Exploring G Protein-coupled Receptor Signaling Networks Using SILAC-based Phosphoproteomics

Overview

Journal Methods

Specialty Biochemistry

Date 2015 Jul 11

PMID 26160508

Citations 12

Authors

Grace R Williams

Jennifer R Bethard

Mary N Berkaw

Alexis K Nagel

Louis M Luttrell

Lauren E Ball

Affiliations

Soon will be listed here.

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.

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References

Schwartz D, Gygi S . An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat Biotechnol. 2005; 23(11):1391-8. DOI: 10.1038/nbt1146. View

Ten Klooster J, Jaffer Z, Chernoff J, Hordijk P . Targeting and activation of Rac1 are mediated by the exchange factor beta-Pix. J Cell Biol. 2006; 172(5):759-69. PMC: 2063707. DOI: 10.1083/jcb.200509096. View

Gesty-Palmer D, Chen M, Reiter E, Ahn S, Nelson C, Wang S . Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation. J Biol Chem. 2006; 281(16):10856-64. DOI: 10.1074/jbc.M513380200. View

Beausoleil S, Villen J, Gerber S, Rush J, Gygi S . A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol. 2006; 24(10):1285-92. DOI: 10.1038/nbt1240. View

Galandrin S, Bouvier M . Distinct signaling profiles of beta1 and beta2 adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy. Mol Pharmacol. 2006; 70(5):1575-84. DOI: 10.1124/mol.106.026716. View

Olsen J, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P . Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 2006; 127(3):635-48. DOI: 10.1016/j.cell.2006.09.026. View

Canas B, Pineiro C, Calvo E, Lopez-Ferrer D, Gallardo J . Trends in sample preparation for classical and second generation proteomics. J Chromatogr A. 2007; 1153(1-2):235-58. DOI: 10.1016/j.chroma.2007.01.045. View

Villen J, Gygi S . The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc. 2008; 3(10):1630-8. PMC: 2728452. DOI: 10.1038/nprot.2008.150. View

Ubersax J, Ferrell Jr J . Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol. 2007; 8(7):530-41. DOI: 10.1038/nrm2203. View

10.

Linding R, Jensen L, Ostheimer G, van Vugt M, Jorgensen C, Miron I . Systematic discovery of in vivo phosphorylation networks. Cell. 2007; 129(7):1415-26. PMC: 2692296. DOI: 10.1016/j.cell.2007.05.052. View

11.

Gilar M, Jaworski A, Olivova P, Gebler J . Peptide retention prediction applied to proteomic data analysis. Rapid Commun Mass Spectrom. 2007; 21(17):2813-21. DOI: 10.1002/rcm.3150. View

12.

Kenakin T . Functional selectivity through protean and biased agonism: who steers the ship?. Mol Pharmacol. 2007; 72(6):1393-401. DOI: 10.1124/mol.107.040352. View

13.

Linding R, Jensen L, Pasculescu A, Olhovsky M, Colwill K, Bork P . NetworKIN: a resource for exploring cellular phosphorylation networks. Nucleic Acids Res. 2007; 36(Database issue):D695-9. PMC: 2238868. DOI: 10.1093/nar/gkm902. View

14.

Chahdi A, Sorokin A . Protein kinase A-dependent phosphorylation modulates beta1Pix guanine nucleotide exchange factor activity through 14-3-3beta binding. Mol Cell Biol. 2007; 28(5):1679-87. PMC: 2258773. DOI: 10.1128/MCB.00898-07. View

15.

Cheung M, Sharma A, Madhunapantula S, Robertson G . Akt3 and mutant V600E B-Raf cooperate to promote early melanoma development. Cancer Res. 2008; 68(9):3429-39. PMC: 2603082. DOI: 10.1158/0008-5472.CAN-07-5867. View

16.

Miedlich S, Abou-Samra A . Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization. Am J Physiol Endocrinol Metab. 2008; 295(3):E665-71. PMC: 2536737. DOI: 10.1152/ajpendo.00036.2008. View

17.

Baljuls A, Schmitz W, Mueller T, Zahedi R, Sickmann A, Hekman M . Positive regulation of A-RAF by phosphorylation of isoform-specific hinge segment and identification of novel phosphorylation sites. J Biol Chem. 2008; 283(40):27239-54. DOI: 10.1074/jbc.M801782200. View

18.

Guan K, Figueroa C, Brtva T, Zhu T, Taylor J, Barber T . Negative regulation of the serine/threonine kinase B-Raf by Akt. J Biol Chem. 2000; 275(35):27354-9. DOI: 10.1074/jbc.M004371200. View

19.

Zhang B, Guan K . Regulation of the Raf kinase by phosphorylation. Exp Lung Res. 2001; 27(3):269-95. DOI: 10.1080/019021401300054046. View

20.

Tawfeek H, Qian F, Abou-Samra A . Phosphorylation of the receptor for PTH and PTHrP is required for internalization and regulates receptor signaling. Mol Endocrinol. 2002; 16(1):1-13. DOI: 10.1210/mend.16.1.0760. View