Caterina Ambrosio

Morphine-like Opiates Selectively Antagonize Receptor-Arrestin Interactions

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of μ and δ receptors with G protein or β-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gβ1. In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was Gα-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at δ and μ receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (δ) or partial agonists (μ) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.

Cell Contact-dependent Functional Selectivity of β2-Adrenergic Receptor Ligands in Stimulating cAMP Accumulation and Extracellular Signal-regulated Kinase Phosphorylation

Background: β2-Adrenegic receptor (β2-AR) mediates cAMP accumulation and ERK phosphorylation via different transducers. Results: Some β2-AR ligands selectively activate cAMP and ERK responses in HEK-293 cells. Selectivity is cell contact-dependent. Conclusion: β2-AR stimulates ERK phosphorylation by cAMP-dependent and Gi/Go-mediated pathways. Cell contact inhibits the cAMP-dependent pathway. Significance: Cell adherence/contact is an important modulatory factor in biased signaling by GPCR ligands. Activation of β2-adrenegic receptor (β2-AR) leads to an increase in intracellular cAMP and activation of ERK. These two signals are activated by the interaction of the receptor with different transducer partners. We showed that the intrinsic activities of β2-AR ligands for stimulating cAMP production and ERK phosphorylation responses in HEK-293 cells were not correlated. The lack of correlation resulted mainly from the discrepancy between the intrinsic activities of two groups of ligands for these two responses: The first group consisted of clenbuterol, cimaterol, procaterol, and terbutaline which acted as full agonists for cAMP production but displayed very weak effect on ERK phosphorylation. The second group comprised adrenaline and noradrenaline which displayed higher intrinsic activity for the ERK phosphorylation than for the cAMP response. Thus, both groups behaved as functionally selective ligands. The functional selectivity of the first group was observable only in adherent cells when confluence was approximately 100%. When cell-cell contact was minimized either by decreasing the density of the adherent cells or by bringing the cells into suspension, the first group of ligands gained the ability to stimulate ERK phosphorylation without a change in their effect on cAMP production. In contrast, selectivity of the second group was independent of the adherence state of the cells. Our results show that the inherent “bias” of ligands in coupling a G protein-coupled receptor to different transducers may not always be revealed as functional selectivity when there is a “cross-talk” between the signaling pathways activated by the same receptor.

Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the β2-adrenergic receptor

We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human β2 adrenergic receptor (β2AR). A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol‐binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild‐type β2AR. Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. We also found that AA mutation‐induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild‐type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the β‐ethanolamine tail with other residues of the β2‐AR binding pocket.

Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

Divergent agonist selectivity in activating β1- and β2-adrenoceptors for G-protein and arrestin coupling.

The functional selectivity of adrenergic ligands for activation of β1- and β2-AR (adrenoceptor) subtypes has been extensively studied in cAMP signalling. Much less is known about ligand selectivity for arrestin-mediated signalling pathways. In the present study we used resonance energy transfer methods to compare the ability of β1- and β2-ARs to form a complex with the G-protein β-subunit or β-arrestin-2 in response to a variety of agonists with various degrees of efficacy. The profiles of β1-/β2-AR selectivity of the ligands for the two receptor-transducer interactions were sharply different. For G-protein coupling, the majority of ligands were more effective in activating the β2-AR, whereas for arrestin coupling the relationship was reversed. These data indicate that the β1-AR interacts more efficiently than β2-AR with arrestin, but less efficiently than β2-AR with G-protein. A group of ligands exhibited β1-AR-selective efficacy in driving the coupling to arrestin. Dobutamine, a member of this group, had 70% of the adrenaline (epinephrine) effect on arrestin via β1-AR, but acted as a competitive antagonist of adrenaline via β2-AR. Thus the structure of such ligands appears to induce an arrestin-interacting form of the receptor only when bound to the β1-AR subtype.

Different Structural Requirements for the Constitutive and the Agonist-induced Activities of the β2-Adrenergic Receptor

We converted Ser-207, located in helix 5 of the β2-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to Gs activation, we expressed the mutants in a Gαs-tethered form. GTP exchange in such constructs is restricted to the fused α-subunit and is a linear function of the receptor concentration. Except S207R, all other mutants were expressed to a suitable level for investigation. All mutations reduced the binding affinities of the catechol agonists, epinephrine and isoproterenol, and the extent of reduction was unrelated to the residue ability to form hydrogen bonds. Instead, both enhancements and reductions of affinity were observed for the partial agonist halostachin and the antagonist pindolol. The mutations also enhanced and diminished ligand-induced receptor activation, but the effects were strictly ligand-specific. Polar residues such as Asp and His exalted the activation by full agonists but suppressed that induced by the partial agonists halostachin and dichloroisoproterenol. In contrast, hydrophobic residues such as Ile and Val augmented partial agonist activation. Only Ile and Lys produced a significant increase of constitutive activity. The effects on binding and activity were not correlated, nor did such parameters show any clear correlation with up to 78 descriptors of amino acid physicochemical properties. Our data question the idea that Ser-207 is exposed to the polar crevice in the unbound receptor. They also suggest that the active receptor form induced by a full agonist might be substantially different from that caused by constitutive activation.

