Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation

Cassandra Renee Koole, Denise Laura Wootten, John Watson Simms, Laurence J Miller, Arthur Christopoulos, Patrick Sexton

Research output: Contribution to journalArticleResearchpeer-review

56 Citations (Scopus)

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH 2 or GLP-1(7-36)-NH2 binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca 2+ (Ca 2+ i) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH2- and GLP-1(7-36)- NH 2-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH 2-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.
Original languageEnglish
Pages (from-to)3642 - 3658
Number of pages17
JournalJournal of Biological Chemistry
Volume287
Issue number6
DOIs
Publication statusPublished - 2012

Cite this

@article{357ba02b2eae41f2bc1c359033f1046d,
title = "Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation",
abstract = "The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH 2 or GLP-1(7-36)-NH2 binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca 2+ (Ca 2+ i) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH2- and GLP-1(7-36)- NH 2-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH 2-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.",
author = "Koole, {Cassandra Renee} and Wootten, {Denise Laura} and Simms, {John Watson} and Miller, {Laurence J} and Arthur Christopoulos and Patrick Sexton",
year = "2012",
doi = "10.1074/jbc.M111.309328",
language = "English",
volume = "287",
pages = "3642 -- 3658",
journal = "Journal of Biological Chemistry",
issn = "1083-351X",
publisher = "American Society for Biochemistry and Molecular Biology",
number = "6",

}

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation. / Koole, Cassandra Renee; Wootten, Denise Laura; Simms, John Watson; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick.

In: Journal of Biological Chemistry, Vol. 287, No. 6, 2012, p. 3642 - 3658.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation

AU - Koole, Cassandra Renee

AU - Wootten, Denise Laura

AU - Simms, John Watson

AU - Miller, Laurence J

AU - Christopoulos, Arthur

AU - Sexton, Patrick

PY - 2012

Y1 - 2012

N2 - The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH 2 or GLP-1(7-36)-NH2 binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca 2+ (Ca 2+ i) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH2- and GLP-1(7-36)- NH 2-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH 2-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.

AB - The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH 2 or GLP-1(7-36)-NH2 binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca 2+ (Ca 2+ i) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH2- and GLP-1(7-36)- NH 2-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH 2-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.

UR - http://www.jbc.org/content/287/6/3642

U2 - 10.1074/jbc.M111.309328

DO - 10.1074/jbc.M111.309328

M3 - Article

VL - 287

SP - 3642

EP - 3658

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 1083-351X

IS - 6

ER -