Leigh A. Stoddart

International Union of Pharmacology. LXXI. Free Fatty Acid Receptors FFA1, -2, and -3: Pharmacology and Pathophysiological Functions

Identification of G protein-coupled receptors that are activated by free fatty acids has led to considerable interest in their pharmacology and function because of the wide range of normal physiology and disease states in which fatty acids have been implicated. Free fatty acid receptor (FFA) 1 is activated by medium- to long-chain fatty acids and is expressed in the insulin-producing β-cells of the pancreas. Activation of FFA1 has been proposed to mediate fatty acid augmentation of glucose-stimulated insulin secretion although it is unclear whether the known long-term detrimental effects of β-cell exposure to high levels of fatty acids are also mediated through this receptor. The related receptors FFA2 and FFA3 are both activated by short-chain fatty acids although they have key differences in the signaling pathways they activate and tissue expression pattern. The aim of this review is to provide a comprehensive overview of the current understanding of the pharmacology and physiological role of these fatty acid receptors.

The Action and Mode of Binding of Thiazolidinedione Ligands at Free Fatty Acid Receptor 1

The endogenous ligands for free fatty acid receptor 1 (FFA1) are medium and longer chain free fatty acids. However, a range of selective, small molecule ligands have recently been developed as tool compounds to explore the therapeutic potential of this receptor, whereas clinically employed thiazolidinedione “glitazone” drugs are also agonists at FFA1. Each of these classes of agonist was able to promote phosphorylation of the ERK1/2 mitogen-activated protein (MAP) kinases in cells able to express human FFA1 on demand. However, although both lauric acid and the synthetic agonist GW9508X produced rapid and transient ERK1/2 MAP kinase phosphorylation, the thiazolidinedione rosiglitazone produced responses that were sustained for a substantially longer period. Despite this difference, the effects of each ligand required FFA1 and were transduced in each case predominantly via G proteins of the Gαq/Gα11 family. Different glitazone drugs also displayed markedly different efficacy and kinetics of sustainability of ERK1/2 MAP kinase phosphorylation. A number of orthosteric binding site mutants of FFA1 were generated, and despite variations in the changes of potency and efficacy of the three ligand classes in different functional end point assays, these were consistent with rosiglitazone also binding at the orthosteric site. Four distinct polymorphic variants of human FFA1 have been described. Despite previous indications that these display differences in function and pharmacology, they all responded in entirely equivalent ways to lauric acid, rosiglitazone, and GW9508X in measures of ERK1/2 MAP kinase phosphorylation, enhancement of binding of [35S]GTPγS (guanosine 5′-O-(3-[35S]thio)triphosphate) to Gαq, and elevation of intracellular [Ca2+], suggesting that individuals expressing each variant are likely to respond equivalently to orthosteric agonists of FFA1.

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.

Application of BRET to monitor ligand binding to GPCRs

Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein–coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs.

Uncovering the Pharmacology of the G Protein-Coupled Receptor GPR40: High Apparent Constitutive Activity in Guanosine 5′-O-(3-[35S]thio)triphosphate Binding Studies Reflects Binding of an Endogenous Agonist

In cells lacking expression of Ca2+-mobilizing G proteins, coexpression of human GPR40 and Gαq allowed medium- and long-chain fatty acids to elevate intracellular [Ca2+]. This was also observed when human embryonic kidney (HEK) 293 cells were transfected with a GPR40-Gαq fusion protein. The kinetic of elevation of intracellular [Ca2+] slowed with increasing fatty acid chain length, suggesting different ligand on-rates, whereas the addition of fatty acid-free bovine serum albumin reduced signals, presumably by binding the fatty acids. To allow effective ligand equilibration, GPR40-Gαq was used in guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assays. After expression of GPR40-Gαq in HEK293 cells and membrane preparation basal binding of [35S]GTPγSinGαq immunoprecipitates was high and not elevated substantially by fatty acids. However, treatment of membranes with fatty acid-free bovine serum albumin reduced the basal [35S]GTPγS binding in a concentration-dependent manner and allowed the responsiveness and pharmacology at GPR40 of each of the fatty acids thiazolidinediones and a novel small-molecule agonist to be uncovered. Membranes of rat INS-1E cells that express GPR40 endogenously provided similar observations. The high apparent constitutive activity of GPR40-Gαq was also reversed by a small-molecule GPR40 antagonist, and basal [35S]GTPγS binding was prevented by the selective Gαq/Gα11 inhibitor YM-254890. The current studies provide novel insights into the pharmacology of GPR40 and indicate that G protein-coupled receptors which respond to fatty acids, and potentially to other lipid ligands, can be occupied by endogenous agonists before assay and that this may mask the pharmacology of the receptor and may be mistaken for high levels of constitutive activity.

