Thierry Durroux

Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput–compatible format. Using this approach, we examined whether G protein–coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers—a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and γ-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABAB receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.

Building a new conceptual framework for receptor heteromers

Receptor heteromers constitute a new area of research that is reshaping our thinking about biochemistry, cell biology, pharmacology and drug discovery. In this commentary, we recommend clear definitions that should facilitate both information exchange and research on this growing class of transmembrane signal transduction units and their complex properties. We also consider research questions underlying the proposed nomenclature, with recommendations for receptor heteromer identification in native tissues and their use as targets for drug development.

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.

Peptide and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents☆

Oxytocin (OT) and vasopressin (AVP) mediate their biological actions by acting on four known receptors: The OT (uterine) and the AVP V(1a) (vasopressor), V(1b) (pituitary), V(2) (renal) receptors and a fifth putative AVP V(1c)? (vasodilating) receptor. This presentation will summarize some highlights of the recent progress, in the design and synthesis of selective peptide agonists, antagonists, radioiodinated ligands, fluorescent ligands and bivalent ligands for these receptors. Here we present published and unpublished pharmacological data on the most widely used agonists, antagonists and labelled ligands. The pharmacological properties of promising new selective OT antagonists and V(1b) agonists are also presented. This review should serve as a useful guide for the selection of the most appropriate ligand for a given study. The current status of non-peptide OT and AVP antagonists and agonists is also summarized. The relative merits of peptide and non-peptide AVP and OT agonists and antagonists as: (1) research tools and (2) therapeutic agents will be evaluated. Many of the receptor selective peptide agonists and antagonists from this and other laboratories are far more widely used as pharmacological tools for studies on the peripheral and central effects of OT and AVP than their non-peptide counterparts. In addition to OT and to a lesser extent AVP (pitressin), a number of OT and AVP analogues; such as carbetocin (OT agonist) dDAVP (desmopressin, V(2) agonist), terlipressin (V(1a) agonist), felypressin (V(1a) agonist) and atosiban (Tractocile OT antagonist) are also in clinical use. Despite much early promise, no non-peptide V(1a) or OT antagonists are currently in clinical trials. While a number of orally active non-peptide V(2) antagonists (Vaptans); notably, Tolvaptan, Lixivaptan and Satavaptan, are currently in Phase III clinical trials; to date, only the mixed V(2)/V(1a), antagonist Conivaptan (Vaprisol), has been approved by the US FDA for clinical use (by i.v. administration), for the treatment of euvolemic and hypervolemic hyponatremia in hospitalized patients. Promising new non-peptide V(1b) and OT antagonists, as well as non-peptide V(2) and OT agonists are now in pre-clinical development.

Oxytocin and Vasopressin Agonists and Antagonists as Research Tools and Potential Therapeutics

We recently reviewed the status of peptide and nonpeptide agonists and antagonists for the V1a, V1b and V2 receptors for arginine vasopressin (AVP) and the oxytocin receptor for oxytocin (OT). In the present review, we update the status of peptides and nonpeptides as: (i) research tools and (ii) therapeutic agents. We also present our recent findings on the design of fluorescent ligands for V1b receptor localisation and for OT receptor dimerisation. We note the exciting discoveries regarding two novel naturally occurring analogues of OT. Recent reports of a selective VP V1a agonist and a selective OT agonist point to the continued therapeutic potential of peptides in this field. To date, only two nonpeptides, the V2/V1a antagonist, conivaptan and the V2 antagonist tolvaptan have received Food and Drug Administration approval for clinical use. The development of nonpeptide AVP V1a, V1b and V2 antagonists and OT agonists and antagonists has recently been abandoned by Merck, Sanofi and Pfizer. A promising OT antagonist, Retosiban, developed at Glaxo SmithKline is currently in a Phase II clinical trial for the prevention of premature labour. A number of the nonpeptide ligands that were not successful in clinical trials are proving to be valuable as research tools. Peptide agonists and antagonists continue to be very widely used as research tools in this field. In this regard, we present receptor data on some of the most widely used peptide and nonpeptide ligands, as a guide for their use, especially with regard to receptor selectivity and species differences.

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

A Fluorescent Ligand-Binding Alternative Using Tag-lite® Technology

G-protein-coupled receptors (GPCRs) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands. Indeed, 40% to 50% of all marketed drugs are thought to modulate GPCR activity, making them the major class of targets in the drug discovery process. Binding assays are widely used to identify high-affinity, selective, and potent GPCR drugs. In this field, the use of radiolabeled ligands has remained so far the gold-standard method. Here the authors report a less hazardous alternative for high-throughput screening (HTS) applications by the setup of a nonradioactive fluorescence-based technology named Tag-lite®. Selective binding of various fluorescent ligands, either peptidic or not, covering a large panel of GPCRs from different classes is illustrated, particularly for chemokine (CXCR4), opioid (δ, µ, and κ), and cholecystokinin (CCK1 and CCK2) receptors. Affinity constants of well-known pharmacological agents of numerous GPCRs are in line with values published in the literature. The authors clearly demonstrate that the Tag-lite binding assay format can be successfully and reproducibly applied by using different cellular materials such as transient or stable recombinant cells lines expressing SNAP-tagged GPCR. Such fluorescent-based binding assays can be performed with adherent cells or cells in suspension, in 96- or 384-well plates. Altogether, this new technology offers great advantages in terms of flexibility, rapidity, and user-friendliness; allows easy miniaturization; and makes it completely suitable for HTS applications.

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.

The oligomeric state sets GABAB receptor signalling efficacy

G protein‐coupled receptors (GPCRs) have key roles in cell–cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABAB receptor is one of the most abundant, being distributed in most brain regions, on either pre‐ or post‐synaptic elements. Here, using specific antibodies labelled with time‐resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABAB receptor can form higher‐ordered oligomers in the brain, as suggested by the close proximity of the GABAB1 subunits. Destabilizing the oligomers using a competitor or a GABAB1 mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABAB receptor oligomers composed of a wild‐type and a non‐functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABAB heterodimers.

Biased Agonist Pharmacochaperones of the AVP V2 Receptor May Treat Congenital Nephrogenic Diabetes Insipidus

X-linked congenital nephrogenic diabetes insipidus (cNDI) results from inactivating mutations of the human arginine vasopressin (AVP) V2 receptor (hV(2)R). Most of these mutations lead to intracellular retention of the hV(2)R, preventing its interaction with AVP and thereby limiting water reabsorption and concentration of urine. Because the majority of cNDI-hV(2)Rs exhibit protein misfolding, molecular chaperones hold promise as therapeutic agents; therefore, we sought to identify pharmacochaperones for hV(2)R that also acted as agonists. Here, we describe high-affinity nonpeptide compounds that promoted maturation and membrane rescue of L44P, A294P, and R337X cNDI mutants and restored a functional AVP-dependent cAMP signal. Contrary to pharmacochaperone antagonists, these compounds directly activated a cAMP signal upon binding to several cNDI mutants. In addition, these molecules displayed original functionally selective properties (biased agonism) toward the hV(2)R, being unable to recruit arrestin, trigger receptor internalization, or stimulate mitogen-activated protein kinases. These characteristics make these hV(2)R agonist pharmacochaperones promising therapeutic candidates for cNDI.