Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

Drug efficacy is typically considered an intrinsic property of a ligand/receptor couple. However, recent observations suggest that efficacy may also be influenced by the signaling effectors engaged by a unique receptor. To directly and systematically test this possibility, we assessed the ability of a panel of ␤-adrenergic ligands to modulate the activity of two effector systems, the adenylyl cyclase (AC) and the mitogen-activated protein kinase (MAPK), via ␤ 1 and ␤ 2 adrenergic receptors. Although some compounds displayed similar efficacies toward the two pathways, others showed complex efficacy profiles. For example, compounds that are inverse agonists for the AC activity were found to be either agonists, neutral antagonists, or inverse agonists for the MAPK pathway. Likewise, agonists for the AC were either agonists or neutral antagonists for MAPK.Given this complexity, we propose a Cartesian representation of the efficacies that takes into account the activities of the different effectors that can be engaged by a given receptor. In addition, compounds considered as nonselective for ␤ 1 and ␤ 2 adrenergic receptors, based on their binding affinities, showed distinct relative efficacy profiles toward AC and MAPK, adding a new dimension to the concept of ligand selectivity. Taken together, the results suggest that binding of different ligands promote distinct conformational changes leading to specific signaling outcomes. Our data therefore clearly illustrate that efficacy is a pluridimensional parameter that is not an intrinsic characteristic of a ligand/receptor couple. This should have important implications for the future design of screening assays used in drug discovery campaigns.

No abstract

Determining the role of lipid raft nanodomains in G proteincoupled receptor signaling remains fraught by the lack of assays directly monitoring rafts in native membranes. We thus combined extensive biochemical and pharmacological approaches to a nanoscale strategy based on bioluminescence resonance energy transfer (BRET) to assess the spatial and functional influence of cholesterol-rich liquid-ordered lipid nanodomains on ␤ 2 adrenergic receptor (␤ 2 AR) signaling. The data revealed that whereas ␤ 2 AR did not partition within liquid-ordered lipid phase, a pool of G protein and adenylyl cyclase (AC) were sequestered in these domains. Destabilization of the liquid-ordered phase by cholesterol depletion led to a lateral redistribution of G␣ s and AC that favored interactions between the receptor and its signaling partners as assessed by BRET. This resulted in an increased basal and agonist-promoted ␤ 2 AR-stimulated cAMP production that was partially dampened as a result of constitutive protein kinase A-dependent phosphorylation and desensitization of the receptor. This restraining influence of nanodomains on ␤ 2 AR signaling was further substantiated by showing that liquid-ordered lipid phase stabilization using caveolin overexpression or increasing membrane cholesterol amount led to an inhibition of ␤ 2 AR-associated signaling. Given the emerging concept that clustering of receptors and effectors into signaling platforms contributes to the efficacy and selectivity of signal transduction, our results support a model whereby cholesterol-promoted liquid-ordered lipid phase-embedding G s and AC allows their lateral separation from the receptor, thus restraining the basal activity and controlling responsiveness of ␤ 2 AR signaling machinery within larger signaling platforms.

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).

