Influence of agonist efficacy and receptor phosphorylation on antagonist affinity measurements: differences between...

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.