title: Purification and identification of G protein-coupled receptor protein complexes under native conditions.
abstract: G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.
authors.affiliation: Department of Cell Biology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université Paris Descartes, France.
authors.forename: Avais M
authors.initials: AM
authors.lastname: Daulat
authors.forename: Pascal
authors.initials: P
authors.lastname: Maurice
authors.forename: Carine
authors.initials: C
authors.lastname: Froment
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Guillaume
authors.forename: Cédric
authors.initials: C
authors.lastname: Broussard
authors.forename: Bernard
authors.initials: B
authors.lastname: Monsarrat
authors.forename: Philippe
authors.initials: P
authors.lastname: Delagrange
authors.forename: Ralf
authors.initials: R
authors.lastname: Jockers
mesh_terms: D002460:Cell Line; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D044122:Receptor, Melatonin, MT1 / Q000737:chemistry / Q000378:metabolism*; D044123:Receptor, Melatonin, MT2 / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D046912:Multiprotein Complexes; D011506:Proteins; D044122:Receptor, Melatonin, MT1; D044123:Receptor, Melatonin, MT2; D011993:Recombinant Fusion Proteins