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

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.Free fatty acid receptor 2 (FFA2), 4 formerly known as GPR43, and free fatty acid receptor 3 (FFA3), formerly known as GPR41, are a pair of closely related, previously orphan G protein-coupled receptors (GPCRs) that respond to a range of short chain fatty acids (SCFA) (1-4). Their expression patterns in adipocytes, gastrointestinal cells, various leukocyte populations and, potentially, in pancreatic islet cells has suggested possible roles in the control of energy storage and in white cell migration and recruitment. Although variation in potencies are evident (2), particularly for acetate (C2), where FFA2 displays higher potency than FFA3, the high overlap between activating ligands for the two receptors has blurred pharmacological separation and clear resolution of their individual functions. However, this also implies that ligand recognition and binding by the two receptors is likely to share a number of similarities. Studies on other GPCRs whose ligands contain carboxylate groups indicate that positively charged amino acids within the transmembrane (TM) regions may be very important in ligand binding and/or function. For example, Tunaru et al. (5) demonstrated that an arginine residue in TM III of the high affinity nicotinic acid receptor, GPR109A (also called HM74A),...

Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET

“…Herein, we have extended this to explore the presence and regulation of dimeric/oligomeric complexes at the surface of cells stably expressing forms of the M 3 muscarinic acetylcholine receptor and compared the results with those obtained via more conventional FRET imaging-based studies. A key feature of the FRET imaging studies was to employ the inducible expression of DNA located at the Flp-In TM locus of Flp-In TM T-REx TM 293 cells (33)(34)(35). This allowed us to regulate expression of one form of the M 3 muscarinic receptor in the presence of an unaltered amount of a second form in the same cells, rather than attempting to control relative expression levels in a substantial series of transient co-transfection studies.…”

The Pharmacology and Function of Receptors for Short-Chain Fatty Acids

“…Previous studies had shown that positively charged amino acids within the TM regions are essential for the binding and function of other GPCRs whose ligands contain a carboxylic acid group (Stitham et al, 2003;He et al, 2004;Tunaru et al, 2005;Sabirsh et al, 2006). This, together with the observation that uncharged ester derivatives of SCFAs are inactive at FFA2 and FFA3 (Le Poul et al, 2003), led Milligan's group to hypothesize that basic residues might also play a crucial role in the binding of SCFAs to their receptors (Stoddart et al, 2008a). Sequence alignment of Fig.…”

“…Alternatively, SCAs with substituted sp FFA2 and FFA3 with FFA1 revealed that five positively charged amino acids were conserved across these fatty acid receptors. Generation of homology models, linked to a mutagenic strategy, was then used to identify the key polar amino acids for ligand recognition contained in the water-filled cavity within the TM domains of FFA2 and FFA3 (Stoddart et al, 2008a). From this, four positively charged amino acid residues were identified: histidine (His) in TM IV (residue position 4.56), arginine (Arg) in TM V (5.39), His in TM VI (6.55), and Arg in TM VII (7.35) (Stoddart et al, 2008a) (for a more detailed consideration of the position and significance of these amino acids in FFA2 and FFA3, see Ulven, 2012).…”

Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer

“…Cells were then harvested and used to make total cell membrane preparations according to a previously described protocol. 30 Membranes were then resuspended in assay buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl 2 , 10 mM MgCl 2 ) and distributed into the wells of a white 96 well plate (5µg membrane protein/well). Membranes were then co-incubated with the indicated concentration of 4 (and 5 for non-specific binding measurements; or competing ligand for competition experiments) for 1 h at 25 °C.…”