Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands.
Bottom Line: Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two.Moreover, different chemical series of antagonist interact preferentially with different arginine residues.A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.
Affiliation: From the Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom and.Show MeSH
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Mentions: To explore the mode of binding of GLPG0974 in more detail, we employed a radiolabeled form of this compound. This ligand, [3H]GLPG0974, displayed excellent characteristics as a radiotracer. In membranes from Flp-InTM T-RExTM 293 cells that had been induced to express hFFA2-eYFP [3H]GLPG0974 bound with high affinity (Kd = 7.5 ± 0.4 nm, mean ± S.E., n = 4) in saturation equilibrium binding assays and with low non-specific to total binding ratios (Fig. 4A). Fitting of the data to both 1-site and 2-site binding models indicated no improved fit to the 2-site model, and therefore, the data are fully consistent with [3H]GLPG0974 binding to a single site on the receptor. As anticipated from the lack of functional effects of GLPG0974 at mFFA2 and hFFA3 (Fig. 2, D and E), [3H]GLPG0974 showed no specific binding to membranes of Flp-InTM T-RExTM 293 cells induced to express hFFA3-eYFP at concentrations up to 100 nm (Fig. 4B), whereas in membranes induced to express mFFA2-eYFP, a very low level of apparent specific binding was detected (Fig. 4B), but useful analysis of the data was not possible as the binding did not saturate, and best estimates predicted Kd >150 nm. This was despite direct comparison of receptor expression, based on eYFP fluorescence, indicating that each of these constructs was expressed in amounts similar to hFFA2-eYFP.
Affiliation: From the Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom and.