Pharmacological characterization of tachykinin tetrabranched derivatives

Peptide welding technology (PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity and reproducibility. Using this technique, we have synthesized and pharmacologically characterized the tetrabranched derivatives of the tachykinins, substance P (SP), neurokinin A (NKA) and B (NKB).

Systematic errors in detecting biased agonism: Analysis of current methods and development of a new model-free approach

Discovering biased agonists requires a method that can reliably distinguish the bias in signalling due to unbalanced activation of diverse transduction proteins from that of differential amplification inherent to the system being studied, which invariably results from the non-linear nature of biological signalling networks and their measurement. We have systematically compared the performance of seven methods of bias diagnostics, all of which are based on the analysis of concentration-response curves of ligands according to classical receptor theory. We computed bias factors for a number of β-adrenergic agonists by comparing BRET assays of receptor-transducer interactions with Gs, Gi and arrestin. Using the same ligands, we also compared responses at signalling steps originated from the same receptor-transducer interaction, among which no biased efficacy is theoretically possible. In either case, we found a high level of false positive results and a general lack of correlation among methods. Altogether this analysis shows that all tested methods, including some of the most widely used in the literature, fail to distinguish true ligand bias from “system bias” with confidence. We also propose two novel semi quantitative methods of bias diagnostics that appear to be more robust and reliable than currently available strategies.

Propranolol enhances cell cycle‐related gene expression in pressure overloaded hearts

BACKGROUND AND PURPOSE Cell cycle regulators are regarded as essential for cardiomyocyte hypertrophic growth. Given that the β‐adrenoceptor antagonist propranolol blunts cardiomyocyte hypertrophic growth, we determined whether propranolol alters the expression of cell cycle‐related genes in mouse hearts subjected to pressure overload.

The effects of agonist stimulation and β2-adrenergic receptor level on cellular distribution of Gsα protein

Abstract This study examines the effects of adrenergic ligands, cholera toxin, forskolin, and varying levels of β2 adrenergic receptors (β2AR) on the cellular distribution of Gsα subunits in CHO cells. Localization of Gsα was evaluated by confocal microscopy and β2AR-mediated signalling was assessed by adenylyl cyclase (AC) activity. In cells expressing 0.2 pmol/mg protein β2ARs (WT18), the localization of Gsα subunit was restricted to the plasma membrane region. Isoproterenol (ISO), cholera toxin or forskolin elicited redistribution of cellular Gsα so that Gsα appeared as intense spots throughout the plasma membrane as well as the cytoplasm. Exposure to a neutral β2AR antagonist, alprenolol, prevented the ISO-stimulated Gsα translocation from peripheral to inner cytoplasm. In cells expressing high level of β2ARs (8.2 pmol/mg) (WT4), basal and ISO-stimulated AC activities were significantly elevated when compared to the values detected in WT18 clone, suggesting a positive correlation between receptor expression and receptor-mediated signalling. Basal Gsα distribution in this group was similar to that observed in ISO-, cholera toxin-, or forskolin-stimulated WT18 clone. ISO, cholera toxin, or forskolin did not change the distribution of Gsα significantly when tested in WT4 clone. No difference in the cellular level of Gsα protein between WT18 and WT4 clones was detected. Alprenolol did not affect the distribution of Gsα in WT4 clone. ICI 118,551, a negative β2AR antagonist, altered Gsα distribution from a dispersed basal pattern to a membrane-confined pattern. The latter appearance was similar to that observed in unstimulated WT18 clone. Taken together, these data suggest that: (1) enhanced β2AR–Gsα coupling induced by agonist stimulation or by increased expression of β2ARs remodel the cellular distribution of Gsα; (2) the alteration in Gsα distribution induced by β2AR overexpression provides evidence for agonist-independent interaction of β2AR and Gsα, that can be inhibited by a negative antagonist but not by a neutral antagonist; and (3) forskolin influences the activity state of Gsα that displays a Gsα distribution pattern comparable to that observed when Gsα is activated via β2AR stimulation or directly by cholera toxin.