Differential impact of amino acid substitutions on critical residues of the human glucagon-like peptide-1 receptor involved in peptide activity and small-molecule allosterys

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

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor systemis highly complex, able to be activated by both endogenous [GLP-1(1-36)NH<inf>2</inf>, GLP-1(1-37), GLP-1(7-36)NH<inf>2</inf>, GLP-1(7-37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tertbutylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptidemediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca<sup>2+</sup> mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.

Original languageEnglish (US)
Pages (from-to)52-63
Number of pages12
JournalJournal of Pharmacology and Experimental Therapeutics
Volume353
Issue number1
DOIs
StatePublished - Apr 1 2015

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Amino Acid Substitution
Peptides
Glucagon-Like Peptide 1
Oxyntomodulin
Amino Acids
Mutation
Peptide Receptors
Mitogen-Activated Protein Kinase 3
Mitogen-Activated Protein Kinase 1
G-Protein-Coupled Receptors
Homeostasis
Nucleotides
Phosphorylation
Insulin
Glucose
Glucagon-Like Peptide-1 Receptor
Proteins
glucagon-like peptide 1 (7-37)

ASJC Scopus subject areas

  • Pharmacology
  • Molecular Medicine
  • Medicine(all)

Cite this

Differential impact of amino acid substitutions on critical residues of the human glucagon-like peptide-1 receptor involved in peptide activity and small-molecule allosterys. / Koole, Cassandra; Wootten, Denise; Simms, John; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 353, No. 1, 01.04.2015, p. 52-63.

Research output: Contribution to journalArticle

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abstract = "The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor systemis highly complex, able to be activated by both endogenous [GLP-1(1-36)NH2, GLP-1(1-37), GLP-1(7-36)NH2, GLP-1(7-37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tertbutylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptidemediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca2+ mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.",
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