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Methods. 2016 Jan 1;92:36-50. doi: 10.1016/j.ymeth.2015.06.022. Epub 2015 Jul 6.

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

Author information

1
Department of Molecular and Cellular Pharmacology, Medical University of South Carolina, Charleston, SC 29425, USA.
2
Department of Molecular and Cellular Pharmacology, Medical University of South Carolina, Charleston, SC 29425, USA; Department of Oral Health Sciences, Medical University of South Carolina, Charleston, SC 29425, USA.
3
Department of Medicine, Medical University of South Carolina, Charleston, SC 29425, USA; Research Service of the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29401, USA.
4
Department of Molecular and Cellular Pharmacology, Medical University of South Carolina, Charleston, SC 29425, USA; Department of Oral Health Sciences, Medical University of South Carolina, Charleston, SC 29425, USA. Electronic address: ballle@musc.edu.

Abstract

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 5min 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.

KEYWORDS:

G protein-coupled receptor; Mass spectrometry; Osteoblast; Parathyroid hormone receptor; Phosphoproteomics

PMID:
26160508
PMCID:
PMC4696908
DOI:
10.1016/j.ymeth.2015.06.022
[Indexed for MEDLINE]
Free PMC Article

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