Different EGF-induced receptor dimer conformations for signaling and internalization.

The structural basis of the activation and internalization of EGF receptors (EGFR) is still a matter of debate despite the importance of this target in cancer treatment. Whether agonists induce dimer formation or act on preformed dimers remains discussed. Here, we provide direct evidence that EGF-induced EGFR dimer formation as best illustrated by the very large increase in FRET between snap-tagged EGFR subunits induced by agonists. We confirm that Erlotinib-related TK (tyrosine kinase) inhibitors also induce dimer formation despite the inactive state of the binding domain. Surprisingly, TK inhibitors do not inhibit EGF-induced EGFR internalization despite their ability to fully block EGFR signaling. Only Erlotinib-related TK inhibitors promoting asymmetric dimers could slow down this process while the lapatinib-related ones have almost no effect. These results reveal that the conformation of the intracellular TK dimer, rather than the known EGFR signaling, is critical for EGFR internalization. These results also illustrate clear differences in the mode of action of TK inhibitors on the EGFR and open novel possibilities to control EGFR signaling for cancer treatment.

A nanobody activating metabotropic glutamate receptor 4 discriminates between homo- and heterodimers.

There is growing interest in developing biologics due to their high target selectivity. The G protein-coupled homo- and heterodimeric metabotropic glutamate (mGlu) receptors regulate many synapses and are promising targets for the treatment of numerous brain diseases. Although subtype-selective allosteric small molecules have been reported, their effects on the recently discovered heterodimeric receptors are often not known. Here, we describe a nanobody that specifically and fully activates homodimeric human mGlu4 receptors. Molecular modeling and mutagenesis studies revealed that the nanobody acts by stabilizing the closed active state of the glutamate binding domain by interacting with both lobes. In contrast, this nanobody does not activate the heterodimeric mGlu2-4 but acts as a pure positive allosteric modulator. These data further reveal how an antibody can fully activate a class C receptor and bring further evidence that nanobodies represent an alternative way to specifically control mGlu receptor subtypes.

Targeting the Brain with Single-Domain Antibodies: Greater Potential Than Stated So Far?

Treatments for central nervous system diseases with therapeutic antibodies have been increasingly investigated over the last decades, leading to some approved monoclonal antibodies for brain disease therapies. The detection of biomarkers for diagnosis purposes with non-invasive antibody-based imaging approaches has also been explored in brain cancers. However, antibodies generally display a low capability of reaching the brain, as they do not efficiently cross the blood-brain barrier. As an alternative, recent studies have focused on single-domain antibodies (sdAbs) that correspond to the antigen-binding fragment. While some reports indicate that the brain uptake of these small antibodies is still low, the number of studies reporting brain-penetrating sdAbs is increasing. In this review, we provide an overview of methods used to assess or evaluate brain penetration of sdAbs and discuss the pros and cons that could affect the identification of brain-penetrating sdAbs of therapeutic or diagnostic interest.

SGIP1 modulates kinetics and interactions of the cannabinoid receptor 1 and G protein-coupled receptor kinase 3 signalosome.

Cannabinoid receptor 1 (CB1R), a G protein-coupled receptor, plays a fundamental role in synaptic plasticity. Abnormal activity and deregulation of CB1R signaling result in a broad spectrum of pathological conditions. CB1R signaling is regulated by receptor desensitization including phosphorylation of residues within the intracellular C terminus by G protein-coupled receptor kinases (GRKs) that may lead to endocytosis. Furthermore, CB1R signaling is regulated by the protein Src homology 3-domain growth factor receptor-bound 2-like (SGIP1) that hinders receptor internalization, while enhancing CB1R association with ß-arrestin. It has been postulated that phosphorylation of two clusters of serine/threonine residues, 425 SMGDS429 and 460 TMSVSTDTS468 , within the CB1R C-tail controls dynamics of the association between receptor and its interaction partners involved in desensitization. Several molecular determinants of these events are still not well understood. We hypothesized that the dynamics of these interactions are modulated by SGIP1. Using a panel of CB1Rs mutated in the aforementioned serine and threonine residues, together with an array of Bioluminescence energy transfer-based (BRET) sensors, we discovered that GRK3 forms complexes with Gßγ subunits of G proteins that largely independent of GRK3's interaction with CB1R. Furthermore, CB1R interacts only with activated GRK3. Interestingly, phosphorylation of two specific residues on CB1R triggers GRK3 dissociation from the desensitized receptor. SGIP1 increases the association of GRK3 with Gßγ subunits of G proteins, and with CB1R. Altogether, our data suggest that the CB1R signalosome complex is dynamically controlled by sequential phosphorylation of the receptor C-tail and is also modified by SGIP1.

