Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation.
Bottom Line: 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.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.
Affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.Show MeSH
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Mentions: We then examined the dissociation rate of 3 nM BODIPY-TMR-CGP at irreversibly constrained stable wild-type/wild-type β1-adrenoceptor homodimers under ID conditions, which was determined to be 0.02 ± 0.01 min−1 (n = 5; Fig. 9A). This was significantly slower than the dissociation rate measured in CHO-β1 cells (P < 0.05, 2-way ANOVA analysis followed by Tukey’s multiple comparisons test). The dissociation of 3 nM BODIPY-TMR-CGP binding was enhanced in the presence of 1 µM CGP 12177 and 1 µM propranolol with dissociation rates of 0.186 ± 0.008 (n = 6) and 0.189 ± 0.007 min−1 (n = 6), respectively (Fig. 9A). This was significantly faster than the dissociation rate determined in the absence of unlabeled ligands (P < 0.05, 2-way ANOVA followed by Tukey’s multiple comparisons test; Fig. 9A). The change in the 3 nM BODIPY-TMR-CGP dissociation rate in the absence and presence of unlabeled ligands was ∼2-fold in CHO-β1 cells but was ∼10-fold in CHO-K1 cells expressing β1YFPN/β1YFPC homodimers.
Affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.