Sensing of amino acids in a dopaminergic circuitry promotes rejection of an incomplete diet in Drosophila.

The brain is the central organizer of food intake, matching the quality and quantity of the food sources with organismal needs. To ensure appropriate amino acid balance, many species reject a diet lacking one or several essential amino acids (EAAs) and seek out a better food source. Here, we show that, in Drosophila larvae, this behavior relies on innate sensing of amino acids in dopaminergic (DA) neurons of the brain. We demonstrate that the amino acid sensor GCN2 acts upstream of GABA signaling in DA neurons to promote avoidance of the EAA-deficient diet. Using real-time calcium imaging in larval brains, we show that amino acid imbalance induces a rapid and reversible activation of three DA neurons that are necessary and sufficient for food rejection. Taken together, these data identify a central amino-acid-sensing mechanism operating in specific DA neurons and controlling food intake.

Structural basis of GABAB receptor-Gi protein coupling.

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABAB receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with Gi1 protein. We found that a single G protein interacts with the GB2 subunit of the GABAB receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABAB receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.

Structures of human mGlu2 and mGlu7 homo- and heterodimers.

The metabotropic glutamate receptors (mGlus) are involved in the modulation of synaptic transmission and neuronal excitability in the central nervous system1. These receptors probably exist as both homo- and heterodimers that have unique pharmacological and functional properties2-4. Here we report four cryo-electron microscopy structures of the human mGlu subtypes mGlu2 and mGlu7, including inactive mGlu2 and mGlu7 homodimers; mGlu2 homodimer bound to an agonist and a positive allosteric modulator; and inactive mGlu2-mGlu7 heterodimer. We observed a subtype-dependent dimerization mode for these mGlus, as a unique dimer interface that is mediated by helix IV (and that is important for limiting receptor activity) exists only in the inactive mGlu2 structure. The structures provide molecular details of the inter- and intra-subunit conformational changes that are required for receptor activation, which distinguish class C G-protein-coupled receptors from those in classes A and B. Furthermore, our structure and functional studies of the mGlu2-mGlu7 heterodimer suggest that the mGlu7 subunit has a dominant role in controlling dimeric association and G-protein activation in the heterodimer. These insights into mGlu homo- and heterodimers highlight the complex landscape of mGlu dimerization and activation.

Structural basis of the activation of a metabotropic GABA receptor.

Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis1. GABAB belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits2. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABAB. Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.

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.

Nanobody-based sensors reveal a high proportion of mGlu heterodimers in the brain.

Membrane proteins, including ion channels, receptors and transporters, are often composed of multiple subunits and can form large complexes. Their specific composition in native tissues is difficult to determine and remains largely unknown. In this study, we developed a method for determining the subunit composition of endogenous cell surface protein complexes from isolated native tissues. Our method relies on nanobody-based sensors, which enable proximity detection between subunits in time-resolved Förster resonance energy transfer (FRET) measurements. Additionally, given conformation-specific nanobodies, the activation of these complexes can be recorded in native brain tissue. Applied to the metabotropic glutamate receptors in different brain regions, this approach revealed the clear existence of functional metabotropic glutamate (mGlu)2-mGlu4 heterodimers in addition to mGlu2 and mGlu4 homodimers. Strikingly, the mGlu4 subunits appear to be mainly heterodimers in the brain. Overall, these versatile biosensors can determine the presence and activity of endogenous membrane proteins in native tissues with high fidelity and convenience.

Epitope Identification of an mGlu5 Receptor Nanobody Using Physics-Based Molecular Modeling and Deep Learning Techniques.

The world has witnessed a revolution in therapeutics with the development of biological medicines such as antibodies and antibody fragments, notably nanobodies. These nanobodies possess unique characteristics including high specificity and modulatory activity, making them promising candidates for therapeutic applications. Identifying their binding mode is essential for their development. Experimental structural techniques are effective to get such information, but they are expensive and time-consuming. Here, we propose a computational approach, aiming to identify the epitope of a nanobody that acts as an agonist and a positive allosteric modulator at the rat metabotropic glutamate receptor 5. We employed multiple structure modeling tools, including various artificial intelligence algorithms for epitope mapping. The computationally identified epitope was experimentally validated, confirming the success of our approach. Additional dynamics studies provided further insights on the modulatory activity of the nanobody. The employed methodologies and approaches initiate a discussion on the efficacy of diverse techniques for epitope mapping and later nanobody engineering.

