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Submitted on August 7, 2006
Revised on November 24, 2006
Accepted on January 9, 2007

Purification and identification of G protein-coupled receptor protein complexes under native conditions

Avais M. Daulat, Pascal Maurice, Carine Froment, Jean-Luc Guillaume, Cédric Broussard, Bernard Monsarrat, Philippe Delagrange, and Ralf Jockers

Dept. of Cell Biology, Institut Cochin, Paris F-75014

Corresponding Author: jockers{at}cochin.inserm.fr

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 towards a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxy-terminal tails) can be used as “bait”. We report here a method based on Tandem Affinity Purification (TAP) 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 MT1 and MT2 melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the TAP-tag at their carboxy-terminal tails and expressed in HEK 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of Gi 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 MT1 and MT2-associated proteins were identified; some of them were common to both receptors, others 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.


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