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

Institute of Cell Signalling, Medical School, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom

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 ₂-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 K_D 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 ₂-adrenoceptors. Longer-term cAMP studies also showed significant differences in K_D 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 ₂-adrenoceptor was used. This study suggests that high-efficacy agonists induce a chemical modification in ₂-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.

Address correspondence to: Prof. S. J. Hill, Institute of Cell Signaling, Queen's Medical Centre, Nottingham NG7 2UH, UK. E-mail: stephen.hill{at}nottingham.ac.uk

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