Marie-Laure Rives

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.

Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

6′-Guanidinonaltrindole (6′-GNTI) Is a G Protein-biased κ-Opioid Receptor Agonist That Inhibits Arrestin Recruitment

Background: Arrestin recruitment to the κ-opioid receptor (KOR) has been linked to several adverse effects of analgesics, such as dysphoria and tolerance. Results: We identified 6′-guanidinonaltrindole (6′-GNTI) as a potent KOR agonist for G protein activation that fails to recruit arrestin. Conclusion: 6′-GNTI is an extreme G protein-biased KOR ligand. Significance: 6′-GNTI is a lead toward analgesics with fewer arrestin-mediated adverse effects. κ-Opioid receptor (KOR) agonists do not activate the reward pathway stimulated by morphine-like μ-opioid receptor (MOR) agonists and thus have been considered to be promising nonaddictive analgesics. However, KOR agonists produce other adverse effects, including dysphoria, diuresis, and constipation. The therapeutic promise of KOR agonists has nonetheless recently been revived by studies showing that their dysphoric effects require arrestin recruitment, whereas their analgesic effects do not. Moreover, KOR agonist-induced antinociceptive tolerance observed in vivo has also been proposed to be correlated to the ability to induce arrestin-dependent phosphorylation, desensitization, and internalization of the receptor. The discovery of functionally selective drugs that are therapeutically effective without the adverse effects triggered by the arrestin pathway is thus an important goal. We have identified such an extreme G protein-biased KOR compound, 6′-guanidinonaltrindole (6′-GNTI), a potent partial agonist at the KOR receptor for the G protein activation pathway that does not recruit arrestin. Indeed, 6′-GNTI functions as an antagonist to block the arrestin recruitment and KOR internalization induced by other nonbiased agonists. As an extremely G protein-biased KOR agonist, 6′-GNTI represents a promising lead compound in the search for nonaddictive opioid analgesic as its signaling profile suggests that it will be without the dysphoria and other adverse effects promoted by arrestin recruitment and its downstream signaling.

Discovery of a Novel Selective Kappa-Opioid Receptor Agonist Using Crystal Structure-Based Virtual Screening

Kappa-opioid (KOP) receptor agonists exhibit analgesic effects without activating reward pathways. In the search for nonaddictive opioid therapeutics and novel chemical tools to study physiological functions regulated by the KOP receptor, we screened in silico its recently released inactive crystal structure. A selective novel KOP receptor agonist emerged as a notable result and is proposed as a new chemotype for the study of the KOP receptor in the etiology of drug addiction, depression, and/or pain.

The atypical antidepressant and neurorestorative agent tianeptine is a μ-opioid receptor agonist

Current pharmacological treatments of depression and related disorders suffer from major problems, such as a low rate of response, slow onset of therapeutic effects, loss of efficacy over time and serious side effects. Therefore, there is an urgent need to explore new therapeutic approaches that address these issues. Interestingly, the atypical antidepressant tianeptine already meets in part these clinical goals. However, in spite of three decades of basic and clinical investigations, the molecular target of tianeptine, as well as its mechanism of action, remains elusive. Herein, we report the characterization of tianeptine as a μ-opioid receptor (MOR) agonist. Using radioligand binding and cell-based functional assays, including bioluminescence resonance energy transfer-based assays for G-protein activation and cAMP accumulation, we identified tianeptine as an efficacious MOR agonist (Ki Human of 383±183 nM and EC50 Human of 194±70 nM  and EC50 Mouse of 641±120 nM for G-protein activation). Tianeptine was also a full δ-opioid receptor (DOR) agonist, although with much lower potency (EC50 Human of 37.4±11.2 μM and EC50 Mouse of 14.5±6.6  μM for G-protein activation). In contrast, tianeptine was inactive at the κ-opioid receptor (KOR, both human and rat). On the basis of these pharmacological data, we propose that activation of MOR (or dual activation of MOR and DOR) could be the initial molecular event responsible for triggering many of the known acute and chronic effects of this agent, including its antidepressant and anxiolytic actions.

Sensing conformational changes in metabotropic glutamate receptors

G protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and are targets of more than 25% of the medications currently on the market. Metabotropic glutamate receptors (mGluRs), family C GPCRs, modulate synaptic transmission and neuronal excitability throughout the central nervous system and thus are promising targets for the treatment of various neurological and psychiatric disorders (1). Understanding the detailed activation mechanisms of mGluRs is therefore crucial to the development of new therapeutics. Although extensively studied over the last decade, the conformational changes involved in receptor activation are still a matter of debate (2). In PNAS, Doumazane et al. (3) describe how they have developed an innovative FRET-based approach to monitor these conformational changes upon ligand binding. Their assay not only helps to resolve controversy regarding the conformational changes involved in receptor activation but also facilitates our understanding of the mode of action of allosteric modulators. This approach therefore provides a powerful tool for drug screening.

Chapter 12:Crosstalk Between Receptors: Challenges of Distinguishing Upstream from Downstream Mechanisms

G protein-coupled receptors (GPCRs) constitute one of the largest families of cell surface proteins that initiate intracellular signalling in response to a diverse array of ligands and play a vital role in cell–cell communication as well as smell, taste and vision. Considerable evidence suggests that GPCRs form homomeric and heteromeric complexes that can lead to novel pharmacological properties. Although receptor coexpression can lead to signal integration and crosstalk, the underlying molecular mechanisms are still not well understood. In this chapter we discuss the various signalling possibilities that result from receptor coexpression, as well as novel approaches that combine the power of protein complementation and resonance energy transfer to allow us to study individual components of a GPCR signalling unit.