Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling.

The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides ß-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger ß-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.

Dosage-dependent regulation of cell proliferation and adhesion through dual ß2-adrenergic receptor/cAMP signals.

The role of ß-adrenergic receptors (ß-ARs) remains controversial in normal and tumor breast. Herein we explore the cAMP signaling involved in ß-AR-dependent control of proliferation and adhesion of nontumor human breast cell line MCF-10A. Low concentrations of a ß-agonist, isoproterenol (ISO), promote cell adhesion (87.5% cells remaining adherent to the plastic dishes following specific detachment vs. 35.0% in control, P<0.001), while increasing concentrations further engages an additional 36% inhibition of Erk1/2 phosphorylation (p-Erk1/2)-dependent cell proliferation (P<0.01). Isoproterenol dose response on cell adhesion was fitted to a 2-site curve (EC50(1): 16.5±11.5 fM, EC50(2): 4.08±3.09 nM), while ISO significantly inhibited p-Erk1/2 according to a 1-site model (EC50: 0.25±0.13 nM). Using ß-AR-selective agonist/antagonists and cAMP analogs/inhibitors, we identified a dosage-dependent signaling in which low ISO concentrations target a ß2-AR population localized in raft microdomains and stimulate a Gs/cAMP/Epac/adhesion-signaling module, while higher concentrations engage a concomitant activation of another ß2-AR population outside rafts and inhibit the proliferation by a Gs/cAMP/PKA-dependent signaling module. Our data provide a new molecular basis for the dose-dependent switch of ß-AR signaling. This study also sheds light on a new cAMP pathway core mechanism with a single receptor triggering dual cAMP signaling controlled by PKA or Epac but with different cellular outputs.

Specific interactions between Epac1, ß-arrestin2 and PDE4D5 regulate ß-adrenergic receptor subtype differential effects on cardiac hypertrophic signaling.

ß1 and ß2 adrenergic receptors (ßARs) are highly homologous but fulfill distinct physiological and pathophysiological roles. Here we show that both ßAR subtypes activate the cAMP-binding protein Epac1, but they differentially affect its signaling. The distinct effects of ßARs on Epac1 downstream effectors, the small G proteins Rap1 and H-Ras, involve different modes of interaction of Epac1 with the scaffolding protein ß-arrestin2 and the cAMP-specific phosphodiesterase (PDE) variant PDE4D5. We found that ß-arrestin2 acts as a scaffold for Epac1 and is necessary for Epac1 coupling to H-Ras. Accordingly, knockdown of ß-arrestin2 prevented Epac1-induced histone deacetylase 4 (HDAC4) nuclear export and cardiac myocyte hypertrophy upon ß1AR activation. Moreover, Epac1 competed with PDE4D5 for interaction with ß-arrestin2 following ß2AR activation. Dissociation of the PDE4D5-ß-arrestin2 complex allowed the recruitment of Epac1 to ß2AR and induced a switch from ß2AR non-hypertrophic signaling to a ß1AR-like pro-hypertrophic signaling cascade. These findings have implications for understanding the molecular basis of cardiac myocyte remodeling and other cellular processes in which ßAR subtypes exert opposing effects.

Deciphering biased-agonism complexity reveals a new active AT1 receptor entity.

Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but ß-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

Probing heterotrimeric G protein activation: applications to biased ligands.

Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands.

Functional selective oxytocin-derived agonists discriminate between individual G protein family subtypes.

We used a bioluminescence resonance energy transfer biosensor to screen for functional selective ligands of the human oxytocin (OT) receptor. We demonstrated that OT promoted the direct engagement and activation of G(q) and all the G(i/o) subtypes at the OT receptor. Other peptidic analogues, chosen because of specific substitutions in key OT structural/functional residues, all showed biased activation of G protein subtypes. No ligand, except OT, activated G(oA) or G(oB), and, with only one exception, all of the peptides that activated G(q) also activated G(i2) and G(i3) but not G(i1), G(oA), or G(oB), indicating a strong bias toward these subunits. Two peptides (DNalOVT and atosiban) activated only G(i1) or G(i3), failed to recruit ß-arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual G(i/o) family members. Both analogs inhibited cell proliferation, showing that a single G(i) subtype-mediated pathway is sufficient to prompt this physiological response. These analogs represent unique tools for examining the contribution of G(i/o) members in complex biological responses and open the way to the development of drugs with peculiar selectivity profiles. This is of particular relevance because OT has been shown to improve symptoms in neurodevelopmental and psychiatric disorders characterized by abnormal social behaviors, such as autism. Functional selective ligands, activating a specific G protein signaling pathway, may possess a higher efficacy and specificity on OT-based therapeutics.

Functional membrane diffusion of G-protein coupled receptors.

G-protein-coupled receptor function involves interactions between the receptor, G-proteins and effectors in the cell plasma membrane. The main biochemical processes have been individually identified but the mechanisms governing the successive protein-protein interactions of this complex multi-molecular machinery have yet to be established. We discuss advances in understanding the functional dynamics of the receptor resulting from diffusion measurements, and in the context of the plasma membrane organization.

CD4 interacts constitutively with multiple CCR5 at the plasma membrane of living cells. A fluorescence recovery after photobleaching at variable radii approach.

The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by Förster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-microm-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.

Diffusion of the mu opioid receptor at the surface of human neuroblastoma SH-SY5Y cells is restricted to permeable domains.

Previous single-molecule studies have shown a long-term diffusion superimposed to a short-term confinement of the human mu opioid (hMOP) receptors at the surface of heterologous cells. However, additional ensemble average measurements are required to reach a complete understanding of the undergoing process. Here, we analyse, by fluorescence recovery after photobleaching measurements, the lateral diffusion of fully functional T7-EGFP-hMOP receptors in neuroblastoma SH-SY5Y cells naturally expressing a low level of the wild-type receptor. Experiments carried out at variable observation radii demonstrate the restriction of the receptors diffusion to sub-micrometer sized domains. Furthermore, consistently with the long-term single-molecule data, the domains are found permeable.

Guanine oxidation by electron transfer: one- versus two-electron oxidation mechanism.

The degeneracy of the guanine radical cation, which is formed in DNA by oxidation of guanine by electron transfer, was studied by a detailed analysis of the oxidation products of guanine on oligonucleotide duplexes and by labeling experiments. It was shown that imidazolone, the major product of guanine oxidation, is formed through a one-electron oxidation process and incorporates one oxygen atom from O2. The formation of 8-oxo-7,8-dihydroguanine by a two-electron oxidation process was a minor pathway. The two-electron oxidation mechanism was also evidenced by the formation of a tris(hydroxymethyl)aminomethane adduct.