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Visualizing trafficking of the adenosine‐A3 receptor using a fluorescent agonist: a tool for delineating intracellular signalling
Author(s) -
Briddon Steve,
Stoddart Leigh,
Vernall Andrea,
Kellam Barrie,
Hill Steve
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1173.6
Subject(s) - agonist , g protein coupled receptor , receptor , microbiology and biotechnology , yellow fluorescent protein , chemistry , intracellular , chinese hamster ovary cell , biophysics , adenosine receptor , second messenger system , signal transduction , biology , biochemistry , gene
Activation of many G protein‐coupled receptors (GPCRs) leads to β‐arrestin binding and sequestration of the receptor from the cell surface. Whilst internalisation of a GPCR leads to termination of some aspects of receptor‐mediated signalling, recent reports have shown that some GPCRs continue to signal even after internalisation. Here, we show using a mutated version of the adenosine A 3 receptor (W243F‐A 3 AR), that persistent signalling to the cAMP pathway requires receptor internalisation. To investigate this further we developed a fluorescent A 3 AR agonist, consisting of the agonist NECA conjugated to BODIPY‐630/650‐X via a dipeptide linker. This fluorescent agonist was highly potent at the A 3 AR ( pEC 50 = 9.06 ± 0.17, n=4) and acted as a full agonist in Chinese Hamster Ovary cells expressing this receptor. Confocal microscopy showed a high degree of co‐localisation of the fluorescent agonist with a YFP‐tagged A 3 AR on the cell surface after a short incubation period (5 min). After longer incubation (30 min), A 3 AR‐YFP was sequestered from the cell surface to intracellular granules where co‐localisation with the fluorescent agonist was still observed. This fluorescent probe thus allows us to visualise A 3 AR trafficking and investigate how the receptor continues to signal to second messenger pathways upon internalisation. We thank the Medical Research Council for financial support.