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Use of an NLuc-BRET System to Investigate Ligand-Binding to the Human β1-adrenoceptor


Use of an NLuc-BRET System to Investigate Ligand-Binding to the Human β1-adrenoceptor
Mark Soave1, Alastair Brown2, Jeanette Woolard1, Stephen J Hill1; 1-School of Life Sciences, University of Nottingham, Nottingham, UK; 2-Heptares Therapeutics Ltd., Bio Park, Welwyn Garden City, AL7 3AX, UK

Introduction. Bioluminescence resonance energy transfer (BRET) is a tool which can measure protein-protein interactions (1). BRET relies on the non-radiative energy transfer from the oxidation of a luciferase substrate to a nearby acceptor fluorophore. A new BRET system has recently been utilised to monitor ligand-receptor interactions in human adenosine A1 and A3 receptors (2). In this report we have determined whether a 19kDa luciferase NanoLuc (NLuc; Promega) from the deep sea shrimp Oplophorus gracilirostris can be expressed on the N-terminus of the human β1-adrenoceptor (β1-AR) and subsequently used to monitor ligand-receptor binding with a fluorescent ligand.

Table 1 - Competition binding with 100nM Prop-BY630
Competing Compound NLuc pKi
(mean + SEM) n Radioligand pKi4,5
ICI 118551 6.55 ± 0.19 4 6.5
Cimaterol 6.44 ± 0.11 7 6.6
Isoprenaline 6.72 ± 0.12 7 6.1
Propranolol 8.11 ± 0.12 4 8.2
CGP 20712a 8.52 ± 0.09 7 8.8
CGP 12177 8.67 ± 0.05 7 9.2
Methods. Experiments were conducted in white 96-well plates containing HEK 293 cells transfected with the human NLuc-β1-AR. Cells were grown to confluence, growth media was removed and then replaced with 100μl HBSS. The fluorescent ligand propranolol-peg8-BY630 (Prop-BY630 (3); 100nM) was added in the presence or absence of competing ligands and incubated at 37°C for one hour. The substrate furimazine was then added to the plate and after 5 minutes read on a BMG PheraStar (filters: 460-80nm, 610nm-LP).

Results. Specific binding of Prop-BY630 (pKD = 7.06 ± 0.06, n=8) was observed to the human NLuc-β1-AR. Both β-AR agonists and antagonists were able to compete with this binding (Table 1).

Discussion. These data demonstrate the ability of BRET-based systems to quantify ligand-receptor interactions at the human β1-AR pharmacology in intact living cells.

(1) Jaeger, W., et al., (2014), Front. Endocrinol., 5, 1-26
(2) Stoddart L., et al. (2015), Nature Methods, 12, 661-663
(3) Baker, J. G., et al., (2011) J. Med. Chem., 54, 6874-6887
(4) Baker (2005) Br. J. Pharmacol, 144, 317-322
(5) Baker (2010) Br. J. Pharmacol., 160, 1048-1062

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