ABSTRACT
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 ß-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.
Subject(s)
Adrenergic Agonists/metabolism , Biological Assay , Chromogranins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Receptors, Adrenergic, beta-2/metabolism , beta-Arrestins/metabolism , Adrenergic Agonists/pharmacology , Bias , Chromogranins/genetics , Clenbuterol/metabolism , Clenbuterol/pharmacology , Dopamine/metabolism , Dopamine/pharmacology , Epinephrine/metabolism , Epinephrine/pharmacology , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , Gene Expression , HEK293 Cells , Humans , Isoetharine/metabolism , Isoetharine/pharmacology , Isoproterenol/metabolism , Isoproterenol/pharmacology , Ligands , Monte Carlo Method , Protein Binding , Receptors, Adrenergic, beta-2/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Regression Analysis , beta-Arrestins/geneticsABSTRACT
Extracellular ligand binding to G protein-coupled receptors (GPCRs) modulates G protein and ß-arrestin signaling by changing the conformational states of the cytoplasmic region of the receptor. Using site-specific (19)F-NMR (fluorine-19 nuclear magnetic resonance) labels in the ß(2)-adrenergic receptor (ß(2)AR) in complexes with various ligands, we observed that the cytoplasmic ends of helices VI and VII adopt two major conformational states. Changes in the NMR signals reveal that agonist binding primarily shifts the equilibrium toward the G protein-specific active state of helix VI. In contrast, ß-arrestin-biased ligands predominantly impact the conformational states of helix VII. The selective effects of different ligands on the conformational equilibria involving helices VI and VII provide insights into the long-range structural plasticity of ß(2)AR in partial and biased agonist signaling.
Subject(s)
Adrenergic beta-2 Receptor Agonists/metabolism , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction , Adrenergic beta-2 Receptor Agonists/chemistry , Adrenergic beta-2 Receptor Agonists/pharmacology , Arrestins/metabolism , Binding Sites , Carbazoles/chemistry , Carbazoles/metabolism , Carbazoles/pharmacology , Carvedilol , Cytoplasm/chemistry , Drug Partial Agonism , Fluorine , Isoetharine/chemistry , Isoetharine/metabolism , Isoetharine/pharmacology , Isoproterenol/metabolism , Ligands , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Propanolamines/chemistry , Propanolamines/metabolism , Propanolamines/pharmacology , Protein Conformation , Protein Structure, Secondary , Structure-Activity Relationship , beta-ArrestinsABSTRACT
Isoprenaline, isoetharine, rimiterol, dobutamine and nadolol were investigated as substrates for purified pig-liver catechol-O-methyltransferase using a sensitive spectrophotometric assay. Kinetic parameters, Km and Vmax, were defined and the apparent first-order rate constant (Vmax/Km) was derived. On the basis of the apparent first-order rate constant, rimiterol was found to be a 1.5-fold and dobutamine a 5-fold better substrate for catechol-O-methyltransferase than isoprenaline; isoetharine shows no improvement over isoprenaline. Nadolol is not a substrate for catechol-O-methyltransferase. O-Methylation of isoprenaline- and noradrenaline-enantiomers was found to be stereoselective: catechol-O-methyltransferase shows selectivity towards the laevo (-) isomer with respect to the (+) form or racemic mixture. The investigation indicated stereochemical and steric determinants important in the interaction of catechol-O-methyltransferase with physiologically and clinically important beta-adrenoceptor agents.
