Negative cooperativity across β ₁ ‐adrenoceptor homodimers provides insights into the nature of the secondary low‐affinity CGP 12177 β1‐adrenoceptor binding conformation

Abstract

ABSTRACT At the β 1 ‐adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high‐affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low‐affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β 1 ‐adrenoceptor as key for this low‐affinity conformation. Others suggested that TM4 has a role in β 1 ‐adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane‐tetramethylrhodamine‐(±)CGP 12177 (BODIPY‐TMR‐CGP)] at the human β 1 ‐adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β 1 ‐adrenoceptor conformations. The dissociation rate of 3 nM BODIPY‐TMR‐CGP was 0.09 ± 0.01 min ‐1 in the absence of competitor ligands, and this was enhanced 2.2‐ and 2.1‐fold in the presence of 1 μM CGP 12177 and 1 μM propranolol, respectively. These effects on the BODIPY‐TMR‐CGP dissociation rate were markedly enhanced in β 1 ‐adrenoceptorhomodimers constrained by bimolecular fluorescence complementation (9.8‐ and 9.9‐fold for 1 μM CGP 12177 and 1 μM propranolol, respectively) and abolished in β 1 ‐adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β 1 ‐adrenoceptor homodimer may be responsible for generating the low‐affinity pharmacology of the secondary β 1 ‐adrenoceptor conformation.—Gherbi, K., May, L. T., Baker, J. G., Briddon, S. J., Hill, S. J. Negative cooperativity across β 1 ‐adrenoceptor homodimers provides insights into the nature of the secondary low‐affinity CGP 12177 β1‐adrenoceptor binding conformation. FASEB J . 29, 2859‐2871 (2015). www.fasebj.org