The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and barrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1,500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

nt signalling pathways regulate cell proliferation, cell fate and morphogenetic movements. Here, we demonstrate that the Frizzled (Fz) family of Wnt receptors, similarly to G-protein-coupled receptors (GPCRs), form specific homo- and hetero-oligomers. Two lines of evidence suggest that oligomerization occurs in the endoplasmic reticulum: first, a mutant allele of Fz4, encoding a truncated protein that is retained in the endoplasmic reticulum, is linked to the autosomal-dominant retinal degenerative disease, familial exudative vitreoretinopathy (FEVR). We show that this mutant form of Fz4 oligomerizes with wild-type Fz4, retains it in the endoplasmic reticulum and inhibits its signalling. Second, a derivative of Fz1 targeted to the endoplasmic reticulum traps wild-type Fz1 in the endoplasmic reticulum and blocks its signalling. These data support the hypothesis that oligomerization of mutant and wild-type Fz proteins occurs in the endoplasmic reticulum and may explain the genetic dominance of this FEVR allele.

The ability of individual G protein-coupled receptors (GPCR) to engage multiple signaling pathways opens opportunities for the development of better drugs. This requires new knowledge and tools to determine the G protein subtypes and arrestins engaged by a given receptor. Here, we used a new BRET-based effector membrane translocation assay (EMTA) that monitors activation of each Gα protein through the recruitment of selective G protein effectors and βarrestins to the plasma membrane. Profiling of 100 therapeutically relevant GPCR revealed a great diversity of coupling profiles with some receptors displaying exquisite selectivity, whereas others promiscuitely engage all four G protein families. Comparison with existing datasets points to commonalities but also to critical differences between studies.Combining a biosensor subset allowed detecting activity of nearly all GPCR thus providing a new tool for safety screens and systems pharmacology. Overall, this work describes unique resources for studying GPCR function and drug discovery. KEYWORDSG protein-coupled receptor (GPCR), enhanced bystander bioluminescence resonance energy transfer (ebBRET), Biosensor, Effector membrane translocation assay (EMTA), Highthroughput assay, G protein activation, Functional selectivity, Systems pharmacology.

In addition to their interactions with hetero-trimeric G proteins, seven-transmembrane domain receptors are now known to form multimeric complexes that can include receptor homoor hetero-oligomers and/or accessory proteins that modulate their activity. The calcitonin gene-related peptide (CGRP) receptor requires the assembly of the seven-transmembrane domain calcitonin receptor-like receptor with the single-transmembrane domain receptor activity-modifying protein-1 to reach the cell surface and be active. However, the relative stoichiometric arrangement of these two proteins within a receptor complex remains unknown. Despite recent advances in the development of protein-protein interactions assays, determining the composition and stoichiometric arrangements of such signaling complexes in living cells remains a challenging task. In the present study, we combined bimolecular fluorescence complementation (BiFC) with bioluminescence resonance energy transfer (BRET) to probe the stoichiometric arrangement of the CGRP receptor complex. Together with BRET competition assays, co-immunoprecipitation experiments, and BiFC imaging, dual BRET/BiFC revealed that functional CGRP receptors result from the association of a homo-oligomer of the calcitonin receptor-like receptor with a monomer of the accessory protein receptor activity-modifying protein-1. In addition to revealing the existence of an unexpected asymmetric oligomeric organization for a G protein-coupled receptor, our study illustrates the usefulness of dual BRET/BiFC as a powerful tool for analyzing constitutive and dynamically regulated multiprotein complexes.Increasing evidence indicates that "signalosomes," regrouping different components of a signaling pathway into multiprotein complexes with well defined stoichiometries, play crucial roles in defining the specificity and efficacy of signal transduction (1, 2). For instance, the calcitonin gene-related peptide (CGRP) 3 receptor results from the association of the calcitonin receptor-like receptor (CRLR), a seven-transmembrane domain (7TM) receptor belonging to the superfamily of G protein-coupled receptors, with a one-transmembrane domain protein, the receptor activity-modifying protein-1 (RAMP1), which is part of a three member family (RAMP1, -2, and -3). When expressed individually, both CRLR and RAMP1 are retained intracellularly in the endoplasmic reticulum (ER), whereas their association permits their common plasma membrane targeting and determines the pharmacological properties of the receptor (3-5). From these data, it was proposed that the formation of a functional CGRP receptor requires a 1:1 stoichiometric association of CRLR and RAMP1. Consistent with this idea, cross-linking experiments, using bivalent reagents, captured complexes consisting of one CRLR and one RAMP1 molecule (6). However, the observation that both CRLR and RAMP1 can form homo-oligomers (often refer to as dimers) when expressed alone (3, 6 -8) adds a new level of complexity that reactualizes the question of the number of CRLR and R...

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild—yet chronic—neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1—but not pertussis toxin, which affects Gi protein-dependent responses—abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis.