A generic approach for the purification of signaling complexes that specifically interact with the carboxyl-terminal domain of G protein-coupled receptors.
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.
Maurice
Pascal
P
Department of Cell Biology, Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris F-75014, France.
Daulat
Avais M
AM
Broussard
Cédric
C
Mozo
Julien
J
Clary
Guilhem
G
Hotellier
Françoise
F
Chafey
Philippe
P
Guillaume
Jean-Luc
JL
Ferry
Gilles
G
Boutin
Jean A
JA
Delagrange
Philippe
P
Camoin
Luc
L
Jockers
Ralf
R
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*
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