Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) differentially regulates orthosteric but not allosteric agonist binding and function

Cassandra Koole, Denise Wootten, John Simms, Emilia E Savage, Laurence J Miller, Arthur Christopoulos, Patrick M Sexton

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)


The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical family B G protein-coupled receptor that exhibits physiologically important pleiotropic coupling and ligand-dependent signal bias. In our accompanying article (Koole, C., Wootten, D., Simms, J., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2012) J. Biol. Chem. 287, 3642-3658), we demonstrate, through alanine-scanning mutagenesis, a key role for extracellular loop (ECL) 2 of the receptor in propagating activation transition mediated by GLP-1 peptides that occurs in a peptide- and pathway-dependent manner for cAMP formation, intracellular (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). In this study, we examine the effect of ECL2 mutations on the binding and signaling of the peptide mimetics, exendin-4 and oxyntomodulin, as well as small molecule allosteric agonist 6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2). Lys-288, Cys-296, Trp-297, and Asn-300 were globally important for peptide signaling and also had critical roles in governing signal bias of the receptor. Peptide-specific effects on relative efficacy and signal bias were most commonly observed for residues 301-305, although R299A mutation also caused significantly different effects for individual peptides. Met-303 was more important for exendin-4 and oxyntomodulin action than those of GLP-1 peptides. Globally, ECL2 mutation was more detrimental to exendin-4-mediated Ca(2+)i release than GLP-1(7-36)-NH(2), providing additional evidence for subtle differences in receptor activation by these two peptides. Unlike peptide activation of the GLP-1R, ECL2 mutations had only limited impact on compound 2 mediated cAMP and pERK responses, consistent with this ligand having a distinct mechanism for receptor activation. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition of the receptor by peptide agonists.

Original languageEnglish
Pages (from-to)3659-3673
Number of pages15
JournalJournal of Biological Chemistry
Issue number6
Publication statusPublished - 3 Feb 2012


  • Allosteric Regulation
  • Amino Acid Substitution
  • Biomimetic Materials
  • Cell Line
  • Glucagon-Like Peptide-1 Receptor
  • Humans
  • MAP Kinase Signaling System
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mutation, Missense
  • Peptides
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Receptors, Glucagon
  • Journal Article
  • Research Support, Non-U.S. Gov't


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