Abstract
FFA2 and FFA3 are closely related G protein-coupled receptors that bind and respond to short chain fatty acids. (FFA2 and FFA3 are the provisional International Union of Pharmacology designations for the receptors previously called GPR43 and GPR41, respectively.) Sequence comparisons between these two receptors and alignments with the related G protein-coupled receptor FFA1, linked to homology modeling based on the atomic level structure of bovine rhodopsin, indicated the potential for polar residues within the transmembrane helix bundle to play important roles in ligand recognition and function. In both FFA2 and FFA3, mutation of either an arginine at the top of transmembrane domain V or a second arginine at the top of transmembrane domain VII eliminated the function of a range of short chain fatty acids. Mutation of a histidine in transmembrane domain VI, predicted to be in proximity to both the arginine residues, also eliminated function in many but not all assay formats. By contrast, mutation of a histidine in transmembrane domain IV, predicted to be lower in the binding pocket, modulated function in some assays of FFA3 function but had limited effects on the function of acetate and propionate at FFA2. Interestingly, wild type FFA3 responded to caproate, whereas FFA2 did not. Mutation of the transmembrane domain IV histidine eliminated responses of FFA3 to caproate but resulted in a gain of function of FFA2 to this six-carbon fatty acid. These data demonstrate the importance of positively charged residues in the recognition and/or function of short chain fatty acids in both FFA2 and FFA3. The development of small molecule ligands that interact selectively with these receptors will allow further details of the binding pockets to be elucidated.
Highlights
Binding of Fatty Acids to Free fatty acid receptor 2 (FFA2)/3 duce mutations into hFFA2-eYFP or hFFA3-eYFP in either pcDNA3 or pcDNA5/FRT/TO
FFA1 binds and responds to medium and long chain free fatty acids, and small molecule agonists have been identified and developed (34 –38). This reflects, in part, that FFA1 is highly expressed in pancreatic islets, that ligands at this receptor modulate glucose-dependent insulin secretion and that FFA1 is, considered an interesting potential target for the treatment of type 2 diabetes [20, 35, 38, 39]
We constructed homology models of the TM domains of hFFA2 and hFFA3 and took advantage of the relatedness of these receptors to FFA1 in considering amino acids likely to contribute to ligand binding and/or function of short chain fatty acids (SCFA) in hFFA2 and hFFA3
Summary
[35S]GTP␥S Binding Assays—[35S]GTP␥S binding experiments were performed using to two separate methods. ͓35SGTP␥ S binding studies were performed on membranes of HEK293 cells transfected to transiently express the hFFA3-Cys351 Ile G␣i3 fusion protein [20]. FLIPR-based Calcium Assay—HEK293 MSRII cells were transiently transfected with either wild type or mutant FFA2eYFP or FFA3-eYFP and seeded at 15,000 cells/well into polyD-lysine-coated 384-well black-wall, clear-bottom microtiter plates (Greiner Bio-One, Kremsmunster, Austria) using a Multidrop dispenser. Cell Lysate Generation and Phospho-ERK1/2 Assays—FFA2 or FFA3 stable Flp-In TREx HEK293 cells were seeded onto 12-well poly-D-lysine-coated plates and induced with 1 g/ml doxycycline for 48 h. Multiple analyses of the significance of differences in ERK MAP kinase phosphorylation studies were compared via one-way analysis of variance with Bonferonni’s multiple comparisons of the mean post hoc test
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