In recent years, several studies have demonstrated that different ligands can have distinct efficacy profiles toward various signaling pathways through a unique receptor. For example, ␤1-adrenergic compounds that are inverse agonists toward the adenylyl cyclase (AC) can display agonist activity for the mitogen-activated protein kinase (MAPK) pathway. Such a phenomenon, often termed functional selectivity, has now been clearly established for many G protein-coupled receptors when considering distinct signaling output. However, the possibility that ligands could selectively engage distinct effectors to activate a single signaling output by promoting specific receptor conformations has not been extensively examined. Here, we took advantage of the fact that isoproterenol, bucindolol and propranolol (full, partial, and inverse agonists for the AC pathway, respectively) all activate MAPK through the ␤1-adrenergic receptor (␤1AR) to probe such conformational-biased signaling. Although the three compounds stimulated MAPK in a srcdependent manner, isoproterenol acted through both G␣ i ␤␥-and G protein-independent pathways, whereas bucindolol and propranolol promoted MAPK activation through the G proteinindependent pathway only. The existence of such distinct signaling cascades linking ␤1AR to MAPK activation was correlated with ligand-specific conformational rearrangements of receptor/G protein complexes measured by bioluminescence resonance energy transfer. Taken together, our data indicate that discrete local conformational changes can selectively promote the recruitment of distinct proximal signaling partners that can engage distinct signaling outputs and/or converge on the same signaling output.G protein-coupled receptors (GPCRs) represent the most prevalent class of transmembrane signaling proteins. They can modulate a large variety of signaling systems to ensure a fine regulation of cell function in response to external stimuli. This signaling diversity is achieved in part by the capacity of one receptor to couple to diverse G proteins and non-G protein effectors (Bockaert et al., 2004). Over the past decade, many studies have demonstrated that different subsets of these effector systems can be selectively modulated by distinct ligands through a unique receptor, a phenomenon often referred to as ligand-biased signaling (Galandrin et al., 2007;Kenakin, 2007). Hence, efficacy of GPCR ligands is increasingly considered as a pluridimensional parameter that should include in its definition, in addition to the ligand/ receptor pair, the signaling pathways considered.Characterizing a panel of ␤-adrenergic ligands for their efficacy profiles toward two of the ␤1-adrenergic receptor (␤1AR)-stimulated signaling pathways, adenylyl cyclase (AC) and mitogen-activated protein kinase (MAPK) path- Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.043893.ABBREVIATIONS: GPCR, G protein-coupled receptor; ␤AR, ␤-adrenergic receptor; AC, adenylyl cyclase; ...

Hyperactivity of the renin–angiotensin–aldosterone system through the angiotensin II (Ang II)/Ang II type 1 receptor (AT1-R) axis constitutes a hallmark of hypertension. Recent findings indicate that only a subset of AT1-R signaling pathways is cardiodeleterious, and their selective inhibition by biased ligands promotes therapeutic benefit. To date, only synthetic biased ligands have been described, and whether natural renin–angiotensin–aldosterone system peptides exhibit functional selectivity at AT1-R remains unknown. In this study, we systematically determined efficacy and potency of Ang II, Ang III, Ang IV, and Ang-(1–7) in AT1-R–expressing HEK293T cells on the activation of cardiodeleterious G-proteins and cardioprotective β-arrestin2. Ang III and Ang IV fully activate similar G-proteins than Ang II, the prototypical AT1-R agonist, despite weaker potency of Ang IV. Interestingly, Ang-(1–7) that binds AT1-R fails to promote G-protein activation but behaves as a competitive antagonist for Ang II/Gi and Ang II/Gq pathways. Conversely, all renin–angiotensin–aldosterone system peptides act as agonists on the AT1-R/β-arrestin2 axis but display biased activities relative to Ang II as indicated by their differences in potency and AT1-R/β-arrestin2 intracellular routing. Importantly, we reveal Ang-(1–7) a known Mas receptor-specific ligand, as an AT1-R–biased agonist, selectively promoting β-arrestin activation while blocking the detrimental Ang II/AT1-R/Gq axis. This original pharmacological profile of Ang-(1–7) at AT1-R, similar to that of synthetic AT1-R–biased agonists, could, in part, contribute to its cardiovascular benefits. Accordingly, in vivo, Ang-(1–7) counteracts the phenylephrine-induced aorta contraction, which was blunted in AT1-R knockout mice. Collectively, these data suggest that Ang-(1–7) natural-biased agonism at AT1-R could fine-tune the physiology of the renin–angiotensin–aldosterone system.

Hypersecretion of norepinephrine (NE) and angiotensin II (AngII) is a hallmark of major prevalent cardiovascular diseases that contribute to cardiac pathophysiology and morbidity. Herein, we explore whether heterodimerization of presynaptic AngII AT1 receptor (AT1-R) and NE α2C-adrenergic receptor (α2C-AR) could underlie their functional cross-talk to control NE secretion. Multiple bioluminescence resonance energy transfer and protein complementation assays allowed us to accurately probe the structures and functions of the α2C-AR–AT1-R dimer promoted by ligand binding to individual protomers. We found that dual agonist occupancy resulted in a conformation of the heterodimer different from that induced by active individual protomers and triggered atypical Gs-cAMP–PKA signaling. This specific pharmacological signaling unit was identified in vivo to promote not only NE hypersecretion in sympathetic neurons but also sympathetic hyperactivity in mice. Thus, we uncovered a new process by which GPCR heterodimerization creates an original functional pharmacological entity and that could constitute a promising new target in cardiovascular therapeutics.