title: Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation.
abstract: 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 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-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 β1-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 β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformation.
authors.affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Karolina
authors.initials: K
authors.lastname: Gherbi
authors.affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Lauren T
authors.initials: LT
authors.lastname: May
authors.affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Jillian G
authors.initials: JG
authors.lastname: Baker
authors.affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Briddon
authors.affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom stephen.hill@nottingham.ac.uk.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D000319:Adrenergic beta-Antagonists / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001896:Boron Compounds / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003517:Cyclopentanes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011412:Propanolamines / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011758:Pyrroles / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000319:Adrenergic beta-Antagonists; C481870:BODIPY-TMR-CGP; D001896:Boron Compounds; D003517:Cyclopentanes; D008024:Ligands; D011412:Propanolamines; D011758:Pyrroles; D018342:Receptors, Adrenergic, beta-1; D011994:Recombinant Proteins; C033394:CGP 12177
keywords: GPCR; allosterism; dissociation; receptor dimerization
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