Kinetic analysis of the dissociation rate of fluorescent CGP 1277 (BODIPY-TMR-CGP) indicates an allosteric mechanism of action of CGP 12177 at the human β 1 -adrenoceptor.

Karolina Salchow, Stephen Briddon, Stephen Hill. University of Nottingham, School of Biomedical Sciences, NG7 2UH, Nottingham, UK

CGP 12177 was initially described as an antagonist of the high affinity catecholamine site of the human β1-adrenoceptor. However, it has since been shown to exert agonist effects through a second, low affinity “CGP 12177” site of the receptor which is largely resistant to classical β-blockers such as propranolol (Baker et al., 2003). We have previously characterised the fluorescent ligand BODIPY-TMR-CGP (a CGP 12177 analogue) at the human β1-adrenoceptor (Salchow et al., 2012). In this study, we aimed to investigate the dynamics of ligand binding to the β1-adrenoceptor by examining the effects of unlabelled ligands on the dissociation rate of BODIPY-TMR-CGP at the single level.

Live cell imaging of Chinese Hamster Ovary (CHO) cells stably expressing the human β1-adrenoceptor (CHO-β1-CS cells) seeded onto 32 mm coverslips was performed on a Zeiss LSM 710 confocal microscope. The dissociation kinetics of BODIPY-TMR-CGP were obtained using the method of infinite dilution on a temperature-controlled perfusion system (May et al., 2010). CHO-β1-CS cells were first exposed to HEPES-buffered saline solution (HBSS; 30 s; baseline fluorescence read), then 3 nM BODIPY-TMR-CGP (4 min; association), followed by HBSS in the absence or presence of propranolol or CGP12177 (4 min; dissociation). Cells were imaged every 2 s at 37 °C. Regions of interest were drawn around the plasma membrane of 10 cells per experiment (n) to measure the fluorescence intensity change over time. Dissociation data were fitted to a monoexponential decay equation using GraphPad Prism 5. Statistical analysis involved 1-way ANOVA followed by the Dunnet’s multiple comparison test, with p<0.05 reflecting statistical significance.

In the absence of competitor ligands the dissociation rate constant (koff) of 3 nM BODIPY-TMR-CGP was 0.08±0.02 min-1 (n=3, mean±s.e.mean). This was increased in the presence of 1 and 10 µM β-adrenoceptor antagonist propranolol (koff 0.20±0.01 min-1, n=5, p<0.05 and 0.23±0.03 min-1, n=4, p<0.05 respectively) as well as 1 and 10 µM CGP 12177 (koff 0.22±0.04 min-1, n=4, p<0.05 and 0.26±0.03 min-1, n=3, p<0.05 respectively). Under infinite dilution conditions, a competitive unlabelled ligand should not affect the dissociation rate of a labelled ligand, if both ligands are competing for the same site. The BODIPY-TMR-CGP concentration used in this study should label predominantly the high affinity site, whilst the concentrations of propranolol and CGP 12177 used were sufficient to label both high and low affinity sites of the β1-adrenoceptor. These data suggest that an increased dissociation rate of BODIPY-TMR-CGP from the high affinity site is due to propranolol/CGP 12177 binding to the low affinity site and thus indicates possible cooperative interactions between the two β1-adrenoceptor binding sites. Further studies will investigate the role of β1-adrenoceptor homodimerisation in these interactions to provide further insights into the nature of the two sites of the human β1-adreneoceptor.

This work was funded by the Medical Research Council.

1. Baker JG, Hall IP, Hill SJ (2003). Agonist actions of “β-blockers” provide evidence for two agonist activation sites or conformations of the human β1-adrenoceptor. Mol Pharmacol 63(6): 1312-21.

2. Salchow K, Briddon S, Hill S (2012). Proceedings of the British Pharmacological Society at http://www.pA2online.org/abstracts/Vol10Issue1abst055P.pdf

3. May LT, Self TJ, Briddon SJ, Hill SJ (2010). The effect of allosteric modulators on the kinetics of agonist-G protein-coupled receptor interactions in single living cells. Mol Pharmacol 78(3): 511-23.