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 Koole, Denise Wootten, John Simms, Laurence J Miller, Arthur Christopoulos, Patrick M Sexton

Research output: Contribution to journalArticle

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)-NH(2) 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)-NH(2)- 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 - 3 Feb 2012

Fingerprint

Glucagon-Like Peptide 1
Peptide Receptors
Chemical activation
Peptides
Mitogen-Activated Protein Kinase 3
Mitogen-Activated Protein Kinase 1
Phosphorylation
Mutation
Alanine
Bioactivity
Ligands
Mutagenesis
G-Protein-Coupled Receptors
Biosynthesis
Type 2 Diabetes Mellitus
Medical problems
Glucagon-Like Peptide-1 Receptor
Insulin
Scanning

Keywords

  • Amino Acid Substitution
  • Animals
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Cyclic AMP
  • Glucagon-Like Peptide 1
  • Glucagon-Like Peptide-1 Receptor
  • Humans
  • MAP Kinase Signaling System
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mutation, Missense
  • Peptides
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Receptors, Glucagon
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

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; Wootten, Denise; Simms, John; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M.

In: Journal of Biological Chemistry, Vol. 287, No. 6, 03.02.2012, p. 3642-3658.

Research output: Contribution to journalArticle

Koole, Cassandra ; Wootten, Denise ; Simms, John ; Miller, Laurence J ; Christopoulos, Arthur ; Sexton, Patrick M. / Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 6. pp. 3642-3658.
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AU - Miller, Laurence J

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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)-NH(2) 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)-NH(2)- 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)-NH(2) 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)-NH(2)- 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.

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KW - CHO Cells

KW - Cricetinae

KW - Cricetulus

KW - Cyclic AMP

KW - Glucagon-Like Peptide 1

KW - Glucagon-Like Peptide-1 Receptor

KW - Humans

KW - MAP Kinase Signaling System

KW - Mitogen-Activated Protein Kinase 1

KW - Mitogen-Activated Protein Kinase 3

KW - Mutation, Missense

KW - Peptides

KW - Protein Binding

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Receptors, Glucagon

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

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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 - 0021-9258

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ER -