Application of BRET to monitor ligand binding to GPCRs

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

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.

Related collections

Most cited references 18

Record: found
Abstract: found
Article: not found

The selectivity of beta-adrenoceptor antagonists at the human beta1, beta2 and beta3 adrenoceptors.

Michael G Baker (2005)

Beta-adrenoceptor antagonists ("beta-blockers") are one of the most widely used classes of drugs in cardiovascular medicine (hypertension, ischaemic heart disease and increasingly in heart failure) as well as in the management of anxiety, migraine and glaucoma. Where known, the mode of action in cardiovascular disease is from antagonism of endogenous catecholamine responses in the heart (mainly at beta1-adrenoceptors), while the worrisome side effects of bronchospasm result from airway beta2-adrenoceptor blockade. The aim of this study was to determine the selectivity of beta-antagonists for the human beta-adrenoceptor subtypes. (3)H-CGP 12177 whole cell-binding studies were undertaken in CHO cell lines stably expressing either the human beta1-, beta2- or the beta3-adrenoceptor in order to determine the affinity of ligands for each receptor subtype in the same cell background. In this study, the selectivity of well-known subtype-selective ligands was clearly demonstrated: thus, the selective beta1 antagonist CGP 20712A was 501-fold selective over beta2 and 4169-fold selective over beta3; the beta2-selective antagonist ICI 118551 was 550- and 661-fold selective over beta1 and beta3, respectively, and the selective beta3 compound CL 316243 was 10-fold selective over beta2 and more than 129-fold selective over beta1. Those beta2-adrenoceptor agonists used clinically for the treatment of asthma and COPD were beta2 selective: 29-, 61- and 2818-fold for salbutamol, terbutaline and salmeterol over beta1, respectively. There was little difference in the affinity of these ligands between beta1 and beta3 adrenoceptors. The clinically used beta-antagonists studied ranged from bisoprolol (14-fold beta1-selective) to timolol (26-fold beta2-selective). However, the majority showed little selectivity for the beta1- over the beta2-adrenoceptor, with many actually being more beta2-selective. This study shows that the beta1/beta2 selectivity of most clinically used beta-blockers is poor in intact cells, and that some compounds that are traditionally classed as "beta1-selective" actually have higher affinity for the beta2-adrenoceptor. There is therefore considerable potential for developing more selective beta-antagonists for clinical use and thereby reducing the side-effect profile of beta-blockers.

0 comments Cited 129 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET).

Kevin D. G. Pfleger, Karin Eidne (2006)

Bioluminescence resonance energy transfer (BRET) is a straightforward biophysical technique for studying protein-protein interactions. It requires: (1) that proteins of interest and suitable controls be labeled with either a donor or acceptor molecule, (2) placement of these labeled proteins in the desired environment for assessing their potential interaction, and (3) use of suitable detection instrumentation to monitor resultant energy transfer. There are now several possible applications, combinations of donor and acceptor molecules, potential assay environments and detection system perturbations. Therefore, this review aims to demystify and clarify the important aspects of the BRET methodology that should be considered when using this technique.

0 comments Cited 122 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring.

David Baker, Kai Johnsson, Luc Reymond … (2014)

For many drugs, finding the balance between efficacy and toxicity requires monitoring their concentrations in the patient's blood. Quantifying drug levels at the bedside or at home would have advantages in terms of therapeutic outcome and convenience, but current techniques require the setting of a diagnostic laboratory. We have developed semisynthetic bioluminescent sensors that permit precise measurements of drug concentrations in patient samples by spotting minimal volumes on paper and recording the signal using a simple point-and-shoot camera. Our sensors have a modular design consisting of a protein-based and a synthetic part and can be engineered to selectively recognize a wide range of drugs, including immunosuppressants, antiepileptics, anticancer agents and antiarrhythmics. This low-cost point-of-care method could make therapies safer, increase the convenience of doctors and patients and make therapeutic drug monitoring available in regions with poor infrastructure.

0 comments Cited 55 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Journal

Journal ID (nlm-journal-id): 101215604

Journal ID (pubmed-jr-id): 32338

Journal ID (nlm-ta): Nat Methods

Journal ID (iso-abbrev): Nat. Methods

Title: Nature methods

ISSN (Print): 1548-7091

ISSN (Electronic): 1548-7105

Publication date Nihms-submitted: 29 April 2015

Publication date (Electronic): 01 June 2015

Publication date (Print): July 2015

Publication date PMC-release: 01 January 2016

Volume: 12

Issue: 7

Pages: 661-663

Affiliations

[1 ]Cell Signalling Research Group, School of Life Sciences, University of Nottingham Medical School, Nottingham, United Kingdom.

[2 ]Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research, Nedlands, Western Australia, Australia.

[3 ]Centre for Medical Research, The University of Western Australia, Crawley, Western Australia, Australia.

[4 ]Promega Corporation, Madison, Wisconsin, United States.

Author notes

Correspondence should be addressed to K.D.G.P. ( kevin.pfleger@ 123456perkins.uwa.edu.au ) or S.J.H. ( stephen.hill@ 123456nottingham.ac.uk )

AUTHOR CONTRIBUTIONS

L.A.S., E.K.M.J., A.J.W., J.G., M.B.R. and T.M. performed the research.

L.A.S., E.K.M.J., M.B.R., K.V.W., S.J.H. and K.D.G.P. designed the research study.

L.A.S., E.K.M.J., A.J.W., J.G. and M.B.R. analysed the data.

L.A.S., S.J.H. and K.D.G.P. wrote the paper.

Article

Manuscript ID: EMS63067

DOI: 10.1038/nmeth.3398

PMC ID: 4488387

PubMed ID: 26030448

SO-VID: 79a4ab3f-7353-4762-802a-a9e7ce3f59db

History

Comments

Comment on this article

scite_

Cited by 69

See all cited by

Application of BRET to monitor ligand binding to GPCRs

Read this article at

Abstract

Related collections

IUCN Red List of Ecosystems

Most cited references 18

The selectivity of beta-adrenoceptor antagonists at the human beta1, beta2 and beta3 adrenoceptors.

Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET).

Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring.

Author and article information

Journal

Affiliations

Author notes

Article

History

Categories

Comments

Comment on this article

Similar content 174

Cited by 69