Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 3642 - 3658 |
| Number of pages | 17 |
| Journal | Journal of Biological Chemistry |
| Volume | 287 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 2012 |
Cite this
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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 journal › Article › Research › peer-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 -