G protein-coupled receptors can control the Hippo/YAP pathway through Gq signaling.

The Hippo pathway is an evolutionarily conserved kinase cascade involved in the control of tissue homeostasis, cellular differentiation, proliferation, and organ size, and is regulated by cell-cell contact, apical cell polarity, and mechanical signals. Miss-regulation of this pathway can lead to cancer. The Hippo pathway acts through the inhibition of the transcriptional coactivators YAP and TAZ through phosphorylation. Among the various signaling mechanisms controlling the hippo pathway, activation of G12/13 by G protein-coupled receptors (GPCR) recently emerged. Here we show that a GPCR, the ghrelin receptor, that activates several types of G proteins, including G12/13, Gi/o, and Gq, can activate YAP through Gq/11 exclusively, independently of G12/13. We revealed that a strong basal YAP activation results from the high constitutive activity of this receptor, which can be further increased upon agonist activation. Thus, acting on ghrelin receptor allowed to modulate up-and-down YAP activity, as activating the receptor increased YAP activity and blocking constitutive activity reduced YAP activity. Our results demonstrate that GPCRs can be used as molecular switches to finely up- or down-regulate YAP activity through a pure Gq pathway.

Nonclassical Ligand-Independent Regulation of Go Protein by an Orphan Class C G-Protein-Coupled Receptor.

The orphan G-protein-coupled receptor (GPCR) GPR158 is expressed in the brain, where it is involved in the osteocalcin effect on cognitive processes, and at the periphery, where it may contribute to glaucoma and cancers. GPR158 forms a complex with RGS7-ß5, leading to the regulation of neighboring GPCR-induced Go protein activity. GPR158 also interacts with αo, although no canonical Go coupling has been reported. GPR158 displays three VCPWE motifs in its C-terminal domain that are putatively involved in G-protein regulation. Here, we addressed the scaffolding function of GPR158 and its VCPWE motifs on Go. We observed that GPR158 interacted with and stabilized the amount of RGS7-ß5 through a 50-residue region downstream of its transmembrane domain and upstream of the VCPWE motifs. We show that two VCPWE motifs are involved in αo binding. Using a Gαo-ßγ bioluminescence resonance energy transfer (BRET) sensor, we found that GPR158 decreases the BRET signal as observed upon G-protein activation; however, no constitutive activity of GPR158 could be detected through the measurement of various G-protein-mediated downstream responses. We propose that the effect of GPR158 on Go is unlikely due to a canonical activation of Go, but rather to the trapping of Gαo by the VCPWE motifs, possibly leading to its dissociation from ßγ Such action of GPR158 is expected to prolong the ßγ activity, as also observed with some activators of G-protein signaling. Taken together, our data revealed a complex functional scaffolding or signaling role for GPR158 controlling Go through an original mechanism.

Post-endocytotic Deubiquitination and Degradation of the Metabotropic γ-Aminobutyric Acid Receptor by the Ubiquitin-specific Protease 14.

Mechanisms controlling the metabotropic γ-aminobutyric acid receptor (GABAB) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly down-regulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABAB(1b)subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABABin a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination.

Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling.

