title: Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) differentially regulates orthosteric but not allosteric agonist binding and function.
abstract: 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.
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: Cassandra
authors.initials: C
authors.lastname: Koole
authors.forename: Denise
authors.initials: D
authors.lastname: Wootten
authors.forename: John
authors.initials: J
authors.lastname: Simms
authors.forename: Emilia E
authors.initials: EE
authors.lastname: Savage
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Miller
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D040761:Biomimetic Materials / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D000067757:Glucagon-Like Peptide-1 Receptor; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018027:Receptors, Glucagon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C000593833:GLP1R protein, human; D000067757:Glucagon-Like Peptide-1 Receptor; D010455:Peptides; D018027:Receptors, Glucagon; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3