PT  - JOURNAL ARTICLE
AU  - Koole, Cassandra
AU  - Wootten, Denise
AU  - Simms, John
AU  - Miller, Laurence J
AU  - Christopoulos, Arthur
AU  - Sexton, Patrick M
TI  - Differential Impact of Amino Acid Substitutions on Critical Residues of the Human Glucagon-Like Peptide-1 Receptor (GLP-1R) Involved in Peptide Activity and Small Molecule Allostery
AID  - 10.1124/jpet.114.220913
DP  - 2015 Jan 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - jpet.114.220913
4099  - http://jpet.aspetjournals.org/content/early/2015/01/28/jpet.114.220913.short
4100  - http://jpet.aspetjournals.org/content/early/2015/01/28/jpet.114.220913.full
AB  - The glucagon-like peptide-1 receptor (GLP-1R) is a Class B G protein-coupled receptor (GPCR) that has a critical role in the regulation of glucose homeostasis, principally though regulation of insulin secretion. The receptor system is highly complex, with the ability 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 (6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2), 4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP)). 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 that have previously been 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 monitored across physiologically significant signaling pathways including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation (pERK1/2) 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 has little impact on peptide-mediated receptor activation, but this activity is unable to be modulated by compound 2 to the same extent as that observed at the wildtype receptor. These results provide insight into the importance of residues 149 and 333 in peptide function as well as highlighting the complexities of allosteric modulation within this receptor system.