The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489-Ser495 and Ser501-Thr506) specifically responsible for the majority of PTH(1-34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1-34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489-Ser495 and the second cluster Ser501-Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.

A proteomic analysis reveals the interaction of GluK1 ionotropic kainate receptor subunits with Go proteins.

Kainate receptors (KARs) are found ubiquitously in the CNS and are present presynaptically and postsynaptically regulating synaptic transmission and excitability. Functional studies have proven that KARs act as ion channels as well as potentially activating G-proteins, thus indicating the existance of a dual signaling system for KARs. Nevertheless, it is not clear how these ion channels activate G-proteins and which of the KAR subunits is involved. Here we performed a proteomic analysis to define proteins that interact with the C-terminal domain of GluK1 and we identified a variety of proteins with many different functions, including a Go α subunit. These interactions were verified through distinct in vitro and in vivo assays, and the activation of the Go protein by GluK1 was validated in bioluminescence resonance energy transfer experiments, while the specificity of this association was confirmed in GluK1-deficient mice. These data reveal components of the KAR interactome, and they show that GluK1 and Go proteins are natural partners, accounting for the metabotropic effects of KARs.

Quantitative phosphoproteomics unravels biased phosphorylation of serotonin 2A receptor at Ser280 by hallucinogenic versus nonhallucinogenic agonists.

The serotonin 5-HT(2A) receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT(2A) receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT(2A) receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT(2A) agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser(280)) located in the third intracellular loop of the 5-HT(2A) receptor, a region important for its desensitization. The specific phosphorylation of Ser(280) by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT(2A) receptors at Ser(280) in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser(280) to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT(2A) receptor in response to hallucinogenic versus nonhallucinogenic agonists, which underlies their distinct capacity to desensitize the receptor.

The oligomeric state sets GABA(B) 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 GABA(B) 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 GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) 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 GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.

Biased signaling regulates the pleiotropic effects of the urotensin II receptor to modulate its cellular behaviors.

Biased agonism by G-protein-coupled receptor ligands has opened up strategies for targeted physiological or therapeutic actions. We hypothesized that urotensin II (UII)-derived peptides displayed unexpected physiological effects because of such biased signaling on the UII human urotensin (hUT) receptor. We determined the coupling to G proteins and ß-arrestins of the UII-activated hUT receptor expressed in HEK293 using bioluminescence resonance energy transfer (BRET) biosensors, as well as the production of IP1-3 and cAMP using homogenous time-resolved Forster resonance energy transfer (FRET) (HTRF)-based assays. The activated receptor coupled to Gi1, GoA, Gq, and G13, excluding Gs, and recruited ß-arrestins 1 and 2. Integration of these pathways led to a 2-phase kinetic phosphorylation of ERK1/2 kinases. The tested peptides induced three different profiles: UII, urotensin-related peptide (URP), and UII4-11 displayed the full profile; [Orn(8)]UII and [Orn(5)]URP activated G proteins, although with pEC50s 5-10× higher, and did not or barely recruited ß-arrestin; urantide also failed to recruit ß-arrestin but displayed a reversed rank order for Gi and Gq vs. Go pEC50s (-8.79±0.20, -8.43±0.21, and -7.86±0.36, respectively, for urantide, -7.87±0.10, -7.23±0.27, and -8.55±0.19, respectively, for [Orn(5)]URP) and was a partial agonist of all G-protein pathways. Interestingly, the peptides differently modulated cell survival but similarly induced cell migration and adhesion. Thus, we demonstrate biased signaling between ß-arrestin and G proteins, and between G-protein subtypes, which dictates the receptor's cellular responses.

The chemokine CXC4 and CC2 receptors form homo- and heterooligomers that can engage their signaling G-protein effectors and ßarrestin.

G-protein-coupled receptors have been shown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo- and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo- and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of Gαi1, Gα13, Gαz, and ßarrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents Gi activation, also affects the CCR2-promoted engagement of Gαi1 and ßarrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo- and heteromultimeric CXCR4 and CCR2 can form functional signaling complexes that have unique properties.

