Direct visualisation of internalization of the adenosine A3 receptor and localization with arrestin3 using a fluorescent agonist

Leigh A Stoddart,Andrea J Vernall,Stephen J Briddon,Barrie Kellam,Stephen J Hill

doi:10.1016/j.neuropharm.2015.04.013

Leigh A Stoddart, Andrea J Vernall + Show 3 more

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https://doi.org/10.1016/j.neuropharm.2015.04.013

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Abstract

Fluorescence based probes provide a novel way to study the dynamic internalization process of G protein-coupled receptors (GPCRs). Recent advances in the rational design of fluorescent ligands for GPCRs have been used here to generate new fluorescent agonists containing tripeptide linkers for the adenosine A3 receptor. The fluorescent agonist BY630-X-(D)-A-(D)-A-G-ABEA was found to be a highly potent agonist at the adenosine A3 receptor in both reporter gene (pEC50 = 8.48 ± 0.09) and internalization assays (pEC50 = 7.47 ± 0.11). Confocal imaging studies showed that BY630-X-(D)-A-(D)-A-G-ABEA was internalized with A3 linked to yellow fluorescent protein, which was blocked by the competitive antagonist MRS1220. Internalization of untagged adenosine A3 could also be visualized with BY630-X-(D)-A-(D)-A-G-ABEA treatment. Further, BY630-X-(D)-A-(D)-A-G-ABEA stimulated the formation of receptor–arrestin3 complexes and was found to localize with these intracellular complexes. This highly potent agonist with excellent imaging properties should be a valuable tool to study receptor internalization.This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology’.

Highlights

The nucleoside adenosine exerts its biological function via four cell surface G protein-coupled receptors (GPCRs); A1, A2A, A2B and A3 (Fredholm et al, 2001; Hill et al, 2014)
Fluorescent ligands for GPCRs are valuable tools to study the localization of receptors in their native environment and to answer important questions on their pharmacology
This was achieved by incorporating peptide linkers into an existing fluorescent adenosine receptor agonist ABEA-X-BY630 (Middleton et al, 2007) based on a strategy which has been successfully employed with adenosine receptor fluorescent antagonists (Vernall et al, 2013) and which resulted in fluorescent XAC derivatives with improved imaging properties

Summary

Introduction

The nucleoside adenosine exerts its biological function via four cell surface G protein-coupled receptors (GPCRs); A1, A2A, A2B and A3 (Fredholm et al, 2001; Hill et al, 2014). The dysregulation of these processes can lead to a range of different pathological states such as Parkinson's (Jenner et al, 2009) and Alzheimer's (Chen et al, 2007) diseases, autoimmune disorders (Ohta and Sitkovsky, 2001) and tissue damage following reperfusion injury (Wan et al, 2008). These receptors represent attractive drug targets (Chen et al, 2013). A greater understanding of the precise downstream signal induced by an agonist, and the subsequent regulation of GPCR. One way to understand the internalization of a GPCR and the termination of the signal is through the use of fluorescent ligands

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