Influence of Agonist Efficacy and Receptor Phosphorylation on Antagonist Affinity Measurements: Differences between Second Messenger and Reporter Gene Responses

• Published in: Molecular pharmacology 2003-09, Vol.64 (3), p.679-688
• Main Authors: Baker, Jillian G, Hall, Ian P, Hill, Stephen J
• Format: Article
• Language: English
• Subjects: Adrenergic Agonists - metabolism; Adrenergic Agonists - pharmacology; Adrenergic Antagonists - metabolism; Adrenergic Antagonists - pharmacology; Adrenergic beta-2 Receptor Agonists; Adrenergic beta-2 Receptor Antagonists; Animals; CHO Cells; Cricetinae; Cyclic AMP - metabolism; Dose-Response Relationship, Drug; Genes, Reporter - drug effects; Genes, Reporter - physiology; Humans; Phosphorylation; Protein Binding - drug effects; Protein Binding - physiology; Receptors, Adrenergic, beta-2 - metabolism; Second Messenger Systems - drug effects; Second Messenger Systems - physiology
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.64.3.679

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 β2-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 β2-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 β2-adrenoceptor was used. This study suggests that high-efficacy agonists induce a chemical modification in β2-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.