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A novel fluorescent ligand binding assay for free fatty acid receptor FFA1
Author(s) -
Watson SarahJane,
Brown Alastair,
Holliday Nick
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.655.5
Subject(s) - ligand (biochemistry) , chemistry , fluorescence , ligand binding assay , receptor , stereochemistry , biochemistry , physics , quantum mechanics
Obtaining affinity estimates at FFA1 receptors using radioligand binding is made difficult by lipophilic ligands. We characterised the properties of a new fluorescent ligand 40Ag‐Cy5 (CellAura) using 293TR cells expressing FLAG‐FFA1. Whole cell competition binding experiments using 40AG‐Cy5 were performed on 96‐well plates, followed by imaging and granularity analysis on a confocal platereader. Curve fitting used GraphPadPrism. Both 40Ag‐Cy5 and its non‐fluroescent precursor 40Ag‐cg were agonists at FFA1 (Fluo4 measurements of intracellular Ca 2+ ), with respective pEC50 values of 5.8±0.2 and 7.9±0.3. In binding experiments 40Ag‐Cy5 (100 nM) was displaced by synthetic ligands such as GW1100 (pIC50 6.0 ± 0.2) and GW9508 (pIC50 6.8 ± 0.2). However oleic acid (up to 100 μM) did not displace 40Ag‐Cy5 binding. By solving the Cheng‐Prusoff correction for GW1100 competition curves using 100nM or 1μM 40Ag‐Cy5 (GW1100 pIC50 5.6), we obtained pKi estimates for 40‐Ag Cy5 (6.2), and GW1100 (6.0; equivalent to its functional pKb). Fluorescence correlation spectroscopy experiments also confirmed 40Ag‐Cy5 binding to FFA1. Thus 40Ag‐Cy5 provides a novel fluorescent ligand which enables estimation of synthetic ligand affinities at FFA1. Supported by EPSRC and AstraZeneca.