GPR120 is a long-chain fatty acid receptor that stimulates incretin hormone release from colonic endocrine cells and is implicated in macrophage and adipocyte function. The functional consequences of long (L) and short (S) human GPR120 splice variants, which differ by insertion of 16 amino acids in the third intracellular loop, are currently unknown. Here we compare signaling and intracellular trafficking of GPR120S and GPR120L receptors, using calcium mobilization and dynamic mass redistribution (DMR) assays, together with quantitative imaging measurements of ␤-arrestin2 association and receptor internalization. FLAG-or SNAP-tagged GPR120S receptors elicited both intracellular calcium mobilization and DMR responses in human embryonic kidney 293 cells, when stimulated with oleic acid, myristic acid, or the agonist 4-[[(3-phenoxyphenyl)methyl]amino]benzenepropanoic acid (GW9508). Responses were insensitive to pertussis toxin, but increases in intracellular calcium were attenuated by 2-aminoethoxydiphenyl borate, an inhibitor of store inositol trisphosphate receptors. Despite equivalent cell surface expression of SNAP-tagged GPR120L receptors, no specific calcium or DMR responses were observed in cells transfected with this isoform. However, agoniststimulated GPR120S and GPR120L receptors both recruited ␤-arrestin2 and underwent robust internalization, with similar agonist potencies in each case. After oleic acid-induced internalization, neither GPR120 isoform recycled rapidly to the cell surface. In both cases, confocal microscopy indicated receptor targeting to lysosomal compartments. Thus, the third intracellular loop insertion in GPR120L prevents G protein-dependent intracellular calcium and DMR responses, but this receptor isoform remains functionally coupled to the ␤-arrestin pathway, providing one of the first examples of a native ␤-arrestin-biased receptor.

Discovery of G protein coupled receptors for long chain free fatty acids (FFAs), FFA1 (GPR40) and GPR120, has expanded our understanding of these nutrients as signaling molecules. These receptors have emerged as important sensors for FFA levels in the circulation or the gut lumen, based on evidence from in vitro and rodent models, and an increasing number of human studies. Here we consider their promise as therapeutic targets for metabolic disease, including type 2 diabetes and obesity. FFA1 directly mediates acute FFA-induced glucose-stimulated insulin secretion in pancreatic beta-cells, while GPR120 and FFA1 trigger release of incretins from intestinal endocrine cells, and so indirectly enhance insulin secretion and promote satiety. GPR120 signaling in adipocytes and macrophages also results in insulin sensitizing and beneficial anti-inflammatory effects. Drug discovery has focused on agonists to replicate acute benefits of FFA receptor signaling, with promising early results for FFA1 agonists in man. Controversy surrounding chronic effects of FFA1 on beta-cells illustrates that long term benefits of antagonists also need exploring. It has proved challenging to generate highly selective potent ligands for FFA1 or GPR120 subtypes, given that both receptors have hydrophobic orthosteric binding sites, which are not completely defined and have modest ligand affinity. Structure activity relationships are also reliant on functional read outs, in the absence of robust binding assays to provide direct affinity estimates. Nevertheless synthetic ligands have already helped dissect specific contributions of FFA1 and GPR120 signaling from the many possible cellular effects of FFAs. Approaches including use of fluorescent ligand binding assays, and targeting allosteric receptor sites, may improve further pre-clinical ligand development at these receptors, to exploit their unique potential to target multiple facets of diabetes.

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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 Ca2+), 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.