Differential Impact of Amino Acid Substitutions on Critical Residues of the Human Glucagon-Like Peptide-1 Receptor Involved in Peptide Activity and Small-Molecule Allostery

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)NH 2 , GLP-1(1-37), GLP-1(7-36)NH 2 , 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 21 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.

Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists

“…The predominant intracellular signalling intermediate that couples GLP-1R activation to its downstream effects is cyclic adenosine monophosphate (cAMP) (5,6). However, an updated view of GLP-1R pharmacology highlights the roles of membrane trafficking (7,8) and additional effector proteins such as the β-arrestins (9,10) in the control of amplitude, duration and subcellular localisation of signalling events to regulate insulin secretion, particularly in the pharmacological setting. Although all clinically approved GLP-1RAs show broadly similar signalling and trafficking characteristics to the endogenous ligand, GLP-1(7-36)NH2, these can be dramatically altered via sequence modifications close to the ligand N-terminus, as demonstrated recently using analogues of the GLP-1 homologue peptide exendin-4 (11,12).…”

Pharmacogenetics of type 2 diabetes mellitus, the route toward tailored medicine

“…The GLP‐1 receptor is an important drug target for the treatment of T2DM, and several non‐synonymous variants of GLP1R have been carefully characterized: rs367543060 Thr149Met variant, identified in one Japanese diabetic subject, induces a significant loss of function in vitro and impairs the insulin secretory response to GLP‐1 in vivo . The polymorphism rs10305493 (Ser333Cys) instead has been proven to preserve peptide response .…”

“…198,201 Taken together, these findings suggest that GIPR variants could potentially modulate the response to DPP-4 inhibitors, nevertheless, to date, this effect has not been revealed by clinical studies. 194,197,202 The GLP-1 receptor is an important drug target for the treatment of T2DM, and several non-synonymous variants of GLP1R have been carefully characterized: rs367543060 Thr149Met variant, identified in one Japanese diabetic subject, 203 induces a significant loss of function in vitro [204][205][206] and impairs the insulin secretory response to GLP-1 in vivo. 203,206 The polymorphism rs10305493 (Ser333Cys) instead has been proven to preserve peptide response.…”

“…194,197,202 The GLP-1 receptor is an important drug target for the treatment of T2DM, and several non-synonymous variants of GLP1R have been carefully characterized: rs367543060 Thr149Met variant, identified in one Japanese diabetic subject, 203 induces a significant loss of function in vitro [204][205][206] and impairs the insulin secretory response to GLP-1 in vivo. 203,206 The polymorphism rs10305493 (Ser333Cys) instead has been proven to preserve peptide response. 205,206 The SNP rs6923761 (Gly168Ser) was nominally associated with reduced insulin secretion in response to GLP-1 infusion during a hyperglycemic clamp in nondiabetic American subjects 207 and with a weaker response to the glucose-lowering effect of DPP-4 inhibitors in patients with T2DM.…”

“…203,206 The polymorphism rs10305493 (Ser333Cys) instead has been proven to preserve peptide response. 205,206 The SNP rs6923761 (Gly168Ser) was nominally associated with reduced insulin secretion in response to GLP-1 infusion during a hyperglycemic clamp in nondiabetic American subjects 207 and with a weaker response to the glucose-lowering effect of DPP-4 inhibitors in patients with T2DM. 197 On the contrary, the same polymorphism was associated with higher efficacy of liraglutide, 208 and it was shown to increase weight and fat mass loss after liraglutide treatment, 209,210 different types of diet, 211 or bilio-pancreatic diversion surgery.…”