Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs.

Atypical antipsychotic drugs, such as clozapine and risperidone, have a high affinity for the serotonin 5-HT(2A) G protein-coupled receptor (GPCR), the 2AR, which signals via a G(q) heterotrimeric G protein. The closely related non-antipsychotic drugs, such as ritanserin and methysergide, also block 2AR function, but they lack comparable neuropsychological effects. Why some but not all 2AR inhibitors exhibit antipsychotic properties remains unresolved. We now show that a heteromeric complex between the 2AR and the G(i)-linked GPCR, metabotropic glutamate 2 receptor (mGluR2), integrates ligand input, modulating signaling output and behavioral changes. Serotonergic and glutamatergic drugs bind the mGluR2/2AR heterocomplex, which then balances Gi- and Gq-dependent signaling. We find that the mGluR2/2AR-mediated changes in Gi and Gq activity predict the psychoactive behavioral effects of a variety of pharmocological compounds. These observations provide mechanistic insight into antipsychotic action that may advance therapeutic strategies for disorders including schizophrenia and dementia.

Functional crosstalk and heteromerization of serotonin 5-HT2A and dopamine D2 receptors.

The serotonin 5-HT(2A) receptor (5-HT(2A)R) and dopamine D(2) receptor (D(2)R) are high-affinity G protein-coupled receptor targets for two different classes of antipsychotic drugs used to treat schizophrenia. Interestingly, the antipsychotic effects are not based on the regulation of same signaling mediators since activation of the 5-HT(2A)R and of the D(2)R regulate G(q/11) protein and G(i/o) protein, respectively. Here we use radioligand binding and second messenger production assays to provide evidence for a functional crosstalk between 5-HT(2A)R and D(2)R in brain and in HEK293 cells. D(2)R activation increases the hallucinogenic agonist affinity for 5-HT(2A)R and decreases the 5-HT(2A)R induced inositol phosphate production. In vivo, 5-HT(2A)R expression is necessary for the full effects of D(2)R antagonist on MK-801-induced locomotor activity. Co-immunoprecipitation studies show that the two receptors can physically interact in HEK293 cells and raise the possibility that a receptor heterocomplex mediates the crosstalk observed. The existence of this 5-HT(2A)R-D(2)R heteromer and crosstalk may have implications for diseases involving alterations of serotonin and dopamine systems and for the development of new classes of therapeutic drugs.

Time resolved FRET strategy with fluorescent ligands to analyze receptor interactions in native tissues: application to GPCR oligomerization.

G protein-coupled receptors (GPCRs) play a key role in the regulation of physiological functions. Deregulation of their activities often results in pathological disorders and therefore these receptors constitute major targets for drug development. The emergence of new concepts such as GPCR oligomerization has modified our understanding of these proteins, and identifying the role of receptor complexes is probably a major challenge for the next decade. Various experimental strategies have been developed to study GPCR oligomers and energy transfer experiments between partners within a complex constitute one of the most convenient approaches. These experimental strategies usually require receptor fusion to tags or fluorescent or luminescent proteins and therefore cannot be easily applied to native tissues. We developed a new experimental approach based on the labeling of receptors with high affinity fluorescent ligands compatible with time-resolved energy transfer measurements. Because of the very high signal-to-noise ratio of the time-resolved fluorescent energy transfer (TR-FRET) signals, this approach constitutes a breakthrough since it allows the direct identification of wild-type GPCR oligomers in native tissues.

Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists.

Hallucinogenic drugs, including mescaline, psilocybin and lysergic acid diethylamide (LSD), act at serotonin 5-HT2A receptors (5-HT2ARs). Metabotropic glutamate receptor 2/3 (mGluR2/3) ligands show efficacy in modulating the responses induced by activation of 5-HT2ARs. The formation of a 5-HT2AR-mGluR2 complex suggests a functional interaction that affects the hallucinogen-regulated cellular signaling pathways. Here, we tested the cellular and behavioral effects of hallucinogenic 5-HT2AR agonists in mGluR2 knockout (mGluR2-KO) mice. Mice were intraperitoneally injected with the hallucinogens DOI (2 mg/kg) and LSD (0.24 mg/kg), or vehicle. Head-twitch behavioral response, expression of c-fos, which is induced by all 5-HT2AR agonists, and expression of egr-2, which is hallucinogen-specific, were determined in wild type and mGluR2-KO mice. [(3)H]Ketanserin binding displacement curves by DOI were performed in mouse frontal cortex membrane preparations. Head twitch behavior was abolished in mGluR2-KO mice. The high-affinity binding site of DOI was undetected in mGluR2-KO mice. The hallucinogen DOI induced c-fos in both wild type and mGluR2-KO mice. However, the induction of egr-2 by DOI was eliminated in mGlu2-KO mice. These findings suggest that the 5-HT2AR-mGluR2 complex is necessary for the neuropsychological responses induced by hallucinogens.