Up-regulation of GABA(B) receptor signaling by constitutive assembly with the K+ channel tetramerization domain-containing protein 12 (KCTD12).

GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.

Heterodimers Revolutionize the Field of Metabotropic Glutamate Receptors.

Identified 40 years ago, the metabotropic glutamate (mGlu) receptors play key roles in modulating many synapses in the brain, and are still considered as important drug targets to treat various brain diseases. Eight genes encoding mGlu subunits have been identified. They code for complex receptors composed of a large extracellular domain where glutamate binds, connected to a G protein activating membrane domain. They are covalently linked dimers, a quaternary structure needed for their activation by glutamate. For many years they have only been considered as homodimers, then limiting the number of mGlu receptors to 8 subtypes composed of twice the same subunit. Twelve years ago, mGlu subunits were shown to also form heterodimers with specific subunits combinations, increasing the family up to 19 different potential dimeric receptors. Since then, a number of studies brought evidence for the existence of such heterodimers in the brain, through various approaches. Structural and molecular dynamic studies helped understand their peculiar activation process. The present review summarizes the approaches used to study their activation process and their pharmacological properties and to demonstrate their existence in vivo. We will highlight how the existence of mGlu heterodimers revolutionizes the mGlu receptor field, opening new possibilities for therapeutic intervention for brain diseases. As illustrated by the number of possible mGlu heterodimers, this study will highlight the need for further research to fully understand their role in physiological and pathological conditions, and to develop more specific therapeutic tools.

The EBI2 receptor is coexpressed with CCR5 in CD4+ T cells and boosts HIV-1 R5 replication.

OBJECTIVE: CCR5, a G protein-coupled receptor (GPCR), is used by most HIV strains as a coreceptor. In this study, we looked for other GPCRs able to modify HIV-1 infection. DESIGN: We analyzed the effects of one GPCR coexpressed with CCR5, EBI2, on HIV-1 replicative cycle. METHODS: We identified GPCRs expressed in primary CD4+CCR5+ T cells by multi-RT-qPCR. We studied GPCR dimerization by FRET technology. Cell lines expressing EBI2 were established by transduction with HIV vectors. HIV-1 entry was quantified with virions harboring ß-lactamase fused to the viral protein vpr, early and late HIV-1 transcriptions by qPCR, NFkB nuclear activation by immunofluorescence and transfection, and viral production by measuring p24 concentration in culture supernatant by ELISA. RESULTS: We showed that EBI2 is naturally expressed in primary CD4+CCR5+ T cells, and that CCR5 and EBI2 heterodimerize. We observed that this coexpression reduced viral entry by 50%. The amount of HIV reverse transcripts was similar in cells expressing or not EBI2. Finally, the presence of EBI2 induced the translocation of NFkB and activated HIV-1 genome expression. Globally, the result was a drastic HIV-1 R5, but not X4, overproduction in EBI2-transduced cells. CONCLUSIONS: EBI2 expression in CD4+CCR5+ cells boosts HIV-1 R5 productive infection. As the natural ligand for EBI2 is present in blood and lymphoid tissues, the constant EBI2 activation might increase HIV replication in CD4+ T cells. It might be of interest to test the effect of EBI2 antagonists on the residual viral production persisting in patients aviremic under treatment.

Design, Synthesis, Pharmacology, and In Silico Studies of (1S,2S,3S)-2-((S)-Amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic Acid (LBG30300): A Picomolar Potency Subtype-Selective mGlu2 Receptor Agonist.

