Sodium is a negative allosteric regulator of the ghrelin receptor.

The functional properties of G protein-coupled receptors (GPCRs) are intimately associated with the different components in their cellular environment. Among them, sodium ions have been proposed to play a substantial role as endogenous allosteric modulators of GPCR-mediated signaling. However, this sodium effect and the underlying mechanisms are still unclear for most GPCRs. Here, we identified sodium as a negative allosteric modulator of the ghrelin receptor GHSR (growth hormone secretagogue receptor). Combining 23Na-nuclear magnetic resonance (NMR), molecular dynamics, and mutagenesis, we provide evidence that, in GHSR, sodium binds to the allosteric site conserved in class A GPCRs. We further leveraged spectroscopic and functional assays to show that sodium binding shifts the conformational equilibrium toward the GHSR-inactive ensemble, thereby decreasing basal and agonist-induced receptor-catalyzed G protein activation. All together, these data point to sodium as an allosteric modulator of GHSR, making this ion an integral component of the ghrelin signaling machinery.

Non-ionic cholesterol-based additives for the stabilization of membrane proteins.

We report herein the synthesis of two non-ionic amphiphiles with a cholesterol hydrophobic moiety that can be used as chemical additives for biochemical studies of membrane proteins. They were designed to show a high similarity with the planar steroid core of cholesterol and small-to-medium polar head groups attached at the C3 position of ring-A on the sterol skeleton. The two Chol-Tris and Chol-DG have a Tris-hydroxymethyl and a branched diglucose polar head group, respectively, which provide them sufficient water solubility when mixed with the "gold standard" detergent n-Dodecyl-ß-D-Maltoside (DDM). The colloidal properties of these mixed micelles were investigated by means of surface tension (SFT) measurements and dynamic light scattering (DLS) experiments and showed the formation of globular micelles of about 8 nm in diameter with a critical micellar concentration of 0.20 mM for DDM:Chol-DG and 0.22 mM for DDM:Chol-Tris. We showed that mixed micelles do not alter the extraction potency of a G-protein coupled receptor (GPCR): the human adenosine A2A receptor (A2AR). The thermostabilizing effect of the mixed micelles was confirmed on two GPCRs, A2AR and the growth hormone secretagogue receptor (GHSR). Finally, these two mixed micelles were found suitable for the purification of an active form of A2AR which remained able to bind two ligands of different class i.e. the specific agonist CGS-21680 and the specific inverse agonist ZM-241385. This suggests that Chol-Tris and Chol-DG may be used as a non-ionic alternative to the cholesteryl hemisuccinate (CHS) stabilizing agent.

MT1 Melatonin Receptor Reconstitution in Nanodiscs.

A way to study G protein-coupled receptors in a minimal system is to reconstruct artificial membrane mimics, made of detergent and/or of lipids in which the purified receptor is maintained. In particular, it is now possible to generate lipid nanoparticles, such as nanodiscs, in which a single receptor molecule is included. Such objects offer the invaluable potential of studying an isolated receptor stabilized in a finely controlled membrane-like environment to evaluate its pharmacology, its function, and its structure at the molecular level. In this chapter, we detail the different steps from the extraction and isolation of a recombinant MT1 melatonin receptor in detergent, down to its reconstitution into nanodiscs. A G protein activation test is further described in order to exemplify how the functionality of such particles may be investigated.

Design and characterization of a triazole-based growth hormone secretagogue receptor modulator inhibiting the glucoregulatory and feeding actions of ghrelin.

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor that regulates essential physiological functions. In particular, activation of GHSR in response to its endogenous agonist ghrelin promotes food intake and blood glucose increase. Therefore, compounds aimed at blocking GHSR signaling constitute potential options against obesity-related metabolic disorders. We have previously developed potent ligands of GHSR based on a triazole scaffold. Here, we report a new 3,4,5-trisubstituted 1,2,4-triazole compound, named JMV 6616, that potently blocks GHSR activity in vitro and in vivo. Specifically, in HEK293T cells JMV 6616 behaves as an inverse agonist since it binds to GHSR and inhibits its ghrelin-independent signaling. Accordingly, using purified labeled GHSR assembled into lipid nanodiscs we found that JMV 6616 decreases GHSR-catalyzed G protein activation and stabilizes an inactive receptor conformation. Importantly, JMV 6616 also acts on native GHSR since it blocks the insulinostatic effect of ghrelin in pancreatic islets. In mice, JMV 6616 inhibits blood glucose-raising effects of ghrelin treatment and the orexigenic actions of acute ghrelin administration. Together, our data suggest that this triazole-derived modulator of GHSR holds promise to mitigate several pathological features associated with eating and metabolic disorders.

Exploration of the dynamic interplay between lipids and membrane proteins by hydrostatic pressure.

