title: Extracellular loop 2 of the free fatty acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric modulator.
abstract: 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 Leu(173) 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.
authors.affiliation: Molecular Pharmacology Group, Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Smith
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
authors.forename: Leigh A
authors.initials: LA
authors.lastname: Stoddart
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hudson
authors.forename: Evi
authors.initials: E
authors.lastname: Kostenis
authors.forename: Trond
authors.initials: T
authors.lastname: Ulven
authors.forename: Joanne C
authors.initials: JC
authors.lastname: Morris
authors.forename: Christian
authors.initials: C
authors.lastname: Tränkle
authors.forename: Irina G
authors.initials: IG
authors.lastname: Tikhonova
authors.forename: David R
authors.initials: DR
authors.lastname: Adams
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000081:Acetamides / Q000378:metabolism*; D000494:Allosteric Regulation; D002478:Cells, Cultured; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000081:Acetamides; D005227:Fatty Acids; D011956:Receptors, Cell Surface