Visit our Expo - Redox and Inflammation signaling 2012

Session II : Receptor signaling and G proteins Poster II, 16
Purification of G protein-coupled receptors and associated protein complexes under
native condition
Avais M. Daulat*, Jean-Luc Guillaume*, Pascal Maurice*, Philippe Delagrange$, Ralf
Jockers*#.
* Institut Cochin, Département Biologie Cellulaire. Inserm, U567. CNRS, UMR 8104.
Université Paris Descartes, Faculté de Médecine René Descartes, UMR-S 8104, Paris, F-
75014 France. # Email jockers@cochin.inserm.fr
$ Institut de Recherche SERVIER, Suresnes, France
G Protein-Coupled Receptors (GPCRs) constitute the largest family of membrane receptors
and are targeted by about half of the drugs prescribed for human diseases. In contrast to other
protein families the “interactom”, the repertoire of proteins that interact with a given protein,
is only poorly known for GPCRs. Faster progress in the field has been mainly hampered by
the fact that “classical” protein-protein interaction approaches such as yeast two-hybrid and
affinity purification techniques, are not well-adapted for these multi-transmembrane-spanning
proteins.
Recently, the tandem affinity purification (TAP) method has been developed to successfully
purify soluble protein complexes. Here, we adapted this method to the purification of GPCRassociated
protein complexes. This technique relies on a two-step purification protocol of
proteins that associate with the TAP-tagged bait protein in living cells. We established two
HEK 293 clones each stably expressing a distinct C-terminally TAP-tagged GPCR. Among
different detergents tested, digitonin and Brij 96 were selected for their ability to solubilize
high amounts of functional receptors. Typical purification yields varied between 20 and 30%.
The successful co-purification of receptor-associated proteins was shown by the presence of
heterotrimeric G proteins as determined by western blot and mass spectrometric analysis.
Large-scale purifications yielded in coomassie blue stainable protein amounts that were
identified by mass spectrometry.
In conclusion, we established a purification procedure for the isolation of protein complexes
that associated with GPCRs in living cells under basal and activated conditions.
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