title: A generic approach for the purification of signaling complexes that specifically interact with the carboxyl-terminal domain of G protein-coupled receptors.
abstract: G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are major drug targets. Recent progress has shown that GPCRs are part of large protein complexes that regulate their activity. We present here a generic approach for identification of these complexes that is based on the use of receptor subdomains and that overcomes the limitations of currently used genetics and proteomics approaches. Our approach consists of a carefully balanced combination of chemically synthesized His6-tagged baits, immobilized metal affinity chromatography, one- and two-dimensional gel electrophoresis separation and mass spectrometric identification. The carboxyl-terminal tails (C-tails) of the human MT1 and MT2 melatonin receptors, two class A GPCRs, were used as models to purify protein complexes from mouse brain lysates. We identified 32 proteins that interacted with the C-tail of MT1, 14 proteins that interacted with the C-tail of MT2, and eight proteins that interacted with both C-tails. Several randomly selected proteins were validated by Western blotting, and the functional relevance of our data was further confirmed by showing the interaction between the full-length MT1 and the regulator of G protein signaling Z1 in transfected HEK 293 cells and native tissue. Taken together, we have established an integrated and generic purification strategy for the identification of high quality and functionally relevant GPCR-associated protein complexes that significantly widens the repertoire of available techniques.
authors.affiliation: Department of Cell Biology, Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris F-75014, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Maurice
authors.forename: Avais M
authors.initials: AM
authors.lastname: Daulat
authors.forename: Cédric
authors.initials: C
authors.lastname: Broussard
authors.forename: Julien
authors.initials: J
authors.lastname: Mozo
authors.forename: Guilhem
authors.initials: G
authors.lastname: Clary
authors.forename: Françoise
authors.initials: F
authors.lastname: Hotellier
authors.forename: Philippe
authors.initials: P
authors.lastname: Chafey
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Guillaume
authors.forename: Gilles
authors.initials: G
authors.lastname: Ferry
authors.forename: Jean A
authors.initials: JA
authors.lastname: Boutin
authors.forename: Philippe
authors.initials: P
authors.lastname: Delagrange
authors.forename: Luc
authors.initials: L
authors.lastname: Camoin
authors.forename: Ralf
authors.initials: R
authors.lastname: Jockers
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D020710:RGS Proteins; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D020710:RGS Proteins; C115120:RGS20 protein, human; D043562:Receptors, G-Protein-Coupled