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• W2209556632 abstract "Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor. Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor. Short chain fatty acids (SCFAs) 4The abbreviations used are: SCFA, short chain fatty acid; BRET, bioluminescence resonance energy transfer; CATPB, (S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)phenyl) butanoic acid; Cmp 1, (R)-3-benzyl-4-(cyclopropyl-(4-(2,5-dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid; eYFP, enhanced yellow fluorescent protein; FFA2, free fatty acid receptor 2; FFA3, free fatty acid receptor 3; GLPG0974, 4-[[1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid; GPCR, G protein-coupled receptor; TMD, transmembrane domain; GTPγS, guanosine 5′-3-O-(thio)triphosphate; h, human; m, mouse; Cmp 71, 4-(1-(benzo[b]thiophene-3-carbonyl)-2-methyl-N-(4-trifluoromethylbenzyl)azetidine-2-carboxamido)butanoic acid; MeCmp 71, methyl 4-(1-(benzo[b]thiophene-3-carbonyl)-2-methyl-N-(4-trifluoromethylbenzyl)azetidine-2-carboxamido)butanoate; Cmp 42, 4-(1-(2-(benzo[b]thiophen-3-yl)acetyl)-N-(4-chlorobenzyl)-2-methylazetidine-2-carboxamido)butanoic acid); MeCmp 42, methyl 4-(1-(2-(benzo[b]thiophen-3-yl)acetyl)-N-(4-chlorobenzyl)-2-methylazetidine-2-carboxamido)butanoate. are produced in large amounts in the gut by microbial fermentation of poorly digestible carbohydrates (1Tan J. McKenzie C. Potamitis M. Thorburn A.N. Mackay C.R. Macia L. The role of short-chain fatty acids in health and disease.Adv. Immunol. 2014; 121: 91-119Crossref PubMed Scopus (1204) Google Scholar, 2Natarajan N. Pluznick J.L. From microbe to man: the role of microbial short chain fatty acid metabolites in host cell biology.Am. J. Physiol. Cell Physiol. 2014; 307: C979-C985Crossref PubMed Scopus (115) Google Scholar3Trompette A. Gollwitzer E.S. Yadava K. Sichelstiel A.K. Sprenger N. Ngom-Bru C. Blanchard C. Junt T. Nicod L.P. Harris N.L. Marsland B.J. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis.Nat. Med. 2014; 20: 159-166Crossref PubMed Scopus (1652) Google Scholar). The predominant products are acetate (C2) and propionate (C3). SCFAs have pleiotropic effects in the body, both locally in the gut and after absorption (1Tan J. McKenzie C. Potamitis M. Thorburn A.N. Mackay C.R. Macia L. The role of short-chain fatty acids in health and disease.Adv. Immunol. 2014; 121: 91-119Crossref PubMed Scopus (1204) Google Scholar, 2Natarajan N. Pluznick J.L. From microbe to man: the role of microbial short chain fatty acid metabolites in host cell biology.Am. J. Physiol. Cell Physiol. 2014; 307: C979-C985Crossref PubMed Scopus (115) Google Scholar3Trompette A. Gollwitzer E.S. Yadava K. Sichelstiel A.K. Sprenger N. Ngom-Bru C. Blanchard C. Junt T. Nicod L.P. Harris N.L. Marsland B.J. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis.Nat. Med. 2014; 20: 159-166Crossref PubMed Scopus (1652) Google Scholar). A broad range of these effects occurs subsequent to activation of one or both of a pair of closely related SCFA-regulated G protein-coupled receptors (GPCRs). These are designated as the free fatty acid 2 (FFA2) and free fatty acid 3 (FFA3) receptors (4Stoddart L.A. Smith N.J. Milligan G. International Union of Pharmacology. LXXI. Free fatty acid receptors FFA1, -2, and -3: pharmacology and pathophysiological functions.Pharmacol. Rev. 2008; 60: 405-417Crossref PubMed Scopus (278) Google Scholar5Milligan G. Stoddart L.A. Smith N.J. Agonism and allosterism: the pharmacology of the free fatty acid receptors FFA2 and FFA3.Br. J. Pharmacol. 