Use of a new proximity assay (NanoBRET) to investigate the ligand‐binding characteristics of three fluorescent ligands to the human β₁‐adrenoceptor expressed in HEK‐293 cells

Abstract

Previous research has indicated that allosteric interactions across the dimer interface of β 1‐adrenoceptors may be responsible for a secondary low affinity binding conformation. Here we have investigated the potential for probe dependence, in the determination of antagonist pKi values at the human β 1‐adenoceptor, which may result from such allosterism interactions. Three fluorescent β 1‐adrenoceptor ligands were used to investigate this using bioluminescence energy transfer (BRET) between the receptor‐bound fluorescent ligand and the N‐terminal NanoLuc tag of a human β 1‐adrenoceptor expressed in HEK 293 cells (NanoBRET). This proximity assay showed high‐affinity‐specific binding to the NanoLuc‐ β 1‐adrenoceptor with each of the three fluorescent ligands yielding K D values of 87.1 ± 10 nmol/L (n = 8), 38.1 ± 12 nmol/L (n = 7), 13.4 ± 2 nmol/L (n = 14) for propranolol‐Peg8‐BY630, propranolol‐ β(Ala‐Ala)‐BY630 and CGP‐12177‐TMR, respectively. Parallel radioligand‐binding studies with 3H‐CGP12177 and TIRF microscopy, to monitor NanoLuc bioluminescence, confirmed a high cell surface expression of the NanoLuc‐ β 1‐adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein−1). Following a 1 h incubation with fluorescent ligands and β 1‐adrenoceptor competing antagonists, there were significant differences (P < 0.001) in the pKi values obtained for CGP20712a and CGP 12177 with the different fluorescent ligands and 3H‐CGP 12177. However, increasing the incubation time to 2 h removed these significant differences. The data obtained show that the NanoBRET assay can be applied successfully to study ligand‐receptor interactions at the human β 1‐adrenoceptor. However, the study also emphasizes the importance of ensuring that both the fluorescent and competing ligands are in true equilibrium before interpretations regarding probe dependence can be made.

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H3 and H4 Receptors on Living Cells

“…The availability of fluorescent ligands in combination with the recently engineered Nluc luciferase allows the development of NanoBRET-based ligand-receptor-binding assays (Stoddart et al, 2015(Stoddart et al, , 2018aSoave et al, 2016). Since NanoBRET only occurs if the fluorescent ligand is bound to the Nluc-tagged GPCR, this assay can be performed in a homogeneous format without additional washing steps to separate unbound from bound fluorescent ligand and is, therefore, well suited to detect real-time ligand binding to GPCRs that are expressed on living cells.…”

“…Recently, a novel proximitybased method was introduced for the histamine H 1 receptor and a number of other G protein-coupled receptors (GPCRs) by genetic fusion of an engineered enzyme, NanoLuc (Nluc), to the N-terminus of these receptors, allowing only the detection of bound fluorescent ligand via bioluminescence resonance energy transfer (nanoBRET). Consequently, this novel method does not require physical separation of free from bound fluorescent ligand (Stoddart et al, 2015;Soave et al, 2016). Hence, this proximity-based (,10 nm) method allows real-time measurement of ligand binding to GPCRs on intact cells.…”

Probe dependence of allosteric enhancers on the binding affinity of adenosine A1‐receptor agonists at rat and human A1‐receptors measured using NanoBRET

“…Human and rat Nluc‐labelled adenosine A 1 ‐receptor (Nluc‐A 1 R) constructs were generated as previously described by Stoddart, Johnstone, et al (). In brief, the full‐length sequence of Nluc luciferase from the pNL1.1 vector (Promega) was amplified and fused in‐frame with the membrane signal sequence of the 5‐HT 3A membrane localization signal sequence (pcDNA3.1 sig‐Nluc; Soave, Stoddart, Brown, Woolard, & Hill, ). This was fused to the full‐length human or rat sequence of the adenosine A 1 ‐receptor (with the methionine start signal removed) to the 3′ end of the sig‐Nluc in pcDNA3.1.…”

Fluorescent ligands: Bringing light to emerging GPCR paradigms

“…Building upon the development of NanoBRET to monitor GPCR–ligand binding in living cells using the bright luciferase NanoLuc, Alcobia et al () sought to implement NanoBRET in vivo. They used a fluorescent analogue of the β‐adrenoceptor antagonist propranolol (Prop‐BY630) that had previously been characterised at β‐adrenoceptors with NanoBRET in vitro (Soave et al, ; Stoddart et al, ). Alcobia et al () used the metastatic triple‐negative breast cancer MDA‐MB‐231 cell line stably transfected with the NLuc‐β 2 ‐adrenoceptor as their model cell since propranolol has been shown to prevent breast cancer metastasis in murine models (Sloan et al, ).…”

“…Methods using fluorescent ligands generally fall into two categories: direct measurement of fluorescence or indirect measurement using resonance energy transfer (RET; Stoddart, White, Nguyen, Hill, & Pfleger, 2016). Both of these techniques have been applied to measure the binding of known ligands for a variety of receptors and in general have found good agreement of affinity constants with radioligand-binding assays (Bosma et al, 2019;Leyris et al, 2011;Nederpelt et al, 2016;Soave, Stoddart, Brown, Woolard, & Hill, 2016). This has allowed fluorescence-based assays to be accepted as a robust way to measure affinity.…”