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.

Asymmetric activation of dimeric GABAB and metabotropic glutamate receptors.

G protein-coupled receptors (GPCRs) represent the largest family of membrane proteins and are important drug targets. GPCRs are allosteric machines that transduce an extracellular signal to the cell by activating heterotrimeric G proteins. Herein, we summarize the recent advancements in the molecular activation mechanism of the γ-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors, the most important class C GPCRs that modulate synaptic transmission in the brain. Both are mandatory dimers, this quaternary structure being needed for their function The structures of these receptors in different conformations and in complexes with G proteins have revealed their asymmetric activation. This asymmetry is further highlighted by the recent discovery of mGlu heterodimers, where the eight mGlu subunits can form specific and functional heterodimers. Finally, the development of allosteric modulators has revealed new possibilities for regulating the function of these receptors by targeting the transmembrane dimer interface. This family of receptors never ceases to astonish and serve as models to better understand the diversity and asymmetric functioning of GPCRs.NEW & NOTEWORTHY γ-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors form constitutive dimers, which are required for their function. They serve as models to better understand the diversity and activation of G protein-coupled receptors (GPCRs). The structures of these receptors in different conformations and in complexes with G proteins have revealed their asymmetric activation. This asymmetry is further highlighted by the recent discovery of specific and functional mGlu heterodimers. Allosteric modulators can be developed to target the transmembrane interface and modulate the asymmetry.

Illuminating the allosteric modulation of the calcium-sensing receptor.

Many membrane receptors are regulated by nutrients. However, how these nutrients control a single receptor remains unknown, even in the case of the well-studied calcium-sensing receptor CaSR, which is regulated by multiple factors, including ions and amino acids. Here, we developed an innovative cell-free Förster resonance energy transfer (FRET)-based conformational CaSR biosensor to clarify the main conformational changes associated with activation. By allowing a perfect control of ambient nutrients, this assay revealed that Ca2+ alone fully stabilizes the active conformation, while amino acids behave as pure positive allosteric modulators. Based on the identification of Ca2+ activation sites, we propose a molecular basis for how these different ligands cooperate to control CaSR activation. Our results provide important information on CaSR function and improve our understanding of the effects of genetic mutations responsible for human diseases. They also provide insights into how a receptor can integrate signals from various nutrients to better adapt to the cell response.

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.

Organization and functions of mGlu and GABAB receptor complexes.

The neurotransmitters glutamate and γ-aminobutyric acid (GABA) transmit synaptic signals by activating fast-acting ligand-gated ion channels and more slowly acting G-protein-coupled receptors (GPCRs). The GPCRs for these neurotransmitters, metabotropic glutamate (mGlu) and GABAB receptors, are atypical GPCRs with a large extracellular domain and a mandatory dimeric structure. Recent studies have revealed how these receptors are activated through multiple allosteric interactions between subunit domains. It emerges that the molecular complexity of these receptors is further increased through association with trafficking, effector and regulatory proteins. The structure and composition of these receptors present opportunities for therapeutic intervention in mental health and neurological disorders.

Structural basis for distinct quality control mechanisms of GABAB receptor during evolution.

Cell surface trafficking of many G protein-coupled receptors is tightly regulated. Among them, the mandatory heterodimer GABAB receptor for the main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), is a model. In mammals, its cell surface trafficking is highly controlled by an endoplasmic reticulum retention signal in the C-terminal intracellular region of the GB1 subunit that is masked through a coiled-coil interaction with the GB2 subunit. Here, we investigate the molecular basis for the export of its homolog in Drosophila melanogaster that regulates the circadian rhythm and sleep. In contrast to mammals, the endoplasmic retention signal is carried by GB2, while GB1 reaches the cell surface alone. NMR analysis showed that the coiled-coil domain that controls GABAB heterodimer formation is structurally conserved between flies and mammals, despite specific features. These findings show the adaptation of a similar quality control system during evolution for maintaining the subunit composition of a functional heterodimeric receptor.

