Abstract
Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.
Highlights
Endogenous ligands of the glucagon-like peptide-1 receptor (GLP-1R) are small peptide hormones including glucagon-like peptide-1 (GLP-1), which is encoded by the glucagon gene [1] and these peptides are secreted from pancreatic beta-cells
In order to compare stabilization of these ligands by the receptor extracellular domain Hydrogen/deuterium exchange (HDX) was performed on the peptide ligands in the presence of N-terminal extracellular domain of GLP-1R (nGLP-1R) (Figure 2)
When HDX was measured for these peptides bound to nGLP-1R, protection to exchange was observed at the N-terminal helix near the bend, the same region in close contact with nGLP-1R in crystal structures [9,10]
Summary
Endogenous ligands of the glucagon-like peptide-1 receptor (GLP-1R) are small peptide hormones including glucagon-like peptide-1 (GLP-1), which is encoded by the glucagon gene [1] and these peptides are secreted from pancreatic beta-cells [2] The incretin hormones are potent stimulators of glucose-induced insulin biosynthesis and secretion. DPP4 resistant mimetics of these endogenous peptides such as exendin-4 and liraglutide, offer extended plasma half-life as compared to endogenous hormone and they are used therapeutically for the treatment of type 2 diabetes mellitus (T2DM) [3]. Like other class B GPCRs, the GLP-1R consists of two main domains, a large extracellular ligand binding domain and a seven transmembrane helical domain [5,6]. This extracellular domain is capable of binding the C-terminal side of peptide ligands, and is believed to orient the N-terminal region of peptide ligands toward the classic orthosteric transmembrane binding pocket [7,8]
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