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 system is 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-tert-butylaminoquinoxaline], 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 peptide-mediated 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(2+) 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 language | English |
---|---|
Pages (from-to) | 52-63 |
Number of pages | 12 |
Journal | The Journal of pharmacology and experimental therapeutics |
Volume | 353 |
Issue number | 1 |
DOIs | |
Publication status | Published - Apr 2015 |
Externally published | Yes |
Keywords
- Allosteric Regulation
- Amino Acid Substitution
- Animals
- CHO Cells
- Calcium
- Cricetulus
- Cyclic AMP
- Extracellular Space
- Glucagon-Like Peptide 1
- Glucagon-Like Peptide-1 Receptor
- Humans
- Mitogen-Activated Protein Kinase 1
- Mitogen-Activated Protein Kinase 3
- Mutation
- Peptide Fragments
- Phosphorylation
- Polymorphism, Single Nucleotide
- Receptors, Glucagon
- Signal Transduction
- Journal Article
- Research Support, Non-U.S. Gov't