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.

Context-Dependent Signaling of CXC Chemokine Receptor 4 and Atypical Chemokine Receptor 3.

G protein-coupled receptors (GPCRs) are regulated by complex molecular mechanisms, both in physiologic and pathologic conditions, and their signaling can be intricate. Many factors influence their signaling behavior, including the type of ligand that activates the GPCR, the presence of interacting partners, the kinetics involved, or their location. The two CXC-type chemokine receptors, CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), both members of the GPCR superfamily, are important and established therapeutic targets in relation to cancer, human immunodeficiency virus infection, and inflammatory diseases. Therefore, it is crucial to understand how the signaling of these receptors works to be able to specifically target them. In this review, we discuss how the signaling pathways activated by CXCR4 and ACKR3 can vary in different situations. G protein signaling of CXCR4 depends on the cellular context, and discrepancies exist depending on the cell lines used. ACKR3, as an atypical chemokine receptor, is generally reported to not activate G proteins but can broaden its signaling spectrum upon heteromerization with other receptors, such as CXCR4, endothelial growth factor receptor, or the α 1-adrenergic receptor (α 1-AR). Also, CXCR4 forms heteromers with CC chemokine receptor (CCR) 2, CCR5, the Na+/H+ exchanger regulatory factor 1, CXCR3, α 1-AR, and the opioid receptors, which results in differential signaling from that of the monomeric subunits. In addition, CXCR4 is present on membrane rafts but can go into the nucleus during cancer progression, probably acquiring different signaling properties. In this review, we also provide an overview of the currently known critical amino acids involved in CXCR4 and ACKR3 signaling.

HTS-compatible FRET-based conformational sensors clarify membrane receptor activation.

Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.

GABAB receptor promotes its own surface expression by recruiting a Rap1-dependent signaling cascade.

G-protein-coupled receptors (GPCRs) are key players in cell signaling, and their cell surface expression is tightly regulated. For many GPCRs such as ß2-AR (ß2-adrenergic receptor), receptor activation leads to downregulation of receptor surface expression, a phenomenon that has been extensively characterized. By contrast, some other GPCRs, such as GABA(B) receptor, remain relatively stable at the cell surface even after prolonged agonist treatment; however, the underlying mechanisms are unclear. Here, we identify the small GTPase Rap1 as a key regulator for promoting GABA(B) receptor surface expression. Agonist stimulation of GABA(B) receptor signals through Gαi/o to inhibit Rap1GAPII (also known as Rap1GAP1b, an isoform of Rap1GAP1), thereby activating Rap1 (which has two isoforms, Rap1a and Rap1b) in cultured cerebellar granule neurons (CGNs). The active form of Rap1 is then recruited to GABA(B) receptor through physical interactions in CGNs. This Rap1-dependent signaling cascade promotes GABA(B) receptor surface expression by stimulating receptor recycling. Our results uncover a new mechanism regulating GPCR surface expression and also provide a potential explanation for the slow, long-lasting inhibitory action of GABA neurotransmitter.

Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer.

G protein-coupled receptors (GPCRs) are major players in cell communication. Although they form functional monomers, increasing evidence indicates that GPCR dimerization has a critical role in cooperative phenomena that are important for cell signal integration. However, the structural bases of these phenomena remain elusive. Here, using well-characterized receptor dimers, the metabotropic glutamate receptors (mGluRs), we show that structural changes at the dimer interface are linked to receptor activation. We demonstrate that the main dimer interface is formed by transmembrane α helix 4 (TM4) and TM5 in the inactive state and by TM6 in the active state. This major change in the dimer interface is required for receptor activity because locking the TM4-TM5 interface prevents activation by agonist, whereas locking the TM6 interface leads to a constitutively active receptor. These data provide important information on the activation mechanism of mGluRs and improve our understanding of the structural basis of the negative cooperativity observed in these GPCR dimers.