Conserved polar residues in transmembrane domains V, VI, and VII of free fatty acid receptor 2 and free fatty acid receptor 3 are required for the binding and function of short chain fatty acids.

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.