A generic approach for the purification of signaling complexes that specifically interact with the carboxyl-terminal domain of G protein-coupled receptors

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.

Fig. 1.

Optimization of the peptide affinity chromatography conditions. A, determination of the optimal concentration of imidazole to minimize nonspecific binding on the beads. Different amounts of brain proteins were added to non-coated beads (20 μl) and incubated overnight at 4 °C in the presence or absence of 10 or 20 mm imidazole. After washes, proteins were eluted from the beads, and the nonspecific binding was quantified. B, determination of the optimal amount of brain protein lysates. Non-coated beads (20 μl) were incubated with 2, 4, 6, 8, or 10 mg of protein lysates in the presence of 20 mm imidazole overnight at 4 °C. After washes, proteins were eluted from the beads, and the nonspecific binding was quantified. C, determination of the binding kinetics of the His₆ (6xHis)-tagged peptides to the beads. 500 μg of His₆-tagged peptides were dissolved in a binding buffer containing 20 mm NaH₂PO₄, 6 m urea, pH 8, at 1 mg/ml. Absorbance of the peptide solutions was measured at 280 nm after 15, 30, 60, and 90 min of incubation with beads. D, determination of the amount of brain proteins specifically recruited by the C-tails of MT₁ and MT₂. 10 mg of brain protein lysate were incubated with 20 μl of beads coated with His₆-tagged baits in the presence of 20 mm imidazole overnight at 4 °C. After washes, the amount of retained proteins was quantified. C, non-coated beads. Results are expressed as mean ± S.E. (n = 5 for non-coated beads, n = 3 for MT₁, and n = 6 for MT₂).

Fig. 2.

Detection of PDZ domain-containing proteins, G_i3α, and GRK2/3 by immunoblotting. 20 μl of beads coated with His₆-tagged MT₁ and MT₂ C-tails were incubated with 10 mg of protein lysates from mouse brain. Beads were washed, and recruited proteins were eluted with 50 μl of 2% SDS in PBS. 10 μl were separated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was performed with antibodies raised against three PDZ domain-containing proteins, polyclonal anti-MUPP1 (1:10,000), polyclonal anti-nNOS (1:1000) and monoclonal anti-PSD-95 (1:2000); against G_i3α (1:1000); and against GRK2/3 (1:1000). C, negative control, beads without peptide; L, brain lysate (20 μg). WB, Western blot.

Fig. 3.

2D and 1D electrophoresis separation of the MT₁ and MT₂ C-tail-associated protein complexes. Mouse brain protein complexes recruited by the Ni-NTA-immobilized C-tail of MT₁ and MT₂ receptors were separated by 2D (A) or 1D electrophoresis on a 10% (B) or 5–9% gradient polyacrylamide gel (C) and silver-stained. A typical gel for each condition is shown. C, negative control, beads without peptide. The arrows indicate the position of RGSZ1.

Fig. 4.

Validation of MALDI-TOF-identified binding proteins by immunoblotting. Immobilized His₆-tagged MT₁ and MT₂ C-tails were incubated with 10 mg of protein lysates from mouse brain. Recruited proteins were eluted with 50 μl of 2% SDS in PBS. 10 μl were separated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was performed with monoclonal anti-tubulin α (1:2000), monoclonal anti-tubulin β (1:2000), monoclonal anti-PP2A catalytic subunit (1:1000), polyclonal anti-PKC ζ (1:1000), monoclonal anti-14-3-3 β (1:1000), polyclonal anti-otubain 1 (1:2000), and polyclonal anti-murine RGSZ1 (1:500) antibodies. C, negative control, non-coated beads; L, brain lysate (20 μg). WB, Western blot.

Fig. 5.

Functional interaction between RGSZ1 and MT₁ in cells. HEK 293 cells transiently expressing either FLAG-MT₁ or FLAG-MT₂ were co-transfected with HA-RGSZ1 (A) or Myc-RGS10 (B). 48 h post-transfection, cells were stimulated, or not, by melatonin (10⁻⁷ m for 15 min) and lysed. The MT₁ and MT₂ receptors were immunoprecipitated by anti-FLAG antibodies, and precipitates were analyzed by Western blot for the presence of co-immunoprecipitated HA-RGSZ1 (A) or Myc-RGS10 (B) using anti-tag antibodies. C, confocal images of HEK 293 cells co-expressing FLAG-MT₁ with RGSZ1-YFP. Receptors were immunostained with anti-FLAG antibodies, and RGSZ1 was revealed by its YFP fluorescence. The co-localization of both proteins was evaluated with the ImageJ co-localization highlighter plug-in. D, [³⁵S]GTPγS binding to MT₁-expressing CHO cells without (black bars) or with 1 μm melatonin (white bars) in the absence or presence of the indicated concentrations of purified RGSZ1. Data represent the mean ± S.E. of three experiments, each conducted in triplicate. Statistical significance was determined using the Mann-Whitney test. *, p < 0.05. E, co-immunoprecipitation of ¹²⁵I-melatonin (¹²⁵I-MLT)-labeled MT₁ receptors with anti-RGSZ1 antibodies. Nonspecific binding was estimated by performing immunoprecipitation using a pool of five non-relevant polyclonal antibodies. Data represent the mean ± S.E. of two experiments. IP, immunoprecipitate; WB, Western blot; NS, nonspecific binding.