2009; 158: 146-153Crossref PubMed Scopus (49) Google Scholar, 6Layden B.T. Angueira A.R. Brodsky M. Durai V. Lowe Jr., W.L. Short chain fatty acids and their receptors: new metabolic targets.Transl. Res. 2013; 161: 131-140Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar7Milligan G. Ulven T. Murdoch H. Hudson B.D. G-protein-coupled receptors for free fatty acids: nutritional and therapeutic targets.Br. J. Nutr. 2014; 111: S3-S7Crossref PubMed Scopus (33) Google Scholar). Mapping of key residues that contribute to the function of the SCFAs at both FFA2 and FFA3 demonstrated that mutation of either of a pair of arginine residues, one in transmembrane domain (TMD) V at position 1805.39 (Ballesteros and Weinstein (8Ballesteros J.A. Weinstein H. Integrated methods for modeling G-protein coupled receptors.Methods Neurosci. 1995; 25: 366-428Crossref Scopus (2465) Google Scholar) positional numbering system in superscript) and the other in TMD VII at position 2557.35, eliminated the agonist action of both C2 and C3 (9Stoddart L.A. Smith N.J. Jenkins L. Brown A.J. Milligan G. 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.J. Biol. Chem. 2008; 283: 32913-32924Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Moreover, a pair of histidine residues in TMDs IV and VI, at positions 1404.56 and 2426.55, also played important roles in defining the binding pocket for the SCFAs or their function (9Stoddart L.A. Smith N.J. Jenkins L. Brown A.J. Milligan G. 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.J. Biol. Chem. 2008; 283: 32913-32924Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Hudson B.D. Due-Hansen M.E. Christiansen E. Hansen A.M. Mackenzie A.E. Murdoch H. Pandey S.K. Ward R.J. Marquez R. Tikhonova I.G. Ulven T. Milligan G. Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.J. Biol. Chem. 2013; 288: 17296-17312Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). There is a degree of selectivity in rank-order of potency for the SCFAs between FFA2 and FFA3 with FFA2 preferentially activated by the shorter SCFAs (4Stoddart L.A. Smith N.J. Milligan G. International Union of Pharmacology. LXXI. Free fatty acid receptors FFA1, -2, and -3: pharmacology and pathophysiological functions.Pharmacol. Rev. 2008; 60: 405-417Crossref PubMed Scopus (278) Google Scholar, 5Milligan G. Stoddart L.A. Smith N.J. Agonism and allosterism: the pharmacology of the free fatty acid receptors FFA2 and FFA3.Br. J. Pharmacol. 2009; 158: 146-153Crossref PubMed Scopus (49) Google Scholar) and, in general, by short carboxylic acids with sp2 or sp-hybridized α-carbon atoms (11Schmidt J. Smith N.J. Christiansen E. Tikhonova I.G. Grundmann M. Hudson B.D. Ward R.J. Drewke C. Milligan G. Kostenis E. Ulven T. Selective orthosteric free fatty acid receptor 2 (FFA2) agonists: identification of the structural and chemical requirements for selective activation of FFA2 versus FFA3.J. Biol. Chem. 2011; 286: 10628-10640Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). However, the selectivity is modest, and C3 is the most potent SCFA on both receptors, limiting the ability to use SCFAs to define the specific roles of FFA2 and FFA3 (12Tolhurst G. Heffron H. Lam Y.S. Parker H.E. Habib A.M. Diakogiannaki E. Cameron J. Grosse J. Reimann F. Gribble F.M. Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.Diabetes. 2012; 61: 364-371Crossref PubMed Scopus (1342) Google Scholar, 13Hudson B.D. Tikhonova I.G. Pandey S.K. Ulven T. Milligan G. Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3.J. Biol. Chem. 2012; 287: 41195-41209Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). This is compounded because the absolute potency of the SCFAs also varies between human and rodent orthologs of the receptors (13Hudson B.D. Tikhonova I.G. Pandey S.K. Ulven T. Milligan G. Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3.J. Biol. Chem. 2012; 287: 41195-41209Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). As such, the availability of higher potency and selective synthetic agonists and antagonists would greatly assist efforts to explore the specific function of FFA2 over FFA3. However, few such ligands have been described to date (14Ulven T. Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41 as new potential therapeutic targets.Front. Endocrinol. 2012; 3: 111Crossref PubMed Google Scholar, 15Hudson B.D. Ulven T. Milligan G. The therapeutic potential of allosteric ligands for free fatty acid-sensitive GPCRs.Curr. Top. Med. Chem. 2013; 13: 14-25Crossref PubMed Scopus (27) Google Scholar). The first reported synthetic FFA2-selective agonists were a group of phenylacetamides exemplified by 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (16Smith N.J. Ward R.J. Stoddart L.A. Hudson B.D. Kostenis E. Ulven T. Morris J.C. Tränkle C. Tikhonova I.G. Adams D.R. Milligan G. Extracellular loop 2 of the free fatty acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric modulator.Mol. Pharmacol. 2011; 80: 163-173Crossref PubMed Scopus (63) Google Scholar, 17Lee T. Schwandner R. Swaminath G. Weiszmann J. Cardozo M. Greenberg J. Jaeckel P. Ge H. Wang Y. Jiao X. Liu J. Kayser F. Tian H. Li Y. Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2.Mol. Pharmacol. 2008; 74: 1599-1609Crossref PubMed Scopus (124) Google Scholar18Wang Y. Jiao X. Kayser F. Liu J. Wang Z. Wanska M. Greenberg J. Weiszmann J. Ge H. Tian H. Wong S. Schwandner R. Lee T. Li Y. The first synthetic agonists of FFA2: discovery and SAR of phenylacetamides as allosteric modulators.Bioorg. Med. Chem. Lett. 2010; 20: 493-498Crossref PubMed Scopus (72) Google Scholar). However, these clearly did not share the same binding site as the SCFAs as they were fully active at forms of FFA2 in which either Arg-1805.39 or Arg-2557.35 was altered to alanine (16Smith N.J. Ward R.J. Stoddart L.A. Hudson B.D. Kostenis E. Ulven T. Morris J.C. Tränkle C. Tikhonova I.G. Adams D.R. Milligan G. Extracellular loop 2 of the free fatty acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric modulator.Mol. Pharmacol. 2011; 80: 163-173Crossref PubMed Scopus (63) Google Scholar). They also increased the observed potency of the SCFAs when co-added (16Smith N.J. Ward R.J. Stoddart L.A. Hudson B.D. Kostenis E. Ulven T. Morris J.C. Tränkle C. Tikhonova I.G. Adams D.R. Milligan G. Extracellular loop 2 of the free fatty acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric modulator.Mol. Pharmacol. 2011; 80: 163-173Crossref PubMed Scopus (63) Google Scholar, 17Lee T. Schwandner R. Swaminath G. Weiszmann J. Cardozo M. Greenberg J. Jaeckel P. Ge H. Wang Y. Jiao X. Liu J. Kayser F. Tian H. Li Y. Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2.Mol. Pharmacol. 2008; 74: 1599-1609Crossref PubMed Scopus (124) Google Scholar18Wang Y. Jiao X. Kayser F. Liu J. Wang Z. Wanska M. Greenberg J. Weiszmann J. Ge H. Tian H. Wong S. Schwandner R. Lee T. Li Y. The first synthetic agonists of FFA2: discovery and SAR of phenylacetamides as allosteric modulators.Bioorg. Med. Chem. Lett. 2010; 20: 493-498Crossref PubMed Scopus (72) Google Scholar) and, as such, acted as both allosteric agonists and positive allosteric modulators of the SCFAs. In efforts to identify FFA2-selective agonists that share the same binding site as the SCFAs and are therefore orthosteric in action, we showed that (R)-3-benzyl-4-(cyclopropyl-(4-(2,5-dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid (Cmp 1) is a relatively potent and highly selective agonist of both hFFA2 and murine (m)FFA2 (10Hudson B.D. Due-Hansen M.E. Christiansen E. Hansen A.M. Mackenzie A.E. Murdoch H. Pandey S.K. Ward R.J. Marquez R. Tikhonova I.G. Ulven T. Milligan G. Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.J. Biol. Chem. 2013; 288: 17296-17312Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Like the SCFAs, this ligand contains a carboxylate function, and therefore, on this basis, it was not unexpected when the activity of Cmp 1 was also shown to be lacking in either the R180A5.39 or R255A7.35 mutants of hFFA2 (10Hudson B.D. Due-Hansen M.E. Christiansen E. Hansen A.M. Mackenzie A.E. Murdoch H. Pandey S.K. Ward R.J. Marquez R. Tikhonova I.G. Ulven T. Milligan G. Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.J. Biol. Chem. 2013; 288: 17296-17312Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The lack of function for Cmp 1 and the SCFAs at mutants R180A5.39 or R255A7.35 has generally been taken to suggest that the carboxylate of these ligands form ionic interactions with these arginine residues, and therefore the lack of function results from a lack of binding. However, as binding assays for FFA2 have not been available, it has not been possible to directly test this hypothesis. Therefore, in this study we have developed a radioligand binding assay for FFA2 based on a recently reported FFA2-selective antagonist, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chlorobenzyl)amino]-butyric acid (GLPG0974) (19Pizzonero M. Dupont S. Babel M. Beaumont S. Bienvenu N. Blanqué R. Cherel L. Christophe T. Crescenzi B. De Lemos E. Delerive P. Deprez P. De Vos S. Djata F. Fletcher S. et al.Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: from hit to clinic.J. Med. Chem. 2014; 57: 10044-10057Crossref PubMed Scopus (67) Google Scholar), and we used this in combination with receptor mutation studies and molecular modeling to define how agonist and antagonist ligands interact with this receptor. Through this we demonstrate that although both Arg-1805.39 and Arg-2557.35 are required for agonist binding, only one of these residues is required for high affinity antagonist binding. FFA2 ligands Cmp 1 and CATPB ((S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)phenyl) butanoic acid) were synthesized as described previously (10Hudson B.D. Due-Hansen M.E. Christiansen E. Hansen A.M. Mackenzie A.E. Murdoch H. Pandey S.K. Ward R.J. Marquez R. Tikhonova I.G. Ulven T. Milligan G. Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.J. Biol. Chem. 2013; 288: 17296-17312Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). MeCATPB (methyl (S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-trifluoromethylphenyl)butanoate) is an intermediate in the synthesis of CATPB (10Hudson B.D. Due-Hansen M.E. Christiansen E. Hansen A.M. Mackenzie A.E. Murdoch H. Pandey S.K. Ward R.J. Marquez R. Tikhonova I.G. Ulven T. Milligan G. Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.J. Biol. Chem. 2013; 288: 17296-17312Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Racemic GLPG0974 (4-[[1-(benzo[b]thiophene-3-carbonyl)-2-methylazetidine-2-carbonyl]-(3-chlorobenzyl)amino]butyric acid) (19Pizzonero M. Dupont S. Babel M. Beaumont S. Bienvenu N. Blanqué R. Cherel L. Christophe T. Crescenzi B. De Lemos E. Delerive P. Deprez P. De Vos S. Djata F. Fletcher S. et al.Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: from hit to clinic.J. Med. Chem. 2014; 57: 10044-10057Crossref PubMed Scopus (67) Google Scholar), Cmp 71 (4-(1-(benzo[b]thiophene-3-carbonyl)-2-methyl-N-(4-trifluoromethylbenzyl)azetidine-2-carboxamido)butanoic acid), MeCmp 71 (methyl 4-(1-(benzo[b]thiophene-3-carbonyl)-2-methyl-N-(4-trifluoromethylbenzyl)azetidine-2-carboxamido)butanoate), Cmp 42 (4-(1-(2-(benzo[b]thiophen-3-yl)acetyl)-N-(4-chlorobenzyl)-2-methylazetidine-2-carboxamido)butanoic acid), and MeCmp 42 (methyl 4-(1-(2-(benzo[b]thiophen-3-yl)acetyl)-N-(4-chlorobenzyl)-2-methylazetidine-2-carboxamido)butanoate) were synthesized essentially as described previously (19Pizzonero M. Dupont S. Babel M. Beaumont S. Bienvenu N. Blanqué R. Cherel L. Christophe T. Crescenzi B. De Lemos E. Delerive P. Deprez P. De Vos S. Djata F. Fletcher S. et al.Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: from hit to clinic.J. Med. Chem. 2014; 57: 10044-10057Crossref PubMed Scopus (67) Google Scholar, 20Hansen S.V. Ulven T. Oxalyl chloride as a practical carbon monoxide source for carbonylation reactions.Org. Lett. 2015; 17: 2832-2835Crossref PubMed Scopus (60) Google Scholar). The identity and >95% purity of each compound was confirmed by NMR, HRMS, and HPLC. Tissue culture reagents were from Invitrogen. Molecular biology enzymes and reagents were from Promega. The radiochemical [35S]GTPγS was from PerkinElmer Life Sciences. [3H]GLPG0974 (129 MBq/ml) was a gift of AstraZeneca (Molndal, Sweden). All other experimental reagents used were from Sigma unless indicated otherwise. The hFFA2 or mFFA3 receptors with enhanced yellow fluorescent protein (eYFP) fused to their C termini were cloned into the pcDNA5/FRT/TO expression vector as described previously (9Stoddart L.A. Smith N.J. Jenkins L. Brown A.J. Milligan G. 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.J. Biol. Chem. 2008; 283: 32913-32924Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Hudson B.D. Due-Hansen M.E. Christiansen E. Hansen A.M. Mackenzie A.E. Murdoch H. Pandey S.K. Ward R.J. Marquez R. Tikhonova I.G. Ulven T. Milligan G. Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.J. Biol. Chem. 2013; 288: 17296-17312Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 13Hudson B.D. Tikhonova I.G. Pandey S.K. Ulven T. Milligan G. Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3.J. Biol. Chem. 2012; 287: 41195-41209Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Site-directed mutagenesis to generate the point mutations described was performed according to the QuikChange method (Stratagene, Cheshire, UK). HEK293T cells were used for experiments employing transient heterologous expression. These cells were maintained in Dulbecco's modification of Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and 1× penicillin/streptomycin mixture (Sigma) at 37 °C and 5% CO2. Transfections were performed using polyethyleneimine, and experiments were carried out 48 h after transfection. The inducible lines are described as Flp-InTM T-Rex 293 cells by Invitrogen. These cell lines were generated as described previously (10Hudson B.D. Due-Hansen M.E. Christiansen E. Hansen A.M. Mackenzie A.E. Murdoch H. Pandey S.K. Ward R.J. Marquez R. Tikhonova I.G. Ulven T. Milligan G. Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.J. Biol. Chem. 2013; 288: 17296-17312Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 21Ward R.J. Alvarez-Curto E. Milligan G. Using the Flp-InTMT-RexTM system to regulate GPCR expression.Methods Mol. Biol. 2011; 746: 21-37Crossref PubMed Scopus (44) Google Scholar) and maintained in DMEM without sodium pyruvate supplemented with 10% fetal bovine serum, 1× penicillin/streptomycin mixture, 5 μg/ml blasticidin, and 200 μg/ml hygromycin B. All experiments carried out using these cells were conducted after a 24-h treatment with 100 ng/ml doxycycline, unless otherwise stated, to induce expression of the receptor construct of interest. HEK293T cells were co-transfected at a 4:1 ratio with plasmids encoding an eYFP-tagged form of the receptor construct of interest and a β-arrestin-2 Renilla luciferase (22Hudson B.D. Shimpukade B. Milligan G. Ulven T. The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120).J. Biol. Chem. 2014; 289: 20345-20358Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 23MacKenzie A.E. Caltabiano G. Kent T.C. Jenkins L. McCallum J.E. Hudson B.D. Nicklin S.A. Fawcett L. Markwick R. Charlton S.J. Milligan G. The antiallergic mast cell stabilizers lodoxamide and bufrolin as the first high and equipotent agonists of human and rat GPR35.Mol. Pharmacol. 