Adenosine A 2A receptor (A 2A R)-dopamine D 2 receptor (D 2 R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A 2A R-D 2 R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D 2 R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A 2A R agonists, but also A 2A R antagonists, decrease the affinity and intrinsic efficacy of D 2 R agonists and the affinity of D 2 R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A 2A R-D 2 R heteromers as heterotetramers, constituted by A 2A R and D 2 R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence com- Most evidence indicates that G protein-coupled receptors (GPCRs) form homodimers and heteromers. Homodimers seem to be a predominant species, and oligomeric entities can be viewed as multiples of dimers (1). It has been proposed that GPCR heteromers are constituted mainly by heteromers of homodimers (1, 2). Allosteric mechanisms determine a multiplicity of unique pharmacologic properties of GPCR homodimers and heteromers (1, 3). First, binding of a ligand to one of the receptors in the heteromer can modify the affinity of ligands for the other receptor (1, 3, 4). The most widely reproduced allosteric modulation of ligand-binding properties in a GPCR heteromer is the ability of adenosine A 2A receptor (A 2A R) agonists to decrease the affinity of dopamine D 2 receptor (D 2 R) agonists in the A 2A R-D 2 R heteromer (5). A 2A R-D 2 R heteromers have been revealed both in transfected cells (6, 7), striatal neurons in culture (6,8) and in situ, in mammalian striatum (9, 10), where they play an important role in the modulation of GABAergic striatopallidal neuronal function (9, 11).In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to the varying intrinsic efficacy of ligands for different signaling pathways (1-3). Intrinsic efficacy refers to the power of the agonist to induce a functional response, independent of its affinity for the receptor. Thus, allosteric modulation of an agonist can potentially involve changes in affinity and/or intrinsic efficacy (1, 3). This principle can be observed in the A 2A R-D 2 R heteromer, where a decrease in D 2 R agonist affinity cannot alone explain the ability of an A 2A R agonist to abolish the decreased excitability of GABAergic striatopallidal neurons induced by high concentration of a D 2 R agonist (9), which should overcome the decrease in affinity. Furthermore, a differential effect of allosteric modulations of different agonist-mediated signaling responses (i.e., functional selectivity) can occur within GPCR heteromers (1, 2, 8 It has been hypothesized that the allos...

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.

It is well known that cocaine blocks the dopamine transporter. This mechanism should lead to a general increase in dopaminergic neurotransmission, and yet dopamine D 1 receptors (D 1 Rs) play a more significant role in the behavioral effects of cocaine than the other dopamine receptor subtypes. Cocaine also binds to σ-1 receptors, the physiological role of which is largely unknown. In the present study, D 1 R and σ 1 R were found to heteromerize in transfected cells, where cocaine robustly potentiated D 1 R-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK activation induced by D 1 R stimulation in a dopamine transporterindependent and σ 1 R-dependent manner. Some of these effects were also demonstrated in murine striatal slices and were absent in σ 1 R KO mice, providing evidence for the existence of σ 1 R-D 1 R heteromers in the brain. Therefore, these results provide a molecular explanation for which D 1 R plays a more significant role in the behavioral effects of cocaine, through σ 1 R-D 1 R heteromerization, and provide a unique perspective toward understanding the molecular basis of cocaine addiction.receptor heteromer | drug addiction A key molecular mechanism contributing to the development of addiction by drugs of abuse consist of the increase of the extracellular levels of dopamine in the striatum, particularly in its ventral portion, the nucleus accumbens (1, 2). Cocaine causes a rapid and strong increase in striatal extracellular dopamine by its ability to bind with high affinity to the dopamine transporter (DAT) and to inhibit its function (3-5). In the striatum, dopamine signaling is mediated mainly by dopamine D 1 and D 2 receptors (D 1 Rs and D 2 Rs, respectively), which are mostly segregated in two phenotypically different subtypes of GABAergic medium-sized spiny neurons (MSNs) (6). Activation of D 1 Rs is an absolute requirement for the induction of many of the cellular and behavioral responses to cocaine, as deduced from studies performed in D 1 R KO mice and from experiments with transgenic mice in which D 1 R-or D 2 R-expressing MSNs are visualized by the expression of fluorescent proteins (7-11).The σ-1 receptor, originally proposed as a subtype of opioid receptors, is now considered to be a nonopioid receptor with two transmembrane domains, one extracellular loop and cytosolic N and C termini (12). The σ 1 R is highly expressed in the brain, including the striatum, and its association with neurons is well established (12, 13). However, its biological function and even its main endogenous neurotransmitter remain enigmatic (12). Cocaine interacts with σ 1 Rs at pharmacologically relevant concentrations (12,14). In fact, reducing brain σ 1 R levels with antisense oligonucleotides attenuates the convulsive and locomotor stimulant actions of cocaine (15, 16), and σ 1 R antagonists mitigate the actions of cocaine in animal models (12,14). A recent study showed that σ 1 R agonists not only potentiate the reinforcing effects of cocaine, but they may be self...

Striatal adenosine A2A receptors (A2ARs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D2 receptors (D2Rs). A2ARs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A1 receptors (A1Rs). It has been hypothesized that postsynaptic A2AR antagonists should be useful in Parkinson's disease, while presynaptic A2AR antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A2AR antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261) showed no clear preference. Radioligand-binding experiments were performed in cells expressing A2AR-D2R and A1R-A2AR heteromers to determine possible differences in the affinity of these compounds for different A2AR heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A2AR when co-expressed with D2R than with A1R. KW-6002 showed the best relative affinity for A2AR co-expressed with D2R than co-expressed with A1R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile. On the basis of their preferential pre- versus postsynaptic actions, SCH-442416 and KW-6002 may be used as lead compounds to obtain more effective antidyskinetic and antiparkinsonian compounds, respectively.