Phosphorylation Sites of the Gastric Inhibitory Polypeptide Receptor (GIPR) Revealed by Trapped-Ion-Mobility Spectrometry Coupled to Time-of-Flight Mass Spectrometry (TIMS-TOF MS)

Abstract

The gastric inhibitory polypeptide receptor (GIPR), a G protein-coupled receptor (GPCR) that regulates glucose metabolism and insulin secretion, is a target for the development of therapeutic agents to address type 2 diabetes and obesity. Signal transduction processes mediated by GPCR activation typically result in receptor phosphorylation, but very little is known about GIPR phosphorylation. Mass spectrometry (MS) is a powerful tool for detecting phosphorylation and other post-translational modifications of proteins and for identifying modification sites. However, applying MS methods to GPCRs is challenging because the native expression levels are low and the hydrophobicity of these proteins complicates isolation and enrichment. Here we use a widely available technique, trapped-ion-mobility spectrometry coupled to time-of-flight mass spectrometry (TIMS-TOF MS), to characterize the phosphorylation status of the GIPR. We identified eight serine residues that are phosphorylated, one in an intracellular loop and the remainder in the C-terminal domain. Stimulation with the native agonist GIP enhanced phosphorylation at four of these sites. For comparison, we evaluated tirzepatide (TZP), a dual agonist of the glucagon-like peptide-1 (GLP-1) receptor and the GIPR that has recently been approved for the treatment of type 2 diabetes. Stimulation with TZP enhanced phosphorylation at the same four sites that were enhanced with GIP; however, TZP also enhanced phosphorylation at a fifth site that is unique to this synthetic agonist. This work establishes an important and accessible tool for the characterization of signal transduction via the GIPR and reveals an unanticipated functional difference between GIP and TZP.

Figure 1.

Schematic for MS-based analysis of GIPR phosphorylation sites.

Figure 2.

(A) Peptide agonist sequences. Dose-response curves for cAMP production mediated by the GIPR (B) and β-arrestin-2 recruitment to the GIPR (C). N=3 biological replicates for each.

Figure 3.

(A) Sequence coverage (71%) mapped on to the GIPR structure using SCV with AlphaFold. Red indicates regions for which peptides were identified. P indicates a phosphorylation site identified in this work. Label regions include N-terminal, ECD (extracellular domain), TMDs (transmembrane domains), and C-terminal. (B) Sequences of the intracellular loop 3-transmembrane helix 6 junction region and the C-terminal domain showing the possible phosphorylation sites (Ser, Thr, and Tyr residues) in red. Circles indicate phosphorylation sites identified in this study.

Figure 4.

Representative annotated MSMS spectrum for a tryptic peptide that revealed the phosphorylation of S433.

Figure 5.

Phosphorylated peptide intensity and total peptide (unmodified + modified) intensity averaged across three biological replicates. Error bars represent +/− SE. A two-way ANOVA test was used to identify significant changes in phosphorylation for peptide-stimulated samples relative to the control. Changes were considered significant with a p-value ≤ 0.05. P-values of ≤ 0.05, ≤ 0.01, and ≤ 0.001 are represented by *, **, ***, respectively.