G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

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.

G-protein-coupled receptors (GPCRs) are key players in cell communication. Although long considered as monomeric, it now appears that these heptahelical proteins can form homo-or heterodimers. Here, we analyzed the conformational changes in each subunit of a receptor dimer resulting from agonist binding to either one or both subunits by measuring the fluorescent properties of a leukotriene B 4 receptor dimer with a single 5-hydroxytryptophan-labeled protomer. We show that a receptor dimer with only a single agonist-occupied subunit can trigger G-protein activation. We also show that the two subunits of the receptor dimer in the G-protein-coupled state differ in their conformation, even when both are liganded by the agonist. No such asymmetric conformational changes are observed in the absence of G-protein, indicating that the interaction of the G-protein with the receptor dimer brings specific constraints that prevent a symmetric functioning of this dimer. These data open new options for the differential signaling properties of GPCR dimers.

Background: GHS-R1a activates multiple signaling pathways mediating feeding and addictive behaviors. Results: Some GHS-R1a ligands activate G q but not G i/o and fail to recruit ␤-arrestin2; others act as selective inverse agonists at G q compared with G 13 . Conclusion: Synthetic ligands can selectively activate or reverse G q -dependent signaling at GHS-R1a. Significance: Ligand-biased signaling can be exploited for the development of selective drugs to treat GHS-R1a-mediated disorders.

The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, μ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.membrane protein | structural repertoire | biased ligands | constitutive activity M ajor progress has been made in past years in understanding the molecular bases of G protein-coupled receptor (GPCR)-mediated signaling. Spectacular developments in GPCR stabilization and crystallization have resulted in many different crystal structures of active and inactive receptors (1) and ultimately led to the structure of the agonist-activated β 2 -adrenergic receptor in complex with its cognate Gs protein (2). However, these structures still represent unique states, whereas the dynamic character of receptors is essential for their physiological functions. Indeed, many reports point at a model where GPCRs are highly dynamic proteins capable of adopting a large number of conformational states that signal with different efficacies to various pathways (3). The most detailed description of the conformational repertoire that a GPCR can adopt has been obtained in the case of the β 2 -adrenergic receptor with an experimental demonstration of a complex conformational landscape where ligands with distinct pharmacological properties shift the conformation of the receptor from one state to another (4-11). In the same way, we showed that ligands with different efficacies stabilize different conformations of the serotonin receptor (12). Despite this wealth of data, understanding of GPCR activation mechanisms, and in particular of the necessary influence of signaling proteins on the receptor conformationa...

How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.GPCR | G protein | preassembly | conformation dynamics | signaling G protein-coupled receptors (GPCRs), one of the largest cell surface receptor families, are involved in many cellular signaling processes (1). Based on this property, as well as their importance as drug targets, the molecular aspects of GPCR functioning have been extensively investigated. In particular, coupling to heterotrimeric G proteins has been the focus of numerous studies. Indeed, delineating the molecular mechanisms responsible for receptor:G protein interaction is absolutely required to better understand how signaling is controlled. Recent years have seen spectacular advances that have culminated in elucidation of the 3D structure of the β 2 -adrenergic receptor:Gs complex (2). Nevertheless, the need for further progress remains, in particular to fully understand the dynamics of this interaction. This is a crucial question, given that how the receptor interacts with its G protein partner governs signaling, and thus biological and pathophysiological responses.To date, two different models for GPCR:G protein interaction have been proposed: collision coupling and preassembly. Originally, it was proposed that receptors and G proteins couple by collision (3, 4). One of the main features of this model is that only activated receptors interact with G proteins. Since then, alternative models of signaling have been developed. One of these, the preassembly model, proposes that the receptor and the G protein make a complex even in the absence of agonist (5-8).

G protein-coupled receptors (GPCRs) regulate numerous physiological functions. The primary difficulty presented by their study in vitro is to obtain them in sufficient amounts under a functional and stable form. Escherichia coli is a host of choice for producing recombinant proteins for structural studies. However, the insertion of GPCRs into its plasma membrane usually results in bacterial death. An alternative approach consists of targeting recombinant receptors to inclusion bodies, where they accumulate without affecting bacterial growth, and then folding them in vitro. This approach, however, stumbles over the very low folding yields typically achieved, whether in detergent solutions or in detergent-lipid mixtures. Here, we show that synthetic polymers known as amphipols provide a highly efficient medium for folding GPCRs. Using a generic protocol, we have folded four class A GPCRs to their functional state, as evidenced by the binding of their respective ligands. This strategy thus appears to have the potential to be generalized to a large number of GPCRs. These data are also of interest from a more fundamental point of view: they indicate that the structural information stored in the sequence of these four receptors allows them to reach their correct three-dimensional structure in an environment that bears no similarity, beyond the amphiphilic character, to lipid bilayers.

The simultaneous activation of many distinct G proteincoupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the G␣ subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the G␣ subunit is feasible and should constitute a new strategy for therapeutic intervention.