Metabotropic glutamate (Glu) receptors (mGlu receptors) play a key role in modulating excitatory neurotransmission in the central nervous system (CNS). In this study, we report the structure-based design and pharmacological evaluation of densely functionalized, conformationally restricted glutamate analogue (1S,2S,3S)-2-((S)-amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic acid (LBG30300). LBG30300 was synthesized in a stereocontrolled fashion in nine steps from a commercially available optically active epoxide. Functional characterization of all eight mGlu receptor subtypes showed that LBG30300 is a picomolar agonist at mGlu2 with excellent selectivity over mGlu3 and the other six mGlu receptor subtypes. Bioavailability studies on mice (IV administration) confirm CNS exposure, and an in silico study predicts a binding mode of LBG30300 which induces a flipping of Tyr144 to allow for a salt bridge interaction of the acetate group with Arg271. The Tyr144 residue now prevents Arg271 from interacting with Asp146, which is a residue of differentiation between mGlu2 and mGlu3 and thus could explain the observed subtype selectivity.

Synthesis and pharmacological characterization of conformationally restricted 2-amino-Adipic acid analogs and carboxycyclopropyl glycines as selective metabotropic glutamate 2 receptor agonists.

The metabotropic glutamate (Glu) receptors (mGluRs) are G-protein coupled receptors, which play a central role in modulating excitatory neurotransmission in the central nervous system (CNS). Thus, the development of tool compounds thereto, continues to interest the scientific community. In this study, we report the design and synthesis of new conformationally restricted 2-aminoadipic acid (2AA) 2-4, and glutamic acid 5, 6 analogs, which share the cyclopropane ring as the restrictor. The analogs were characterized at rat mGlu1-8 in an IP-One functional assay. While the 2AA analogs 3a, 4a and CCG-I analog 5a were shown to be selective mGlu2 agonists with low micromolar potencies, CCG-II analog 5b was shown to be a potent full agonist at mGlu2 (EC50 = 82 nM) with ∼15-fold selectivity over mGlu3, >25-fold selectivity over group III, and >60-fold selectivity over group I subtypes. An in silico study was performed to address this significant change (>3500 fold) in potency upon introduction of this methyl group (L-CCG-II vs 5b).

Photoswitchable positive allosteric modulators of metabotropic glutamate receptor 4 to improve selectivity.

Metabotropic glutamate receptors (mGlu) regulate multiple functions in the nervous systems and are involved in several neurological disorders. However, selectively targeting individual mGlu subtypes with spatiotemporal precision is still an unmet need. Photopharmacology can address this concern through the utilization of photoswitchable compounds such as optogluram, which is a positive allosteric modulator (PAM) of mGlu4 that enables the precise control of physiological responses using light but does not have an optimal selectivity profile. Optogluram analogs were developed to obtain photoswitchable PAMs of mGlu4 receptor with an improved selectivity. Among them, optogluram-2 emerged as a photoswitchable ligand for mGlu4 receptor with activity as both PAM and allosteric agonists. It presents a higher selectivity and offers improved photoswitching of mGlu4 activity. These improved properties make optogluram-2 an excellent candidate to study the role of mGlu4 with a high spatiotemporal precision in systems where mGlu4 can be co-expressed with other mGlu receptors.

Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration.

G protein-coupled receptors (GPCRs) have critical functions in intercellular communication. Although a wide range of different receptors have been identified in the same cells, the mechanism by which signals are integrated remains elusive. The ability of GPCRs to form dimers or larger hetero-oligomers is thought to generate such signal integration. We examined the molecular mechanisms responsible for the GABA(B) receptor-mediated potentiation of the mGlu receptor signalling reported in Purkinje neurons. We showed that this effect does not require a physical interaction between both receptors. Instead, it is the result of a more general mechanism in which the betagamma subunits produced by the Gi-coupled GABA(B) receptor enhance the mGlu-mediated Gq response. Most importantly, this mechanism could be generally applied to other pairs of Gi- and Gq-coupled receptors and the signal integration varied depending on the time delay between activation of each receptor. Such a mechanism helps explain specific properties of cells expressing two different Gi- and Gq-coupled receptors activated by a single transmitter, or properties of GPCRs naturally coupled to both types of the G protein.