Abstract
The first tandem affinity purification (TAP) protocol was described in 1999. Originally designed for the purification of protein complexes in yeast RNA splicing, its application rapidly expanded towards whole proteome analysis in yeast and mammalian cells. More recently, TAP has been applied to the purification of G protein-coupled receptor (GPCR)-associated protein complexes (GAPCs). This approach is particularly attractive for GPCRs, as the native, seven transmembrane structure is used as bait to purify GAPCs from mammalian cells expressing receptors at physiological levels. Here, a detailed protocol of the TAP method applied to GPCRs is presented.
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References
Rosenbaum, D. M., Rasmussen, S. G., and Kobilka, B. K. (2009) The structure and function of G-protein-coupled receptors. Nature 459, 356–363
Nakatani, Y., and Ogryzko, V. (2003) Immunoaffinity purification of mammalian protein complexes. Methods Enzymol 370, 430–444
Angers, S., Thorpe, C. J., Biechele, T. L., Goldenberg, S. J., Zheng, N., MacCoss, M. J., and Moon, R. T. (2006) The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation. Nat Cell Biol 8, 348–357
Bouwmeester, T., Bauch, A., Ruffner, H., Angrand, P. O., Bergamini, G., Croughton, K., Cruciat, C., Eberhard, D., Gagneur, J., Ghidelli, S., Hopf, C., Huhse, B., Mangano, R., Michon, A. M., Schirle, M., Schlegl, J., Schwab, M., Stein, M. A., Bauer, A., Casari, G., Drewes, G., Gavin, A. C., Jackson, D. B., Joberty, G., Neubauer, G., Rick, J., Kuster, B., and Superti-Furga, G. (2004) A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway. Nat Cell Biol 6, 97–105
Gavin, A. C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J. M., Michon, A. M., Cruciat, C. M., Remor, M., Hofert, C., Schelder, M., Brajenovic, M., Ruffner, H., Merino, A., Klein, K., Hudak, M., Dickson, D., Rudi, T., Gnau, V., Bauch, A., Bastuck, S., Huhse, B., Leutwein, C., Heurtier, M. A., Copley, R. R., Edelmann, A., Querfurth, E., Rybin, V., Drewes, G., Raida, M., Bouwmeester, T., Bork, P., Seraphin, B., Kuster, B., Neubauer, G., and Superti-Furga, G. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147
Daulat, A. M., Maurice, P., Froment, C., Guillaume, J. L., Broussard, C., Monsarrat, B., Delagrange, P., and Jockers, R. (2007) Purification and identification of G protein-coupled receptor protein complexes under native conditions. Mol Cell Proteomics 6, 835–844
Lyssand, J. S., DeFino, M. C., Tang, X. B., Hertz, A. L., Feller, D. B., Wacker, J. L., Adams, M. E., and Hague, C. (2008) Blood pressure is regulated by an alpha1D-adrenergic receptor/dystrophin signalosome. J Biol Chem 283, 18792–18800
Wacker, J. L., Feller, D. B., Tang, X. B., Defino, M. C., Namkung, Y., Lyssand, J. S., Mhyre, A. J., Tan, X., Jensen, J. B., and Hague, C. (2008) Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function. J Biol Chem 283, 31068–31078
Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17, 1030–1032
Rabilloud, T. (1999) Silver staining of 2-D electrophoresis gels. Methods Mol Biol 112, 297–305
Wisniewski, J. R., Zougman, A., Nagaraj, N., and Mann, M. (2009) Universal sample preparation method for proteome analysis. Nat Methods 6, 359–362
Daulat, A. M., Maurice, P., and Jockers, R. (2009) Recent methodological advances in the discovery of GPCR-associated protein complexes. Trends Pharmacol Sci 30, 72–78
Maurice, P., Daulat, A. M., Broussard, C., Mozo, J., Clary, G., Hotellier, F., Chafey, P., Guillaume, J. L., Ferry, G., Boutin, J. A., Delagrange, P., Camoin, L., and Jockers, R. (2008) A generic approach for the purification of signaling complexes that specifically interact with the carboxyl-terminal domain of G protein-coupled receptors. Mol Cell Proteomics 7, 1556–1569
Acknowledgments
We thank Dr. Luc Camoin for help in establishing proteomics procedures in our laboratory and Patty Chen for help in preparing the manuscript. AMD held an EGID fellowship.
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Daulat, A.M., Maurice, P., Jockers, R. (2011). Tandem Affinity Purification and Identification of GPCR-Associated Protein Complexes. In: Willars, G., Challiss, R. (eds) Receptor Signal Transduction Protocols. Methods in Molecular Biology, vol 746. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-126-0_23
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DOI: https://doi.org/10.1007/978-1-61779-126-0_23
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