Cell membranes represent a complex and variable medium in time and space of lipids and proteins. Their physico-chemical properties are determined by lipid components which can in turn influence the biological function of membranes. Here, we used hydrostatic pressure to study the close dynamic relationships between lipids and membrane proteins. Experiments on the ß-barrel OmpX and the α-helical BLT2 G Protein-Coupled Receptor in nanodiscs of different lipid compositions reveal conformational landscapes intimately linked to pressure and lipids. Pressure can modify the conformational landscape of the membrane protein per se, but also increases the gelation of lipids, both being monitored simultaneously at high atomic resolution by NMR. Our study also clearly shows that a membrane protein can modulate, at least locally, the fluidity of the bilayer. The strategy proposed herein opens new perspectives to scrutinize the dynamic interplay between membrane proteins and their surrounding lipids.

Structural basis of human ghrelin receptor signaling by ghrelin and the synthetic agonist ibutamoren.

The hunger hormone ghrelin activates the ghrelin receptor GHSR to stimulate food intake and growth hormone secretion and regulate reward signaling. Acylation of ghrelin at Ser3 is required for its agonistic action on GHSR. Synthetic agonists of GHSR are under clinical evaluation for disorders related to appetite and growth hormone dysregulation. Here, we report high-resolution cryo-EM structures of the GHSR-Gi signaling complex with ghrelin and the non-peptide agonist ibutamoren as an investigational new drug. Our structures together with mutagenesis data reveal the molecular basis for the binding of ghrelin and ibutamoren. Structural comparison suggests a salt bridge and an aromatic cluster near the agonist-binding pocket as important structural motifs in receptor activation. Notable structural variations of the Gi and GHSR coupling are observed in our cryo-EM analysis. Our results provide a framework for understanding GHSR signaling and developing new GHSR agonist drugs.

Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor.

There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

LEAP2 Impairs the Capability of the Growth Hormone Secretagogue Receptor to Regulate the Dopamine 2 Receptor Signaling.

The growth hormone secretagogue receptor (GHSR) signals in response to ghrelin, but also acts via ligand-independent mechanisms that include either constitutive activation or interaction with other G protein-coupled receptors, such as the dopamine 2 receptor (D2R). A key target of GHSR in neurons is voltage-gated calcium channels type 2.2 (CaV2.2). Recently, the liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as a novel GHSR ligand, but the mechanism of action of LEAP2 on GHSR is not well understood. Here, we investigated the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on CaV2.2 function. Using a heterologous expression system and patch-clamp recordings, we found that LEAP2 impairs the reduction of CaV2.2 currents induced by ghrelin-evoked and constitutive GHSR activities, acting as a GHSR antagonist and inverse agonist, respectively. We also found that LEAP2 prevents GHSR from modulating the effects of D2R signaling on CaV2.2 currents, and that the GHSR-binding N-terminal region LEAP2 underlies these effects. Using purified labeled receptors assembled into lipid nanodiscs and Forster Resonance Energy Transfer (FRET) assessments, we found that the N-terminal region of LEAP2 stabilizes an inactive conformation of GHSR that is dissociated from Gq protein and, consequently, reverses the effect of GHSR on D2R-dependent Gi activation. Thus, our results provide critical molecular insights into the mechanism mediating LEAP2 modulation of GHSR.

Allosteric modulation of ghrelin receptor signaling by lipids.

The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated.

Development of Nonpeptidic Inverse Agonists of the Ghrelin Receptor (GHSR) Based on the 1,2,4-Triazole Scaffold.

GHSR controls, among others, growth hormone and insulin secretion, adiposity, feeding, and glucose metabolism. Therefore, an inverse agonist ligand capable of selectively targeting GHSR and reducing its high constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In this context, we present a study that led to the development of several highly potent and selective inverse agonists of GHSR based on the 1,2,4-triazole scaffold. We demonstrate that, depending on the nature of the substituents on positions 3, 4, and 5, this scaffold leads to ligands that exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. Thanks to an in vivo evaluation, we also show that one of the most promising ligands not only exerts an effect on insulin secretion in rat pancreatic islets but also affects the orexigenic effects of ghrelin in mice.

Biotinylated non-ionic amphipols for GPCR ligands screening.

We present herein the synthesis of biotin-functionalized polymers (BNAPols) that have been developed for the fixation of membrane proteins (MPs) onto surfaces. BNAPols were synthesized by free-radical polymerization of a tris(hydroxymethyl)acrylamidomethane (THAM)-derived amphiphilic monomer in the presence of a thiol-based transfer agent with an azido group. Then a Huisgen-cycloaddition reaction was performed with Biotin-(PEG)8-alkyne that resulted in formation of the biotinylated polymers. The designed structure of BNAPols was confirmed by NMR spectroscopy, and a HABA/avidin assay was used for estimating the percentage of biotin grafted on the polymer end chain. The colloidal characterization of these biotin-functionalized polymers was done using both dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. BNAPols were used to stabilize a model G protein-coupled receptor (GPCR), the human Growth Hormone Secretagogue Receptor (GHSR), out of its membrane environment. Subsequent immobilization of the BNAPols:GHSR complex onto a streptavidin-coated surface allowed screening of ligands based on their ability to bind the immobilized receptor. This opens the way to the use of biotinylated NAPols to immobilize functional, unmodified, membrane proteins, providing original sensor devices for multiple applications including innovative ligand screening assays.

