Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a novel BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a novel bioluminescent protein (NanoLuc) that can be readily expressed on the N-terminus of GPCRs.

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The A1-adenosine receptor (A1-AR) is a G protein-coupled receptor that mediates many of the physiological effects of adenosine in the brain, heart, kidney, and adipocytes. Currently, ligand interactions with the A 1-AR can be quantified on large cell populations only by using radioligand binding. To increase the resolution of these measurements, we have designed and characterized a previously unde-

␤-Blockers have beneficial effects in heart failure, although the underlying mechanism is unknown. ␤ 2 -Adrenoceptors, however, are proportionally higher in the failing human heart. This study shows several clinically used ␤-blockers are agonists at the human ␤ 2 -adrenoceptor. Although these agonist effects were small at the cAMP level, they were substantial at the level of cAMP response element (CRE)-mediated gene transcription. Some of the effects of "␤-blockers" seen in heart failure may be related to the ␤ 2 -agonist actions of these compounds. CREgene transcription responses to ␤ 2 -agonists, forskolin, and cAMP-analogs were sensitive to p42/44-mitogen-activated protein (MAP) kinase pathway inhibitors. p42/44-MAP kinase activation was also shown directly by western blotting and enzyme-linked immunosorbent assay techniques. N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89; a protein kinase A inhibitor) stimulated cAMP accumulation and CRE gene transcription via the ␤ 2 -adrenoceptor at concentrations at which protein kinase A was inhibited, providing evidence for an alternative pathway. Propranolol, however, produced paradoxical effects; it reduced basal cAMP accumulation (via ␤ 2 -mediated inverse agonism) but stimulated ␤ 2 -mediated CRE gene transcription. This cannot be explained by a sequential pathway from Gs-adenylyl cyclase-cAMP to CRE binding protein phosphorylation. Both responses to propranolol were insensitive to pertussis toxin, thus excluding Gi-protein involvement. Propranolol CRE gene transcription responses were attenuated by p42/44-MAP kinase inhibitors and propranolol was also found to directly stimulate the p42/44-MAP kinase pathway. Studies of inositol phosphate accumulation and of protein kinase C or Rho kinase inhibitors on CRE-gene transcription provided no evidence for G q/11 or G 12/13 involvement. These data suggest that propranolol can simultaneously act as an inverse agonist through a Gs-coupled mechanism while stimulating the p42/ 44-MAP kinase pathway through an alternative G-protein-independent mechanism.

Previous work with 4-

To realize the full scientific and clinical potential of human embryonic stem cell (hESC)-cardiomyocytes, strategies to overcome the high degree of heterogeneity of differentiated populations are required. Here we demonstrate the utility of two transgenic approaches in enrichment of cardiomyocytes derived from HUES-7 cells: (i) negative selection of proliferating cells with the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system; and (ii) positive selection of cardiomyocytes expressing a bicistronic reporter [green fluorescent protein (GFP)-internal ribosome entry site (IRES)-puromycin-N-acetyltransferase (PAC)] from the human alphamyosin heavy chain promoter. Parental and transgenic HUES-7 cells were similar with regard to morphology, pluripotency marker expression, differentiation, and cardiomyocyte electrophysiology. Whereas immunostaining of dissociated cardiomyocyte preparations expressing HSVtk or PAC contained <7% cardiomyocytes, parallel cultures treated with GCV or puromycin, respectively, contained 33.4 +/- 2.1% or 91.5 +/- 4.3% cardiomyocytes corresponding to an enrichment factor of 6.7- or 14.5-fold. Drug-selected cardiomyocytes responded to chronotropic stimulation and displayed cardiac-specific action potentials, demonstrating that functionality was retained. Both transgenic strategies will be generically applicable and should readily translate to the enrichment of many other differentiated lineages derived from hESCs.

Ligand binding is a vital component of any pharmacologist's toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (Kd) and competition (Ki) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor‐associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time‐resolved FRET (TR‐FRET). Finally, we have recently developed a novel bioluminescence resonance energy transfer (BRET) ligand binding assay utilizing a small (19 kDa), super‐bright luciferase subunit (NanoLuc) from a deep sea shrimp. In combination with fluorescent ligands, measurement of RET now provides an array of methodologies to study ligand binding. While each method has its own advantages and drawbacks, binding studies using fluorescent ligands are now a viable alternative to the use of radioligands.Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of G Protein‐Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc

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