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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M700435-MCP200v1 7/8/1556    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Google Scholar Articles by Maurice, P. Articles by Jockers, R. PubMed PubMed Citation Articles by Maurice, P. Articles by Jockers, R. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 7:1556-1569, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A Generic Approach for the Purification of Signaling Complexes That Specifically Interact with the Carboxyl-terminal Domain of G Protein-coupled Receptors^*,S

Pascal Maurice^,, Avais M. Daulat^,,¶, Cédric Broussard^,||, Julien Mozo^**, Guilhem Clary^,||, Françoise Hotellier^,||, Philippe Chafey^,||, Jean-Luc Guillaume^,, Gilles Ferry^**, Jean A. Boutin^**, Philippe Delagrange^**, Luc Camoin^,|| and Ralf Jockers^,,

From the Department of Cell Biology and ^|| Laboratoire de Protéomique, Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris F-75014, France, INSERM U567, Paris F-75014, France, and ^** Institut de Recherches SERVIER, Suresnes F-92150, France

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.

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