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,Ralf Jockers

doi:10.1074/mcp.m600298-mcp200

Abstract

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 toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification 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 MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) 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 MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were 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.

Highlights

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance
Both receptors were tagged with the tandem affinity purification (TAP) tag at their carboxyl-terminal tail (C-tail), and corresponding stable clones were established in HEK 293 cells
The human Melatonin receptor type 1A (MT1) and MT2 melatonin receptors were tagged with the TAP tag at their C-tails, and corresponding stable clones were established in HEK 293 cells

Summary

EXPERIMENTAL PROCEDURES

Receptor Constructs—MT1-Rluc and MT2-Rluc constructs have been described elsewhere [19]. Luminescence Measurements—Crude membranes were prepared from HEK 293 cell lines stably expressing MT1-Rluc or MT2-Rluc and solubilized in solubilization buffer supplemented with increasing concentrations of CHAPS, Brij96V, dodecylmaltoside, or digitonin. Coverslips were washed three times with PBS and incubated with a FITC-coupled secondary antibody at 1:1000 dilution in PBS, 3% BSA (Jackson ImmunoResearch Laboratories) for 30 min at room temperature. SDS-PAGE and Western Blotting—Whole cells (ERK activation) or crude membranes (receptor detection) were denatured overnight at room temperature in SDS-PAGE loading buffer (62.5 mM Tris/HCl, pH 6.8, 5% SDS, 10% glycerol, 0.5% bromphenol blue). Crude membranes were prepared from ϳ1 ϫ 109 HEK 293 cells and solubilized overnight in solubilization buffer with 0.5% digitonin or 0.25% Brij96V at a concentration of 2 mg of protein/ml.

NT kDa kDa kDa

RESULTS AND DISCUSSION

TEV Eluate Unretained Eluate

Intracellular membrane