Phosphorylation motif dictates GPCR C-terminal domain conformation and arrestin interaction.

Arrestin-dependent G protein-coupled receptor (GPCR) signaling pathway is regulated by the phosphorylation state of GPCR's C-terminal domain, but the molecular bases of arrestin:receptor interaction are to be further illuminated. Here we investigated the impact of phosphorylation on the conformational features of the C-terminal region from three rhodopsin-like GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR), and the ß2-adernergic receptor (ß2AR). Using phosphomimetic variants, we identified pre-formed secondary structure elements, or short linear motifs (SLiMs), that undergo specific conformational transitions upon phosphorylation. Of importance, such conformational transitions appear to favor arrestin-2 binding. Hence, our results suggest a model in which the phosphorylation-dependent structuration of the GPCR C-terminal regions would modulate arrestin binding and therefore signaling outcomes in arrestin-dependent pathways.

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.

Fluorescent P-Hydroxyphosphole for Peptide Labeling through P-N Bond Formation.

Synthesis of fluorescent P-hydroxybinaphtylphosphole-oxide or -sulfide was achieved by trapping a binaphtyl dianion with methyl dichlorophosphite or P-(N,N-diethylamino)dichlorophosphine, followed by oxidation or sulfuration of the P-center. After saponification or acid hydrolysis, the P-hydroxyphospholes were coupled to peptides using the coupling agent BOP, under the conditions required for the synthesis in solution or on a solid support. This new method was illustrated by the labeling of the JMV2959, a potent antagonist of the Growth Hormone Secretagogue Receptor type 1a (GHS-R1a). The labeled conjugates were used to characterize GHSR ligands by competition assays, based on Fluorescence Resonance Energy Transfer (FRET). Such P-hydroxyphosphole-oxide or -sulfide constitute a promising new class of compact fluorophores with large Stokes shift, for labeling biomolecules by grafting through the phosphorus atom.

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.

Structural Insights into the Intrinsically Disordered GPCR C-Terminal Region, Major Actor in Arrestin-GPCR Interaction.

Arrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the ß2-adernergic receptor (ß2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognition.

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.

Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.

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.

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.

The ups and downs of growth hormone secretagogue receptor signaling.

The growth hormone secretagogue receptor (GHSR) has emerged as one of the most fascinating molecules from the perspective of neuroendocrine control. GHSR is mainly expressed in the pituitary and the brain, and plays key roles regulating not only growth hormone secretion but also food intake, adiposity, body weight, glucose homeostasis and other complex functions. Quite atypically, GHSR signaling displays a basal constitutive activity that can be up- or downregulated by two digestive system-derived hormones: the octanoylated-peptide ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2), which was recently recognized as an endogenous GHSR ligand. The existence of two ligands with contrary actions indicates that GHSR activity can be tightly regulated and that the receptor displays the capability to integrate such opposing inputs in order to provide a balanced intracellular signal. This article provides a summary of the current understanding of the biology of ghrelin, LEAP2 and GHSR and discusses the reconceptualization of the cellular and physiological implications of the ligand-regulated GHSR signaling, based on the latest findings.

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.

IDPs and their complexes in GPCR and nuclear receptor signaling.

G protein-coupled receptors (GPCRs) and Nuclear Receptors (NRs) are two signaling machineries that are involved in major physiological processes and, as a consequence, in a substantial number of diseases. Therefore, they actually represent two major targets for drugs with potential applications in almost all public health issues. Full exploitation of these targets for therapeutic purposes nevertheless requires opening original avenues in drug design, and this in turn implies a better understanding of the molecular mechanisms underlying their functioning. However, full comprehension of how these complex systems function and how they are deregulated in a physiopathological context is obscured by the fact that these proteins include a substantial number of disordered regions that are central to their mechanism of action but whose structural and functional properties are still largely unexplored. In this chapter, we describe how these intrinsically disordered regions (IDR) or proteins (IDP) intervene, control and finely modulate the thermodynamics of complexes involved in GPCR and NR regulation, which in turn triggers a multitude of cascade of events that are exquisitely orchestrated to ultimately control the biological output.

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.