A Generic Approach for the Purification of Signaling Complexes That Specifically Interact with the Carboxyl-terminal Domain of G Protein-coupled Receptors
| Autor: | Philippe Delagrange, Pascal Maurice, Gilles Ferry, Guilhem Clary, Julien Mozo, Cédric Broussard, Philippe Chafey, Ralf Jockers, Jean-Luc Guillaume, Luc Camoin, Jean A. Boutin, Françoise Hotellier, Avais M. Daulat |
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| Přispěvatelé: | MAURICE, Pascal, Institut Cochin (UMR_S567 / UMR 8104), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut de Recherches Internationales Servier [Suresnes] (IRIS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5) |
| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
Proteomics
[SDV]Life Sciences [q-bio] Nerve Tissue Proteins Plasma protein binding Biology Biochemistry Chromatography Affinity Cell Line Receptors G-Protein-Coupled Analytical Chemistry Mice 03 medical and health sciences 0302 clinical medicine Regulator of G protein signaling Protein structure Animals Humans Receptor Molecular Biology 030304 developmental biology G protein-coupled receptor 0303 health sciences Research GTPase-Activating Proteins HEK 293 cells Membrane Proteins Protein Structure Tertiary [SDV] Life Sciences [q-bio] Multiprotein Complexes RGS Proteins 030217 neurology & neurosurgery Protein Binding |
| Zdroj: | Molecular and Cellular Proteomics Molecular and Cellular Proteomics, 2008, 7 (8), pp.1556-69. ⟨10.1074/mcp.M700435-MCP200⟩ Molecular and Cellular Proteomics, American Society for Biochemistry and Molecular Biology, 2008, 7 (8), pp.1556-69. ⟨10.1074/mcp.M700435-MCP200⟩ |
| ISSN: | 1535-9476 1535-9484 |
| DOI: | 10.1074/mcp.M700435-MCP200⟩ |
| Popis: | International audience; 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 subdo-mains and that overcomes the limitations of currently used genetics and proteomics approaches. Our approach consists of a carefully balanced combination of chemically synthesized His 6-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 MT 1 and MT 2 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 MT 1 , 14 proteins that interacted with the C-tail of MT 2 , 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 MT 1 and the regulator of G protein signaling Z1 in trans-fected 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 |
| Databáze: | OpenAIRE |
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