Probing the stoichiometry of β2-adrenergic receptor phosphorylation by targeted mass spectrometry

Journal of Molecular Signaling

Hsien-Yu Wang²  Craig Malbon¹  Shujuan Gao¹ 

[1] Department of Pharmacology, Health Sciences Center, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA;Department of Physiology & Biophysics, Health Sciences Center, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA

关键词: G-protein coupled receptor kinase (GRK);    Molar quantification;    Targeted mass spectrometry;    Liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS);    Protein phosphorylation;    β2-adrenergic receptor;    G-protein-coupled receptors;   

Others  :  802238
DOI  :  10.1186/1750-2187-9-3

 received in 2014-01-13, accepted in 2014-03-18,  发布年份 2014

【 摘 要 】

Background

Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (β2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation.

Method

We stimulated HEK293 cells stably expressing Flag-tagged β2AR-eGFP with 10 μM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells.

Results

Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied β2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the β2AR is phosphorylated at these sites.

Conclusions

Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of β2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with β2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.

【 授权许可】

   
2014 Gao et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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【 参考文献 】

• [1]Morris AJ, Malbon CC: Physiological regulation of G protein-linked signaling. Physiol Rev 1999, 79:1373-1430.
• [2]Lagerstrom MC, Schioth HB: Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 2008, 7:339-357.
• [3]Kolb P, Rosenbaum DM, Irwin JJ, Fung JJ, Kobilka BK, Shoichet BK: Structure-based discovery of beta2-adrenergic receptor ligands. Proc Natl Acad Sci USA 2009, 106:6843-6848.
• [4]Jensen BC, O’Connell TD, Simpson PC: Alpha-1-adrenergic receptors: targets for agonist drugs to treat heart failure. J Mol Cell Cardiol 2011, 51:518-528.
• [5]Proskocil BJ, Fryer AD: Beta2-agonist and anticholinergic drugs in the treatment of lung disease. Proc Am Thorac Soc 2005, 2:305-310.
• [6]Warmack TS, Estes MA, Heldenbrand S, Franks AM: Beta-adrenergic antagonists in hypertension: a review of the evidence. Ann Pharmacother 2009, 43:2031-2043.
• [7]Deribe YL, Pawson T, Dikic I: Post-translational modifications in signal integration. Nat Struct Mol Biol 2010, 17:666-672.
• [8]Tobin AB, Butcher AJ, Kong KC: Location, location, location…site-specific GPCR phosphorylation offers a mechanism for cell-type-specific signalling. Trends Pharmacol Sci 2008, 29:413-420.
• [9]Benovic JL, Pike LJ, Cerione RA, Staniszewski C, Yoshimasa T, Codina J, Caron MG, Lefkowitz RJ: Phosphorylation of the mammalian beta-adrenergic receptor by cyclic AMP-dependent protein kinase. Regulation of the rate of receptor phosphorylation and dephosphorylation by agonist occupancy and effects on coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. J Biol Chem 1985, 260:7094-7101.
• [10]Benovic JL, Strasser RH, Caron MG, Lefkowitz RJ: Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci USA 1986, 83:2797-2801.
• [11]Fredericks ZL, Pitcher JA, Lefkowitz RJ: Identification of the G protein-coupled receptor kinase phosphorylation sites in the human beta2-adrenergic receptor. J Biol Chem 1996, 271:13796-13803.
• [12]Lefkowitz RJ: G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J Biol Chem 1998, 273:18677-18680.
• [13]Tran TM, Friedman J, Qunaibi E, Baameur F, Moore RH, Clark RB: Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the beta2-adrenergic receptor using phosphoserine-specific antibodies. Mol Pharmacol 2004, 65:196-206.
• [14]Vayttaden SJ, Friedman J, Tran TM, Rich TC, Dessauer CW, Clark RB: Quantitative modeling of GRK-mediated beta2AR regulation. PLoS Comput Biol 2010, 6:e1000647.