2014; 85: 91-104Crossref PubMed Scopus (41) Google Scholar). Cells were transferred into white 96-well microtiter plates at 24 h post-transfection. At 48 h post-transfection, cells were washed, and the culture medium was replaced with Hanks' balanced salt solution immediately prior to conducting the assay. To assess the inhibitory ability of prospective antagonist ligands, test compounds were added to the cells followed by incubation for 5 min at 37 °C. To measure β-arrestin-2 recruitment to the receptor, the Renilla luciferase substrate coelenterazine h (Nanolight Tech, Pinetop, CA) was added to a final concentration of 2.5 μm, and cells were incubated for a further 5 min at 37 °C. Next, an EC80 concentration (where EC80 concentration is an 80% maximally effective concentration of an agonist ligand) of an appropriate agonist was added, and cells were incubated for an additional 10 min at 37 °C. BRET resulting from receptor-β-arrestin-2 interaction was assessed by measuring the ratio of luminescence at 535 and 475 nm using a PHERAstar FS plate reader fitted with the BRET1 optic module (BMG Labtech, Aylesbury, UK). All Ca2+ experiments were carried out using Flp-InTM T-RExTM stable-inducible cell lines (24Ward R.J. Pediani J.D. Milligan G. Heteromultimerization of cannabinoid CB(1) receptor and orexin OX(1) receptor generates a unique complex in which both protomers are regulated by orexin A.J. Biol. Chem. 2011; 286: 37414-37428Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 25Alvarez-Curto E. Ward R.J. Pediani J.D. Milligan G. 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.J. Biol. Chem. 2010; 285: 23318-23330Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Cells were plated at 70,000/well in black 96-well plates with a clear bottom and then allowed to adhere for 3–6 h. Doxycycline was then added at 100 ng/ml concentration to induce receptor expression, and cells were maintained in culture overnight. Prior to the assay, cells were labeled for 45 min with the calcium-sensitive dye Fura-2 AM and then washed and incubated for 20 min with Hanks' balanced salt solution containing the indicated concentration of antagonist. Fura-2 fluorescent emission at 510 nm resulting from 340 or 380 nm excitation was then monitored using a Flexstation (Molecular Devices, Sunnyvale, CA) plate reader. Baseline fluorescence was measured for 16 s; test compounds were then added, and fluorescence was measured for an additional 74 s. The baseline-subtracted maximum 340/380 nm ratio obtained after the compound addition was used to plot concentration-response data. Cell membranes were generated as described previously (9Stoddart L.A. Smith N.J. Jenkins L. Brown A.J. Milligan G. 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.J. Biol. Chem. 2008; 283: 32913-32924Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) from Flp-InTM T-RExTM cells either uninduced or treated with doxycycline (100 ng/ml unless otherwise indicated) to induce expression of the receptor construct of interest. [35S]GTPγS binding assays (26Milligan G. Principles: extending the utility of [35S]GTPγS binding assays.Trends Pharmacol. Sci. 2003; 24: 87-90Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 27Cooper T. McMurchie E.J. Leifert W.R. [35S]GTPγS binding in G protein-coupled receptor assays.Methods Mol. Biol. 2009; 552: 143-151Crossref PubMed Scopus (2) Google Scholar) were performed in reactions with 5 μg of cell membrane protein pre-incubated for 15 min at 25 °C in assay buffer (50 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 100 mm NaCl, 1 mm EDTA, 1 μm GDP, and 0.1% fatty acid-free bovine serum albumin) containing the indicated concentrations of ligands. The reaction was initiated with addition of [35S]GTPγS at 50 nCi per tube, and the reaction was terminated after 1 h of incubation at 25 °C by rapid filtration through GF/C glass filters using a 24-well Brandel cell harvester (Alpha Biotech, Glasgow, UK). Unbound radioligand was removed from filters by washing three times with ice-cold wash buffer (50 mm Tris-HCl, pH 7.4, and 10 mm MgCl2), and [35S]GTPγS binding was determined by liquid scintillation spectrometry. All cAMP experiments were performed using Flp-InTM T-RExTM 293 cells able to express receptors of interest in an inducible manner. Experiments were carried out using a homogeneous time-resolved FRET-based detection kit (CisBio Bioassays, Codolet, France) according