Exploring G protein-coupled receptor signaling networks using SILAC-based phosphoproteomics

• Dynamic phosphorylation events in osteoblasts stimulated with hPTH(1-34) were measured.
• Acute stimulation of the PTH1R engaged canonical GPCR signaling pathways.
• Novel signaling intermediates stimulated by PTH[1-34] were revealed.
• Quantitative LC–MS/MS methods were established to assess PTH1R signaling networks.

The type 1 parathyroid hormone receptor (PTH1R) is a key regulator of calcium homeostasis and bone turnover. Here, we employed SILAC-based quantitative mass spectrometry and bioinformatic pathways analysis to examine global changes in protein phosphorylation following short-term stimulation of endogenously expressed PTH1R in osteoblastic cells in vitro. Following 5 min exposure to the conventional agonist, PTH(1-34), we detected significant changes in the phosphorylation of 224 distinct proteins. Kinase substrate motif enrichment demonstrated that consensus motifs for PKA and CAMK2 were the most heavily upregulated within the phosphoproteome, while consensus motifs for mitogen-activated protein kinases were strongly downregulated. Signaling pathways analysis identified ERK1/2 and AKT as important nodal kinases in the downstream network and revealed strong regulation of small GTPases involved in cytoskeletal rearrangement, cell motility, and focal adhesion complex signaling. Our data illustrate the utility of quantitative mass spectrometry in measuring dynamic changes in protein phosphorylation following GPCR activation.