Detection of the secondary, low-affinity β1-adrenoceptor site in living cells using the fluorescent CGP 12177 derivative BODIPY-TMR-CGP

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Abstract

Background and Purpose

CGP 12177 not only inhibits agonist effects mediated through the catecholamine site of the β ₁-adrenoceptor with high affinity, but also exhibits agonist effects of its own at higher concentrations through a secondary, low-affinity β ₁-adrenoceptor site or conformation. β-blocker affinities for this ‘CGP 12177’ site of the human β ₁-adrenoceptor have thus far only been characterized in functional studies. Here, we used the fluorescent CGP 12177 analogue BODIPY-TMR-CGP to directly investigate receptor–ligand interactions at the secondary binding site of the β ₁-adrenoceptor.

Experimental Approach

The human β ₁-adrenoceptor was stably expressed in CHO cells containing a cAMP response element (CRE)-secreted placental alkaline phosphatase (SPAP) reporter gene construct. Functional responses of BODIPY-TMR-CGP were determined in the CRE-SPAP reporter gene assay, and manual and automated confocal microscopy platforms used to investigate the binding properties of BODIPY-TMR-CGP.

Key Results

BODIPY-TMR-CGP displayed a pharmacological profile similar to that of CGP 12177, retaining agonist activity at the secondary β ₁-adrenoceptor site. In confocal microscopy studies, specific BODIPY-TMR-CGP binding allowed clear visualization of β ₁-adrenoceptors in live cells. Using a wider concentration range of labelled ligand in a high-content fluorescence-based binding assay than is possible in radioligand binding assays, two-site inhibition binding curves of β-adrenoceptor antagonists were revealed in CHO cells expressing the human β ₁-adrenoceptor, but not the β ₂-adrenoceptor.

Conclusions and Implications

The fluorescent CGP 12177 analogue allowed the detection of the β ₁-adrenoceptor secondary site in both functional and binding studies. This suggests that BODIPY-TMR-CGP presents an important and novel fluorescent tool to investigate the nature of the secondary β ₁-adrenoceptor site.

Related collections

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Author and article information

Journal

Journal ID (nlm-ta): Br J Pharmacol

Journal ID (iso-abbrev): Br. J. Pharmacol

Journal ID (publisher-id): bph

Title: British Journal of Pharmacology

Publisher: BlackWell Publishing Ltd (Oxford, UK )

ISSN (Print): 0007-1188

ISSN (Electronic): 1476-5381

Publication date (Print): December 2014

Publication date (Electronic): 24 November 2014

Volume: 171

Issue: 23

Pages: 5431-5445

Affiliations

Cell Signalling Research Group, School of Life Sciences, University of Nottingham Nottingham, UK

Author notes

Correspondence, Stephen John Hill, University of Nottingham, Cell Signalling Research Group, School of Life Sciences, Nottingham, NG7 2UH, UK. E-mail: stephen.hill@ 123456nottingham.ac.uk

Article

DOI: 10.1111/bph.12858

PMC ID: 4261997

PubMed ID: 25052258

SO-VID: cb4e2946-a54c-4eee-9c17-8f9a168841b9

License:

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

History

Date received : 20 March 2014

Date revision received : 11 July 2014

Date accepted : 18 July 2014