Structure of the agonist 12-HHT in its BLT2 receptor-bound state.

G Protein-Coupled receptors represent the main communicating pathway for signals from the outside to the inside of most of eukaryotic cells. They define the largest family of integral membrane receptors at the surface of the cells and constitute the main target of the current drugs on the market. The low affinity leukotriene receptor BLT2 is a receptor involved in pro- and anti-inflammatory pathways and can be activated by various unsaturated fatty acid compounds. We present here the NMR structure of the agonist 12-HHT in its BLT2-bound state and a model of interaction of the ligand with the receptor based on a conformational homology modeling associated with docking simulations. Put into perspective with the data obtained with leukotriene B4, our results illuminate the ligand selectivity of BLT2 and may help define new molecules to modulate the activity of this receptor.

Development of a novel fluorescent ligand of growth hormone secretagogue receptor based on the N-Terminal Leap2 region.

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently recognized as an endogenous ligand for the growth hormone secretagogue receptor (GHSR), which also is a receptor for the hormone ghrelin. LEAP2 blocks ghrelin-induced activation of GHSR and inhibits GHSR constitutive activity. Since fluorescence-based imaging and pharmacological analyses to investigate the biology of GHSR require reliable probes, we developed a novel fluorescent GHSR ligand based on the N-terminal LEAP2 sequence, hereafter named F-LEAP2. In vitro, F-LEAP2 displayed binding affinity and inverse agonism to GHSR similar to LEAP2. In a heterologous expression system, F-LEAP2 labeling was specifically observed in the surface of GHSR-expressing cells, in contrast to fluorescent ghrelin labeling that was mainly observed inside the GHSR-expressing cells. In mice, centrally-injected F-LEAP2 reduced ghrelin-induced food intake, in a similar fashion to LEAP2, and specifically labeled cells in GHSR-expressing brain areas. Thus, F-LEAP2 represents a valuable tool to study the biology of GHSR in vitro and in vivo.

Structure and dynamics of G protein-coupled receptor-bound ghrelin reveal the critical role of the octanoyl chain.

Ghrelin plays a central role in controlling major biological processes. As for other G protein-coupled receptor (GPCR) peptide agonists, the structure and dynamics of ghrelin bound to its receptor remain obscure. Using a combination of solution-state NMR and molecular modeling, we demonstrate that binding to the growth hormone secretagogue receptor is accompanied by a conformational change in ghrelin that structures its central region, involving the formation of a well-defined hydrophobic core. By comparing its acylated and nonacylated forms, we conclude that the ghrelin octanoyl chain is essential to form the hydrophobic core and promote access of ghrelin to the receptor ligand-binding pocket. The combination of coarse-grained molecular dynamics studies and NMR should prove useful in improving our mechanistic understanding of the complex conformational space explored by a natural peptide agonist when binding to its GPCR. Such information should also facilitate the design of new ghrelin receptor-selective drugs.

N-Terminal Liver-Expressed Antimicrobial Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the Ghrelin Receptor.

The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.

GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation.

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

Illuminating the Energy Landscape of GPCRs: The Key Contribution of Solution-State NMR Associated with Escherichia coli as an Expression Host.

Conformational dynamics of GPCRs are central to their function but are difficult to explore at the atomic scale. Solution-state NMR has provided the major contribution in that area of study during the past decade, despite nonoptimized labeling schemes due to the use of insect cells and, to a lesser extent, yeast as the main expression hosts. Indeed, the most efficient isotope-labeling scheme ever to address energy landscape issues for large proteins or protein complexes relies on the use of 13CH3 probes immersed in a perdeuterated dipolar environment, which is essentially out of reach of eukaryotic expression systems. In contrast, although its contribution has been underestimated because of technical issues, Escherichia coli is by far the best-adapted host for such labeling. As it is now tightly controlled, we show in this review that bacterial expression can provide an NMR spectral resolution never achieved in the GPCR field.

Fluorescence Study of Imidazoquinoxalines.

The fluorescence properties of eleven novel derivatives based on the imidazo[1,2-a]quinoxaline structures have been studied. The absorption and emission spectra of these compounds have been recorded in dimethylsulfoxide solution. The phenyl substituting group on position 1 gives them particular properties thanks to the diverse hydroxy or methoxy decorating moieties, especially when they are multiplied or mixed. The investigated fluorescence auto-quenching revealed that the decreasing fluorescence intensity correlated only with the chemical structures of the aromatic compounds.

Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer.

Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.

New ligands of the ghrelin receptor based on the 1,2,4-triazole scaffold by introduction of a second chiral center.

Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the 'C-terminal part' of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog.