ABSTRACT
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.
Subject(s)
GTP-Binding Proteins/chemistry , Receptors, GABA-B/chemistry , Cryoelectron Microscopy , Humans , Protein Binding , Protein Domains , Protein Multimerization , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
Receptors, Metabotropic Glutamate/chemistry , Cryoelectron Microscopy , Humans , Protein Multimerization , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
Cryoelectron Microscopy , Receptors, GABA-B/chemistry , Receptors, GABA-B/ultrastructure , Allosteric Regulation/drug effects , Apoproteins/chemistry , Apoproteins/metabolism , Apoproteins/ultrastructure , Binding Sites/drug effects , GABA-B Receptor Agonists/chemistry , GABA-B Receptor Agonists/metabolism , GABA-B Receptor Agonists/pharmacology , Humans , Models, Molecular , Protein Domains/drug effects , Protein Multimerization/drug effects , Receptors, GABA-B/metabolism , Signal Transduction , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Glutamic Acid , Receptors, Metabotropic Glutamate , Brain/metabolism , Fluorescence Resonance Energy Transfer/methods , Receptors, Metabotropic Glutamate/metabolismABSTRACT
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.
Subject(s)
Deep Learning , Epitopes , Receptor, Metabotropic Glutamate 5 , Single-Domain Antibodies , Receptor, Metabotropic Glutamate 5/chemistry , Receptor, Metabotropic Glutamate 5/metabolism , Receptor, Metabotropic Glutamate 5/immunology , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunology , Epitopes/immunology , Epitopes/chemistry , Animals , Rats , Models, Molecular , Epitope Mapping/methods , Molecular Dynamics Simulation , Protein ConformationABSTRACT
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.
Subject(s)
Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/metabolism , Single-Domain Antibodies , Gene Expression Regulation/drug effects , Humans , Models, Biological , Mutation , Protein Binding , Protein Conformation , Receptors, Metabotropic Glutamate/geneticsABSTRACT
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.
Subject(s)
Receptors, Metabotropic Glutamate , Receptors, Metabotropic Glutamate/genetics , Receptors, Metabotropic Glutamate/chemistry , Receptors, Metabotropic Glutamate/metabolism , Allosteric Regulation , Receptors, G-Protein-Coupled , Synaptic Transmission , Glutamic Acid , Receptors, GABA-B/genetics , Receptors, GABA-B/metabolismABSTRACT
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.
Subject(s)
Calcium/metabolism , Receptors, Calcium-Sensing/metabolism , Receptors, Calcium-Sensing/ultrastructure , Allosteric Regulation/physiology , Binding Sites/genetics , Calcium/physiology , Fluorescence Resonance Energy Transfer/methods , Humans , Ligands , Molecular Conformation , Receptors, Calcium-Sensing/physiology , Receptors, G-Protein-Coupled/metabolism , Signal TransductionABSTRACT
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.
Subject(s)
Single-Domain Antibodies , Diagnostic Imaging , BrainABSTRACT
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.
Subject(s)
Receptors, GABA/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Membrane/metabolism , Circadian Rhythm/physiology , Dimerization , Drosophila melanogaster/metabolism , Endoplasmic Reticulum/metabolism , Fishes/metabolism , HEK293 Cells , Humans , Mammals/metabolism , Protein Subunits , Protein Transport/physiology , Quality Control , Rats , Sleep/physiology , gamma-Aminobutyric Acid/metabolismABSTRACT
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.
Subject(s)
Biosensing Techniques , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , HEK293 Cells , Humans , RatsABSTRACT
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.
Subject(s)
Protein Multimerization , Receptors, Metabotropic Glutamate/chemistry , Alanine/genetics , Animals , COS Cells , Chlorocebus aethiops , Cysteine/genetics , HEK293 Cells , Humans , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Tertiary , Protein Subunits , Rats , Receptor, Metabotropic Glutamate 5/agonists , Receptor, Metabotropic Glutamate 5/chemistry , Receptor, Metabotropic Glutamate 5/genetics , Receptors, GABA-B/chemistry , Receptors, GABA-B/genetics , Receptors, GABA-B/metabolism , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/genetics , Transcriptional Activation , TransfectionABSTRACT
In multimeric cell-surface receptors, the conformational changes of the extracellular ligand-binding domains (ECDs) associated with receptor activation remain largely unknown. This is the case for the dimeric metabotropic glutamate receptors even though a number of ECD structures have been solved. Here, using an innovative approach based on cell-surface labeling and FRET, we demonstrate that a reorientation of the ECDs is associated with receptor and G-protein activation. Our approach helps identify partial agonists and highlights allosteric interactions between the effector and binding domains. Any approach expected to stabilize the active conformation of the effector domain increased the agonist potency in stabilizing the active ECDs conformation. These data provide key information on the structural dynamics and drug action at metabotropic glutamate receptors and validate an approach for tackling such analysis on other receptors.
