Peptide Affinity Purification for the Isolation and Identification of GPCR-Associated Protein Complexes
Protein networks and their dynamic regulation play a fundamental role in biological systems. Seven transmembrane-spanning G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors controlling the flow of information from the extracellular environment into cells by inducing intracellular signaling pathways. Several GPCR-associated protein complexes (GAPCs), particularly those binding to the intracellular carboxyl-terminus (C-terminus), have been identified over the last 20 years. Recent optimizations in purification protocols and advances in mass spectrometry-based protein identification techniques have considerably accelerated the identification of GAPCs. We will concentrate here on a description of the latest version of the peptide affinity purification approach dedicated to the purification of GAPCs interacting with GPCR C-termini or any other soluble receptor subdomain.
Key wordsG protein-coupled receptor (GPCR) Immobilized metal affinity chromatography (IMAC) Protein complexes Proteomics
We thank Patty Chen (Institut Cochin, Paris) for comments on the manuscript.
- 2.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 carboxyterminal domain of G protein-coupled receptors. Mol. Cell. Proteomics 7, 1556–1569.PubMedCrossRefGoogle Scholar
- 3.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.Google Scholar
- 4.Becamel, C., Alonso, G., Galeotti, N., Demey, E., Jouin, P., Ullmer, C., Dumuis, A., Bockaert, J., and Marin, P. (2002) Synaptic multiprotein complexes associated with 5-HT2C receptors: a proteomic approach. EMBO J. 21, 2332–2342.Google Scholar
- 5.Becamel, C., Gavarini, S., Chanrion, B., Alonso, G., Galeotti, N., Dumuis, A., Bockaert, J., and Marin, P. (2004) The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins. J. Biol. Chem. 279, 20257–20266.Google Scholar
- 6.Joubert, L., Hanson, B., Barthet, G., Sebben, M., Claeysen, S., Hong, W., Marin, P., Dumuis, A., and Bockaert, J. (2004) New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting. J. Cell Sci. 117, 5367–5379.Google Scholar
- 7.Enz, R. (2007) The trick of the tail: protein-protein interactions of metabotropic glutamate receptors. Bioessays 29, 60–73.Google Scholar
- 8.Daulat, A. M., Maurice, P., and Jockers, R. (2009) Recent methodological advances in the discovery of GPCR-associated protein complexes (GAPCs). Trends Pharmacol. Sci. 30, 72–78. Google Scholar
- 9.Wisniewski, J. R., Zougman, A., Nagaraj, N., and Mann, M. (2009) Universal sample preparation method for proteome analysis. Nat Methods 6, 359–62.Google Scholar