to the manufacturer's protocol. Cells were plated at 2000 cells/well in low-volume 384-well plates. The ability of agonists to inhibit 1 μm forskolin-induced cAMP production was assessed following a co-incubation for 30 min with agonist compounds, which was preceded by a 15-min pre-incubation with antagonist to allow for equilibration. Experiments were performed using a homogeneous time-resolved FRET-based detection kit (CisBio Bioassays) according to the manufacturer's protocol. Cells were plated at 15,000 cells/well in low-volume 384-well plates. After a 1-h preincubation with antagonist, agonist was added for a further 30 min, and then ERK1/2 phosphorylation was measured. All receptor radioligand binding experiments using [3H]GLPG0974 were conducted in glass tubes, in binding buffer (50 mm Tris-HCl, 100 mm NaCl, 10 mm MgCl2, 1 mm EDTA, pH 7.4). Membrane protein was generated from Flp-InTM T-RExTM cells induced to express the receptor construct of interest with 100 ng/ml doxycycline (unless otherwise stated). Nonspecific binding of the radioligand was determined in the presence of 10 μm CATPB. After the indicated incubation period at 25 °C, bound and free [3H]GLPG0974 were separated by rapid vacuum filtration through GF/C glass filters using a 24-well Brandel cell harvester (Alpha Biotech, Glasgow, UK), and unbound radioligand was washed from filters by three washes" @default.
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• W2209556632 date "2016-01-01" @default.
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• W2209556632 title "Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands" @default.
• W2209556632 cites W1487006512 @default.
• W2209556632 cites W1602174959 @default.
• W2209556632 cites W163048175 @default.
• W2209556632 cites W1688407452 @default.
• W2209556632 cites W1833104430 @default.
• W2209556632 cites W1910086575 @default.
• W2209556632 cites W1948266836 @default.
• W2209556632 cites W1964006025 @default.
• W2209556632 cites W1966345506 @default.
• W2209556632 cites W1974405795 @default.
• W2209556632 cites W1975732928 @default.
• W2209556632 cites W1975740938 @default.
• W2209556632 cites W1990264028 @default.
• W2209556632 cites W1991006409 @default.
• W2209556632 cites W1995358487 @default.
• W2209556632 cites W1998253541 @default.
• W2209556632 cites W1999076037 @default.
• W2209556632 cites W2001938165 @default.
• W2209556632 cites W2004934052 @default.
• W2209556632 cites W2016202053 @default.
• W2209556632 cites W2019419945 @default.
• W2209556632 cites W2021766250 @default.
• W2209556632 cites W2031069848 @default.
• W2209556632 cites W2035421462 @default.
• W2209556632 cites W2037404289 @default.
• W2209556632 cites W2045729780 @default.
• W2209556632 cites W2048469790 @default.
• W2209556632 cites W2056502744 @default.
• W2209556632 cites W2068858277 @default.
• W2209556632 cites W2069247721 @default.
• W2209556632 cites W2070275823 @default.
• W2209556632 cites W2080842149 @default.
• W2209556632 cites W2098863183 @default.
• W2209556632 cites W2104477830 @default.
• W2209556632 cites W2110877685 @default.
• W2209556632 cites W2112379709 @default.
• W2209556632 cites W2125360614 @default.
• W2209556632 cites W2133641393 @default.
• W2209556632 cites W2134066378 @default.
• W2209556632 cites W2140650554 @default.
• W2209556632 cites W2148919838 @default.
• W2209556632 cites W2149531726 @default.
• W2209556632 cites W2150854800 @default.
• W2209556632 cites W2162704444 @default.
• W2209556632 cites W2168842536 @default.
• W2209556632 cites W2169673925 @default.
• W2209556632 cites W2195116424 @default.
• W2209556632 cites W2258399945 @default.
• W2209556632 cites W2330558293 @default.
• W2209556632 cites W2330799739 @default.
• W2209556632 cites W41101067 @default.
• W2209556632 doi "https://doi.org/10.1074/jbc.m115.687939" @default.
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