A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand

J Biol Chem. 2008 Oct 24;283(43):29312-21. doi: 10.1074/jbc.M803801200. Epub 2008 Aug 22.

Abstract

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride / pharmacology*
  • Allosteric Site
  • Carbamates / chemistry
  • Cell Line
  • Humans
  • Kinetics
  • Ligands
  • Models, Biological
  • Models, Chemical
  • Molecular Conformation
  • Mutagenesis
  • Phosphorylation
  • Point Mutation
  • Receptors, G-Protein-Coupled / metabolism*
  • Receptors, Muscarinic / metabolism*

Substances

  • Carbamates
  • Ligands
  • Receptors, G-Protein-Coupled
  • Receptors, Muscarinic
  • (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride