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Negative cooperativity across ?1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 ?1-adrenoceptor binding conformation.

ABSTRACT: At the ?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 ?1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in ?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 ?1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct ?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 ?1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in ?1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a ?1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary ?1-adrenoceptor conformation.

SUBMITTER: Gherbi K

PROVIDER: S-EPMC4478806 | biostudies-other | 2015 Jul

REPOSITORIES: biostudies-other

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Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation.

Gherbi Karolina K May Lauren T LT Baker Jillian G JG Briddon Stephen J SJ Hill Stephen J SJ

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20150402 7

At the β1-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 β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP ...[more]

PMID: 25837585

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Project description:The β1-adrenoceptor exists in two agonist conformations/states: 1) a high-affinity state where responses to catecholamines and other agonists (e.g., cimaterol) are potently inhibited by β1-adrenoceptor antagonists, and 2) a low-affinity secondary conformation where agonist responses, particularly CGP12177 [(-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one] are relatively resistant to inhibition by β1-adrenoceptor antagonists. Although both states have been demonstrated in many species (including human), the precise nature of the secondary state is unknown and does not occur in the closely related β2-adrenoceptor. Here, using site-directed mutagenesis and functional measurements of production of a cyclic AMP response element upstream of a secreted placental alkaline phosphatase reporter gene and accumulation of (3)H-cAMP, we examined the pharmacological consequences of swapping transmembrane (TM) regions of the human β1- and β2-adrenoceptors, followed by single point mutations, to determine the key residues involved in the β1-adrenoceptor secondary conformation. We found that TM4 (particularly amino acids L195 and W199) had a major role in the generation of the secondary β1-adrenoceptor conformation. Thus, unlike at the human β1-wild-type adrenoceptor, at β1-TM4 mutant receptors, cimaterol and CGP12177 responses were both potently inhibited by antagonists. CGP12177 acted as a simple partial agonist with similar KB and EC50 values in the β1-TM4 but not β1-wild-type receptors. Furthermore pindolol switched from a biphasic concentration response at human β1-wild-type adrenoceptors to a monophasic concentration response in the β1-TM4 mutant receptors. Mutation of these amino acids to those found in the β2-adrenoceptor (L195Q and W199Y), or mutation of a single residue (W199D) in the human β1-adrenoceptor thus abolished this secondary conformation and created a β1-adrenoceptor with only one high-affinity agonist conformation.

Dataset Information

Negative cooperativity across ?1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 ?1-adrenoceptor binding conformation.

Publications

Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation.

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