• [15]Goodman OB Jr, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW, Keen JH, Benovic JL: Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor. Nature 1996, 383:447-450.
• [16]Kang DS, Tian X, Benovic JL: beta-Arrestins and G protein-coupled receptor trafficking. Methods Enzymol 2013, 521:91-108.
• [17]Lohse MJ, Benovic JL, Codina J, Caron MG, Lefkowitz RJ: beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science 1990, 248:1547-1550.
• [18]Reiter E, Ahn S, Shukla AK, Lefkowitz RJ: Molecular mechanism of beta-arrestin-biased agonism at seven-transmembrane receptors. Annu Rev Pharmacol Toxicol 2012, 52:179-197.
• [19]Butcher AJ, Prihandoko R, Kong KC, McWilliams P, Edwards JM, Bottrill A, Mistry S, Tobin AB: Differential G-protein-coupled receptor phosphorylation provides evidence for a signaling bar code. J Biol Chem 2011, 286:11506-11518.
• [20]Butcher AJ, Kong KC, Prihandoko R, Tobin AB: Physiological role of G-protein coupled receptor phosphorylation. Handb Exp Pharmacol 2012, 208:79-94.
• [21]Tobin AB: G-protein-coupled receptor phosphorylation: where, when and by whom. Br J Pharmacol 2008, 153(Suppl 1):S167-S176.
• [22]Macek B, Mann M, Olsen JV: Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 2009, 49:199-221.
• [23]Ong SE, Mann M: Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol 2005, 1:252-262.
• [24]Christensen GL, Kelstrup CD, Lyngso C, Sarwar U, Bogebo R, Sheikh SP, Gammeltoft S, Olsen JV, Hansen JL: Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists. Mol Cell Proteomics 2010, 9:1540-1553.
• [25]Eyrich B, Sickmann A, Zahedi RP: Catch me if you can: mass spectrometry-based phosphoproteomics and quantification strategies. Proteomics 2011, 11:554-570.
• [26]Ibarrola N, Kalume DE, Gronborg M, Iwahori A, Pandey A: A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture. Anal Chem 2003, 75:6043-6049.
• [27]Domon B, Aebersold R: Options and considerations when selecting a quantitative proteomics strategy. Nat Biotechnol 2010, 28:710-721.
• [28]Kiyonami R, Schoen A, Prakash A, Peterman S, Zabrouskov V, Picotti P, Aebersold R, Huhmer A, Domon B: Increased selectivity, analytical precision, and throughput in targeted proteomics. Mol Cell Proteomics 2011, 10:M110.
• [29]Baker JG: The selectivity of beta-adrenoceptor antagonists at the human beta1, beta2 and beta3 adrenoceptors. Br J Pharmacol 2005, 144:317-322.
• [30]Gurevich EV, Tesmer JJ, Mushegian A, Gurevich VV: G protein-coupled receptor kinases: more than just kinases and not only for GPCRs. Pharmacol Ther 2012, 133:40-69.
• [31]Kohout TA, Lefkowitz RJ: Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol Pharmacol 2003, 63:9-18.
• [32]Ribas C, Penela P, Murga C, Salcedo A, Garcia-Hoz C, Jurado-Pueyo M, Aymerich I, Mayor F Jr: The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in GPCR regulation and signaling. Biochim Biophys Acta 2007, 1768:913-922.
• [33]Nobles KN, Xiao K, Ahn S, Shukla AK, Lam CM, Rajagopal S, Strachan RT, Huang TY, Bressler EA, Hara MR, Shenoy SK, Gyqi SP, Lefkowitz RJ: Distinct phosphorylation sites on the beta (2)-adrenergic receptor establish a barcode that encodes differential functions of beta-arrestin. Sci Signal 2011, 4:ra51.
• [34]Trester-Zedlitz M, Burlingame A, Kobilka B, von Zastrow M: Mass spectrometric analysis of agonist effects on posttranslational modifications of the beta-2 adrenoceptor in mammalian cells. Biochemistry 2005, 44:6133-6143.
• [35]Schlesinger DH, Hay DI: Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva. J Biol Chem 1977, 252:1689-1695.
• [36]Khambati HK, Moraes TF, Singh J, Shouldice SR, Yu RH, Schryvers AB: The role of vicinal tyrosine residues in the function of Haemophilus influenzae ferric-binding protein A. Biochem J 2010, 432:57-64.
• [37]Xiao K, Shenoy SK: Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains. J Biol Chem 2011, 286:12785-12795.
• [38]Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M: Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 1996, 93:14440-14445.