title: Influence of agonist efficacy and receptor phosphorylation on antagonist affinity measurements: differences between second messenger and reporter gene responses.
abstract: The ability of an antagonist to bind to a receptor is an innate property of that ligand-receptor chemical interaction. Provided no change in the antagonist or receptor chemical nature occurs, this affinity should remain constant for a given antagonist-receptor interaction, regardless of the agonists used. This fundamental assumption underpins the classification of receptors. Here, measurements of beta2-adrenoceptor-mediated cAMP accumulation and cAMP response-element (CRE)-mediated reporter-gene transcription revealed differences in antagonist affinity that depended upon agonist incubation time and the efficacy of the competing agonist. In cAMP accumulation studies (10-min agonist incubation), antagonist affinities were the same regardless of the agonist used. The CRE-reporter gene assay (5 h of incubation) antagonist affinities were 10-fold lower in the presence of isoprenaline and adrenaline than when salbutamol or terbutaline were present (e.g., log KD propranolol -8.65 +/- 0.08, n = 22, and -9.68 +/- 0.07, n = 17, for isoprenaline and salbutamol-induced responses, respectively). Isoprenaline and adrenaline were more efficacious in functional studies, and their ability to internalize GFP-tagged human beta2-adrenoceptors. Longer-term cAMP studies also showed significant differences in KD values moving toward that seen with gene transcription. Agonist-dependent differences in antagonist affinity were reduced for reporter-gene responses when a phosphorylation-deficient mutant of the beta2-adrenoceptor was used. This study suggests that high-efficacy agonists induce a chemical modification in beta2-adrenoceptors (via phosphorylation) that reduces antagonist affinities. Because reporter-gene assays are used for high-throughput screening in drug discovery, less efficacious or partial agonists may be more reliable than highly efficacious agonists when reporter-gene techniques are used to estimate antagonist affinity.
authors.affiliation: Institute of Cell Signaling, Queen's Medical Centre, Nottingham NG7 2UH, UK. stephen.hill@nottingham.ac.uk
authors.forename: Jillian G
authors.initials: JG
authors.lastname: Baker
authors.forename: Ian P
authors.initials: IP
authors.lastname: Hall
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D000322:Adrenergic Agonists / Q000378:metabolism* / Q000494:pharmacology; D018674:Adrenergic Antagonists / Q000378:metabolism* / Q000494:pharmacology; D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects* / Q000502:physiology*
chemical_list: D000322:Adrenergic Agonists; D018674:Adrenergic Antagonists; D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D018343:Receptors, Adrenergic, beta-2; D000242:Cyclic AMP