Extracellular Loop 2 of the Free Fatty Acid Receptor 2 Mediates Allosterism of a Phenylacetamide Ago-Allosteric Modulator

Nicola J. Smith, Richard J. Ward, Leigh A. Stoddart, Brian D. Hudson, Evi Kostenis, Trond Ulven, Joanne C Morris, Christian Traenkle, Irina G. Tikhonova, David R. Adams, Graeme Milligan

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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-alpha-(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.

Original languageEnglish
Pages (from-to)163-173
Number of pages11
JournalMolecular Pharmacology
Volume80
Issue number1
DOIs
Publication statusPublished - Jul 2011

Keywords

  • PROTEIN-COUPLED RECEPTOR
  • STRUCTURAL BASIS
  • FUNCTIONAL-CHARACTERIZATION
  • DRUG DISCOVERY
  • ACTIVATION
  • BINDING
  • IDENTIFICATION
  • PHARMACOLOGY
  • RESIDUES
  • AGONISM

Cite this

Smith, N. J., Ward, R. J., Stoddart, L. A., Hudson, B. D., Kostenis, E., Ulven, T., Morris, J. C., Traenkle, C., Tikhonova, I. G., Adams, D. R., & Milligan, G. (2011). Extracellular Loop 2 of the Free Fatty Acid Receptor 2 Mediates Allosterism of a Phenylacetamide Ago-Allosteric Modulator. Molecular Pharmacology, 80(1), 163-173. https://doi.org/10.1124/mol.110.070789