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

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

doi:10.1074/mcp.m700435-mcp200

Abstract

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.

Highlights

Gprotein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are major drug targets
We identified 58 proteins that were retained by the non-coated beads. These proteins, having affinity for NiNTA-agarose beads, were considered as nonspecific binders and systematically removed when found in the lists of MT1and MT2-interacting proteins. 40 proteins that bind to the carboxyl-terminal tails (C-tails) of MT1 and 22 proteins that bind to the C-tail of MT2 were identified
We present an improved proteomics approach coupling peptide affinity chromatography with 1D and 2D electrophoresis separation and mass spectrometry for the identification of proteins interacting with the C-tail of GPCRs

Summary

EXPERIMENTAL PROCEDURES

Peptide Affinity Chromatography—Peptides encompassing the Ctails of the MT1 and MT2 receptors were chemically synthesized by NeoMPS (Strasbourg, France) with a His tag at the amino terminus. Receptor immunoprecipitation was performed on 1 ml of supernatant using 4 ␮g of polyclonal anti-FLAG antibodies preadsorbed on protein G-Sepharose beads (Sigma). After blocking with 3% BSA in PBS for 30 min, cells were incubated with monoclonal anti-FLAG M2 (1:500) antibodies in PBS containing 0.3% BSA for 1 h at room temperature. Coverslips were washed three times with PBS and incubated with Cy3-conjugated goat anti-mouse antibodies (1:800) in PBS containing 0.3% BSA for 40 min at room temperature. Radioligand binding was performed with pars tuberalis membranes (1.5–2 mg of protein) in 1 ml of TEM buffer (75 mM Tris, 12 mM MgCl2, 2 mM EDTA, protease inhibitor mixture EDTA-free, pH 7.4) containing 400 pM 2-[125I]melatonin (PerkinElmer Life Sciences) for 90 min at 37 °C. Radioactivity was measured after rapid filtration through GF/F glass fiber filters (Whatman)

RESULTS

Clathrin heavy polypeptide

DISCUSSION

IP basal