7) receptor in nociception secondary to a sensitizing stimulus in mice. For this purpose, the effects of relevant ligands (5-HT(7) receptor agonists: AS-19, MSD-5a, E-55888; 5-HT(7) receptor antagonists: SB-258719, SB-269970; 5-HT(1A) receptor agonist: F-13640; 5-HT(1A) receptor antagonist: WAY-100635) were assessed on capsaicin-induced mechanical hypersensitivity, a pain behavior involving hypersensitivity of dorsal horn neurons (central sensitization). For the 5-HT(7) receptor agonists used, binding profile and intrinsic efficacy to stimulate cAMP formation in HEK-293F cells expressing the human 5-HT(7) receptor were also evaluated. AS-19 and E-55888 were selective for 5-HT(7) receptors. E-55888 was a full agonist whereas AS-19 and MSD-5a behaved as partial agonists, with maximal effects corresponding to 77% and 61%, respectively, of the cAMP response evoked by the full agonist 5-HT. Our in vivo results revealed that systemic administration of 5-HT(7) receptor agonists exerted a clear-cut dose-dependent antinociceptive effect that was prevented by 5-HT(7) receptor antagonists, but not by the 5-HT(1A) receptor antagonist. The order of efficacy (E-55888>AS-19>MSD-5a) matched their in vitro efficacy as 5-HT(7) receptor agonists. Contrary to agonists, a dose-dependent promotion of mechanical hypersensitivity was observed after administration of 5-HT(7) receptor antagonists, substantiating the involvement of the 5-HT(7) receptor in the control of capsaicin-induced mechanical hypersensitivity. These findings suggest that serotonin exerts an inhibitory role in the control of nociception through activation of 5-HT(7) receptors, and point to a new potential therapeutic use of 5-HT(7) receptor agonists in the field of analgesia.

Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo-and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G proteincoupled receptors, namely the metabotropic glutamate type 1␣ (mGlu 1␣ receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu 1␣ and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu 1␣ and A1 receptors in receptorevoked [Ca 2؉ ] i signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu 1␣ and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.Glutamate is the major excitatory neurotransmitter in the central nervous system (1), and its function through ionotropic and metabotropic (mGlu) 1 glutamate receptors can be modulated by other neurotransmitters/neuromodulators (2). Eight members of the mGlu receptor family have been identified and categorized into three subgroups on the basis of their sequence homology, agonist selectivity, and signal transduction pathway. Group I contains mGlu 1 and mGlu 5 subtypes, which are coupled to phospholipase C in transfected cells, and have quisqualic acid as their most potent agonist. Five splice variants of mGlu1 receptor have been described, mGlu 1␣ , mGlu 1␤ , mGlu 1c , mGlu 1d , and mGlu 1e receptors (3, 4), all of them differing in the length of their C-terminal tail. The functional significance of the different splice variants has not yet been fully explored. It has been suggested that the C-terminal tail, which is intracellular, might play a role in the subcellular targeting of the receptor (5). Recently, we have reported that the C terminus of mGlu 1␣ receptor interacts with tubulin (6) and that it can regulate the cell surface expression of the receptor (7) and its plasma membrane anchoring (8, 9).Adenosine is an important neuromodulator implicated in a variety of brain activities, particularly those related to sleep and ischemic-hypoxic episodes (10). This ubiquitous nucleoside exerts its actions via specific receptors, four of which (A1, A2A, A2B, and A3) have been cloned (11). The A1R is functionally coupled to members of the pertussis toxin-sensitive family of G proteins (G i1 , G i2 , G i3 , and G o ), and its activation regulates several membrane and intracellular proteins such as adenylate cyclase, Ca 2ϩ channels, K ϩ channels, and phospholipase C (11). Of the multiple neurophys...

The synthesis and pharmacological activity of a new series of 1-arylpyrazoles as potent σ(1) receptor (σ(1)R) antagonists are reported. The new compounds were evaluated in vitro in human σ(1)R and guinea pig σ(2) receptor (σ(2)R) binding assays. The nature of the pyrazole substituents was crucial for activity, and a basic amine was shown to be necessary, in accordance with known receptor pharmacophores. A wide variety of amines and spacer lengths between the amino and pyrazole groups were tolerated, but only the ethylenoxy spacer and small cyclic amines provided compounds with sufficient selectivity for σ(1)R vs σ(2)R. The most selective compounds were further profiled, and compound 28, 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862), which showed high activity in the mouse capsaicin model of neurogenic pain, emerged as the most interesting candidate. In addition, compound 28 exerted dose-dependent antinociceptive effects in several neuropathic pain models. This, together with its good physicochemical, safety, and ADME properties, led compound 28 to be selected as clinical candidate.