Studying ligand-receptor interactions at the human β1-adrenoceptor using BODIPY-TMR-CGP in single living cells

Karolina Salchow, Stephen Briddon, Stephen Hill. University of Nottingham, Queen's Medical Centre, School of Biomedical Sciences, NG7 2UH, UK

Recent evidence points to a second ligand binding site on the human β1-adrenoceptor, at which the purported antagonist CGP 12177 exerts agonist activity. This low affinity “CGP 12177” binding site is largely resistant to classical β-blockers such as propranolol [1]. The use of BODIPY-TMR-CGP (a fluorescent CGP 12177 analogue) together with confocal microscopy will allow the investigation of ligand-receptor interactions at this second binding site of the β1-adrenoceptor at the single cell level.

Firstly, we have characterised the pharmacology of BODIPY-TMR-CGP at the human β1-adrenoceptor. Functional responses were investigated in a CRE-SPAP gene reporter assay. Like CGP 12177, BODIPY-TMR-CGP stimulated an increase in CRE-mediated gene transcription, albeit with slightly reduced potency (pEC50 = 7.10±0.12 and 7.73±0.11, mean±s.e.m., n = 9 and 14, BY-CGP and CGP 12177, respectively) and efficacy (EMAX = 24.6±2.9% and 47.8±3.6% of cimaterol response, mean±s.e.m.). Affinity values determined by Schild analysis showed reduced affinity of the fluorescent ligand for the β1-adrenoceptor compared to its parent compound (pA2 = 9.23±0.06 and 9.61±0.06, mean±s.e.m., n = 10, respectively). However, BODIPY-TMR-CGP allowed visualisation of receptor distribution, as confocal imaging showed clear concentration-dependent membrane labelling of CHO-β1-CS cells (3-100 nM, 10 min), which could be displaced by the β-adrenoceptor antagonist CGP 20712A (100 nM). Additionally, using up to 100 nM BODIPY-TMR-CGP in competition binding studies performed in a high-content screening assay, further confirmed the two-site binding hypothesis for the β1-adrenoceptor.

To investigate ligand-receptor interactions at the single cell level, association and dissociation kinetics of BODIPY-TMR-CGP were determined using confocal microscopy in conjunction with a novel perfusion system. Membrane-localized ligand binding was measured at 10, 30 and 100 nM BODIPY-TMR-CGP. In addition, co-operativity across two binding sites can be detected by assessing the dissociation rates of a fluorescent ligand under infinite dilution conditions in the absence and presence of unlabelled ligands [2].

In conclusion, BODIPY-TMR-CGP can be used to visualise the human β1-adrenoceptor in single living cells. Using BODIPY-TMR-CGP and unlabelled β-adrenoceptor ligands in this system will give further insight into the nature of the low affinity “CGP 12177” binding site and will further our understanding of the dynamics of ligand-receptor interactions at the human β1-adrenoceptor.

This work was funded by the Medical Research Council.

1. Baker JG et al. (2003) Mol Pharmacol 63(6): 1312-2

2. May LT et al. (2010) Mol Pharmacol 78(3): 511-23