Fragment Screening at Adenosine-A3 Receptors in Living Cells Using a Fluorescence-Based Binding Assay

Summary G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is determined in the correct cellular environment and preferably using an unmodified receptor. We developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to determine binding affinity constants of competing ligands at human adenosine-A1 and -A3 receptors. This method was validated as a tool to screen a library of low molecular weight fragments, and identified a hit with submicromolar binding affinity (KD). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A3 receptor. This technology represents a significant advance that will allow the determination of ligand and fragment affinities at receptors in their native membrane environment.

Extracellular Loop 2 Of The Free Fatty Acid Receptor 2 Mediates Allosterism Of A Phenylacetamide Ago-Allosteric Modulator

Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu173 or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB.

Agonism and allosterism: the pharmacology of the free fatty acid receptors FFA2 and FFA3

The free fatty acid receptors FFA2 and FFA3 are recently de‐orphanized G protein‐coupled receptors that share a group of short‐chain free fatty acids as endogenous ligands. The expression of FFA2 and FFA3 by immune cells, in parts of the gastro‐intestinal tract and by white adipocytes has suggested their potential as therapeutic targets in conditions including inflammation and obesity. However, although FFA2 and FFA3 display distinct structure–activity relationships for stimulation by short‐chain free fatty acids, the overlap between these endogenous agonists and the lack of synthetic small molecule ligands that display selectivity between these two receptors has, until recently, hindered efforts to resolve their individual functions. Recently, chloro‐α‐(1‐methylethyl)‐N‐2‐thiazolylbenzeneacetamide has been described as an FFA2 selective ago‐allosteric ligand, not only being a direct agonist but also acting as a positive allosteric modulator of the function of short‐chain free fatty acids at FFA2. Mutation of a pair of key arginine residues near the top of transmembrane domains V and VII of both FFA2 and FFA3 eliminates the function of short‐chain free fatty acids but is without effect on the direct agonist action of chloro‐α‐(1‐methylethyl)‐N‐2‐thiazolylbenzeneacetamide at FFA2, confirming the distinct nature of the binding site of the ago‐allosteric regulator from the orthosteric binding site for free fatty acids. An understanding of structure–activity relationships for ligands related to chloro‐α‐(1‐methylethyl)‐N‐2‐thiazolylbenzeneacetamide is likely to provide greater insight into the mode of action and site of binding of this ligand, but further FFA2 and FFA3 selective ligands, preferably with higher potency/affinity, will be required to fully explore the physiological function of these receptors.

Functional homomers and heteromers of dopamine D2L and D3 receptors co-exist at the cell surface

Background: Dopamine D2 and D3 receptor subtypes are often co-expressed. Results: Systems were established to allow concurrent detection of receptor homomers and heteromers. Conclusion: Co-expressed D2 and D3 receptors form both homomers and heteromers and all are functional and present concurrently. Significance: These observations are relevant to the pathogenesis and treatment of disorders in which D2 and D3 receptors are implicated. Human dopamine D2long and D3 receptors were modified by N-terminal addition of SNAP or CLIP forms of O6-alkylguanine-DNA-alkyltransferase plus a peptide epitope tag. Cells able to express each of these four constructs only upon addition of an antibiotic were established and used to confirm regulated and inducible control of expression, the specificity of SNAP and CLIP tag covalent labeling reagents, and based on homogenous time-resolved fluorescence resonance energy transfer, the presence of cell surface D2long and D3 receptor homomers. Following constitutive expression of reciprocal constructs, potentially capable of forming and reporting the presence of cell surface D2long-D3 heteromers, individual clones were assessed for levels of expression of the constitutively expressed protomer. This was unaffected by induction of the partner protomer and the level of expression of the partner required to generate detectable cell surface D2long–D3 heteromers was defined. Such homomers and heteromers were found to co-exist and using a reconstitution of function approach both homomers and heteromers of D2long and D3 receptors were shown to be functional, potentially via trans-activation of associated G protein. These studies demonstrate the ability of dopamine D2long and D3 receptors to form both homomers and heteromers, and show that in cells expressing each subtype a complex mixture of homomers and heteromers co-exists at steady state. These data are of potential importance both to disorders in which D2long and D3 receptors are implicated, like schizophrenia and Parkinson disease, and also to drugs exerting their actions via these sites.

Fluorescence‐ and bioluminescence‐based approaches to study GPCR ligand binding

Ligand binding is a vital component of any pharmacologists toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (Kd) and competition (Ki) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor‐associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time‐resolved FRET (TR‐FRET). Finally, we have recently developed a novel bioluminescence resonance energy transfer (BRET) ligand binding assay utilizing a small (19 kDa), super‐bright luciferase subunit (NanoLuc) from a deep sea shrimp. In combination with fluorescent ligands, measurement of RET now provides an array of methodologies to study ligand binding. While each method has its own advantages and drawbacks, binding studies using fluorescent ligands are now a viable alternative to the use of radioligands.