Heteromerization of G protein-coupled receptors: relevance to neurological disorders and neurotherapeutics.

Because G protein-coupled receptors (GPCRs) are numerous, widely expressed and involved in major physiological responses, they represent a relevant therapeutic target for drug discovery, particularly regarding pharmacological treatments of neurological disorders. Among the biological phenomena regulating receptor function, GPCR heteromerization is an important emerging area of interest and investigation. There is increasing evidence showing that heteromerization contributes to the pharmacological heterogeneity of GPCRs by modulating receptor ontogeny, activation and recycling. Although in many cases the physiological relevance of receptor heteromerization has not been fully established, the unique pharmacological and functional properties of heteromers are likely to lead to new strategies in clinical medicine. This review describes the main GPCR heteromers and their implications for major neurological disorders such as Parkinson's disease, schizophrenia and addiction. A better understanding of molecular mechanisms underlying drug interactions related to the targeting of receptor heteromers could provide more specific and efficient therapeutic agents for the treatment of brain diseases.

Time-resolved FRET between GPCR ligands reveals oligomers in native tissues.

G protein-coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.

Differential coupling of the vasopressin V1b receptor through compartmentalization within the plasma membrane.

We show here that the rat vasopressin V(1b) receptor simultaneously activates both the G(q/11)-inositol phosphate (IP) and G(s)-cAMP pathways when transiently expressed in Chinese hamster ovary, human embryonic kidney (HEK) 293, and COS-7 cells and stimulated with arginine-vasopressin. Higher concentrations of the hormone, however, were needed to trigger the cAMP pathway. The nonmammalian analog arginine-vasotocin and the selective V(1b) agonist d[Cha(4)]vasopressin also activated the cAMP and IP pathways, although d[Cha(4)]-vasopressin elicited the two responses with equivalent potencies. We determined that the V(1b) receptor is present as a homodimer at the plasma membrane. Treatment of V(1b)-transfected HEK-293 cells with methyl-beta-cyclodextrin, a drug known to dissociate cholesterol-rich domains of the plasma membrane, shifted the EC(50) of the vasopressin-induced cAMP accumulation to lower concentrations and, remarkably, increased the hormone efficacy related to the activation of this second messenger system. In parallel, the vasopressin-mediated activation of the IP pathway was slightly reduced without modification of its EC(50). These results suggest that, as with many other G protein-coupled receptors, when transfected in heterologous cell systems, the V(1b) receptor forms dimers that signal differentially through the G(q/11) and G(s) proteins depending on the nature of the ligand as well as on its localization within specialized compartments of the plasma membrane. The present study thus illustrates how signal transduction associated with the activation of a G protein-coupled receptor can be versatile and highly dependent on both the cell context and the chemical nature of the extracellular signaling messenger.

Toward efficient drug screening by homogeneous assays based on the development of new fluorescent vasopressin and oxytocin receptor ligands.

A series of fluorescent ligands designed for vasopressin and oxytocin G protein-coupled receptors was synthesized and characterized to develop fluorescence polarization or homogeneous time-resolved fluorescence (HTRF) binding assays. These ligands, labeled with europium pyridine-bis-bipyridine cryptate or with Alexa 488,546,647 selectively bound to the vasopressin V1a and oxytocin receptors with high affinities and exhibited antagonistic properties. The affinities of several unlabeled ligands determined by our homogeneous assays on membrane preparations or on intact cells into 96- and 384-well plate formats were similar to those determined by usual radioligand binding methods. Compared to other binding assays, the polarization and HTRF binding assays are nonradiaoactive, therefore safer to perform, yet very sensitive and homogeneous, therefore easier and faster to automate. These methods are thus suitable for efficient drug high-throughput screening procedures and can easily be applied to other G protein-coupled receptor models.

Probing the existence of G protein-coupled receptor dimers by positive and negative ligand-dependent cooperative binding.

An increasing amount of ligand binding data on G protein-coupled receptors (GPCRs) is not compatible with the prediction of the simple mass action law. This may be related to the propensity of most GPCRs, if not all, to oligomerize. Indeed, one of the consequences of receptor oligomerization could be a possible cross-talk between the protomers, which in turn could lead to negative or positive cooperative ligand binding. We prove here that this can be demonstrated experimentally. Saturation, dissociation, and competition binding experiments were performed on vasopressin and oxytocin receptors expressed in Chinese hamster ovary or COS-7 cells. Linear, concave, and convex Scatchard plots were then obtained, depending on the ligand used. Moreover, some competition curves exhibited an increase of the radiotracer binding for low concentrations of competitors, suggesting a cooperative binding process. These data demonstrate that various vasopressin analogs display either positive or negative cooperative binding. Because positive cooperative binding cannot be explained without considering receptor as multivalent, these binding data support the concept of GPCR dimerization process. The results, which are in good accordance with the predictions of previous mathematical models, suggest that binding experiments can be used to probe the existence of receptor dimers.