Signal Transduction
Second Extracellular Loop of Human Glucagon-like Peptide-1 Receptor (GLP-1R) Differentially Regulates Orthosteric but Not Allosteric Agonist Binding and Function*

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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 (Ca2+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 Ca2+i release than GLP-1(7–36)-NH2, 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.

Cell Signaling
G Protein-coupled Receptors (GPCR)
Peptide Hormones
Receptor Structure-Function
Signal Transduction
Allosteric Agonist
Exendin
Glucagon-like Peptide-1 Receptor
Ligand-directed Signaling Bias
Oxyntomodulin

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*

This work was supported in part by National Health and Medical Research Council of Australia Project Grant 1002180 and Program Grant 519461 and by a National Health and Medical Research Council Principal Research Fellowship (to P. M. S.) and a Senior Research Fellowship (to A. C.).

This article contains supplemental Figs. S1–S3.

1

Senior Research Fellow of the National Health and Medical Research Council.