Subject(s)
GTP-Binding Proteins/metabolism , Glutamic Acid/chemistry , Receptors, Metabotropic Glutamate/chemistry , Allosteric Site , Animals , Calcium/metabolism , Cell Membrane/metabolism , Crystallography, X-Ray , DNA Mutational Analysis , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Ligands , Mutation , Plasmids/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Multimerization , RatsABSTRACT
Seven-transmembrane domain (7TM) receptors have important functions in cell-cell communication and can assemble into dimers or oligomers. Such complexes may allow specific functional cross-talk through trans-activation of interacting 7TMs, but this hypothesis requires further validation. Herein, we used the GABAB receptor, which is composed of two distinct subunits, GABAB1, which binds the agonist, and GABAB2, which activates G proteins, as a model system. By using a novel orthogonal-labelling approach compatible with time-resolved FRET and based on ACP- and SNAP-tag technologies to verify the heterodimerization of wild-type and mutated GABAB subunits, we demonstrate the existence of a direct allosteric coupling between the 7TMs of GABAB heterodimers. Indeed, a GABAB receptor, in which the GABAB2 extracellular domain was deleted, was still capable of activating G proteins. Furthermore, synthetic ligands for the GABAB2 7TM could increase agonist affinity at the GABAB1 subunit in this mutated receptor. In addition to bringing new information on GABAB receptor activation, these data clearly demonstrate the existence of direct trans-activation between the 7TM of two interacting proteins.
Subject(s)
Receptors, GABA-B/chemistry , Receptors, GABA-B/metabolism , Allosteric Regulation , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Humans , Mutation , Protein Multimerization , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Receptors, GABA-B/genetics , TransfectionABSTRACT
Various missense mutations in the gene coding for prokineticin receptor 2 (PROKR2), a G-protein-coupled receptor, have been identified in patients with Kallmann syndrome. However, the functional consequences of these mutations on the different signaling pathways of this receptor have not been studied. We first showed that the wild-type PROKR2 can activate different G-protein subtypes (Gq, Gs, and Gi/o) and recruit ß-arrestins in transfected HEK-293 cells. We then examined, for each of these signaling pathways, the effects of 9 mutations that did not significantly impair cell surface targeting or ligand binding of the receptor. Four mutant receptors showing defective Gq signaling (R85C, R85H, R164Q, and V331M) could still recruit ß-arrestins on ligand activation, which may cause biased signaling in vivo. Conversely, the R80C receptor could activate the 3 types of G proteins but could not recruit ß-arrestins. Finally, the R268C receptor could recruit ß-arrestins and activate the Gq and Gs signaling pathways but could not activate the Gi/o signaling pathway. Our results validate the concept that mutations in the genes encoding membrane receptors can bias downstream signaling in various ways, possibly leading to pathogenic and, perhaps in some cases, protective (e.g., R268C) effects.
Subject(s)
Arrestins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Kallmann Syndrome/genetics , Mutation, Missense , Receptors, G-Protein-Coupled/physiology , Receptors, Peptide/physiology , Signal Transduction/physiology , Calcium Signaling/genetics , Calcium Signaling/physiology , Gastrointestinal Hormones/metabolism , HEK293 Cells , Humans , Models, Molecular , Mutagenesis, Site-Directed , Neuropeptides/metabolism , Protein Binding , Protein Conformation , Protein Transport , Receptors, G-Protein-Coupled/genetics , Receptors, Peptide/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/genetics , Transfection , beta-ArrestinsABSTRACT
The eight metabotropic glutamate receptors (mGluRs) are key modulators of synaptic transmission and are considered promising targets for the treatment of various brain disorders. Whereas glutamate acts at a large extracellular domain, allosteric modulators have been identified that bind to the seven transmembrane domain (7TM) of these dimeric G-protein-coupled receptors (GPCRs). We show here that the dimeric organization of mGluRs is required for the modulation of active and inactive states of the 7TM by agonists, but is not necessary for G-protein activation. Monomeric mGlu2, either as an isolated 7TM or in full-length, purified and reconstituted into nanodiscs, couples to G proteins upon direct activation by a positive allosteric modulator. However, only a reconstituted full-length dimeric mGlu2 activates G protein upon glutamate binding, suggesting that dimerization is required for glutamate induced activation. These data show that, even for such well characterized GPCR dimers like mGluR2, a single 7TM is sufficient for G-protein coupling. Despite this observation, the necessity of dimeric architecture for signaling induced by the endogenous ligand glutamate confirms that the central core of signaling complex is dimeric.
Subject(s)
GTP-Binding Proteins/metabolism , Glutamic Acid/metabolism , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/metabolism , Receptors, Metabotropic Glutamate/chemistry , Receptors, Metabotropic Glutamate/metabolism , Dimerization , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Inositol Phosphates/metabolism , Microscopy, Electron, Transmission , Nanostructures/ultrastructureABSTRACT
Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation.
Subject(s)
Receptors, Metabotropic Glutamate/chemistry , Receptors, Metabotropic Glutamate/metabolism , Alanine/genetics , Allosteric Regulation , Animals , Cysteine/genetics , Disulfides/metabolism , Humans , Models, Molecular , Movement , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , Rats , Structure-Activity RelationshipABSTRACT
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.
ABSTRACT
G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors.