Receptor-regulated interaction of activator of G-protein signaling-4 and Galphai

Abstract

Activator of G-protein signaling-4 (AGS4), via its three G-protein regulatory motifs, is well positioned to modulate G-protein signal processing by virtue of its ability to bind Galpha(i)-GDP subunits free of Gbetagamma. Apart from initial observations on the biochemical activity of the G-protein regulatory motifs of AGS4, very little is known about the nature of the AGS4-G-protein interaction, how this interaction is regulated, or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with Galpha(i1) in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Galpha(i1) reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and Galpha(i1)-YFP were reduced by G-protein-coupled receptor activation, and this agonist-induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4-RLuc and alpha(2)-adrenergic receptor-Venus, which were Galpha(i)-dependent and reduced by agonist, indicating that AGS4-Galpha(i) complexes are receptor-proximal. These data suggest that AGS4-Galpha(i) complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.

FIGURE 1.

Interaction of AGS4 and Gα_i in living cells using BRET. A, emission spectra for luminescence in HEK cells transfected with 10 ng of phRLuc-AGS4 or phRLuc-AGS4-Q/A and 750 ng of pcDNA3-Gα_i-YFP. RLU, relative luminescence units. B, HEK cells were transfected with a fixed amount (2 ng) of phRLuc-AGS4 (open squares) or phRLuc-AGS4-Q/A (open triangles) and increasing amounts of pcDNA3-Gα_i-YFP (0–750 ng) without or with 500 ng of pcDNA3-Gβ₁ and 500 ng of pcDNA3-Gγ₂ (Gβγ, open circles), and BRET signals were measured as described under “Experimental Procedures.” C, net BRET signals generated from HEK cells transfected with 2 ng of phRLuc_N3-AGS4 and 750 ng of pcDNA3-Gα_i1-YFP in the presence or absence of 100 ng/ml pertussis toxin (PTX) pretreatment for 18 h or of Gα_i1-N149I-YFP or Gα_i1-Q204L-YFP. Mean acceptor/donor ratios are: Gα_i1, 2.2; Gα_i + pertussis toxin, 1.6; Gα_i1-N149I, 1.6; Gα_i1-Q204L, 2.3. *, p < 0.05 as compared with Gα_i1. D, net BRET signals generated from HEK cells transfected with 2 ng of phRLuc_N3-AGS4, AGS4-NT (Met¹–Ser⁵⁶), AGS4-CT (Leu⁵⁷–Cys¹⁶⁰), or AGS4-Q/A and 750 ng of pcDNA3-Gα_i1-YFP. Mean acceptor/donor ratios are: AGS4, 2.2; AGS4-NT, 1.8; AGS4-CT, 1.5; AGS4-Q/A, 1.6. *, p < 0.05 as compared with AGS4-RLuc. WT, wild type. E, net BRET signals generated from HEK cells transfected with 750 ng of pcDNA3-Gα_i1-YFP and 2 ng of phRLuc_N3 containing AGS4 or AGS4 variants with single residue mutations in each of the three AGS4-GPR motifs (GPRI-Q80A; GPRII-Q122A; GPRIII-Q151A) that inhibit GPR motif-Gα_i binding. Mean acceptor/donor ratios are: wild type, 1.6; GPRI–III, 1.3; GPRI, 1.6; GPRII, 1.3; GPRIII, 1.4; GPRI–II, 1.1, GPRI,III, 1.2; GPRII–III, 1.0. *, p < 0.05 as compared with AGS4-WT. **, p < 0.05 as compared with GPRI. ***, p < 0.05 as compared with GPRI–III. #, p < 0.05 as compared with GPRI–II. All data presented are representative of 3–9 experiments, and individual values were the average of triplicate determinations. BRET saturation curves were fitted using a non-linear regression equation assuming one-site binding. Error bars in B–E indicate S.E.

FIGURE 2.

Receptor-mediated regulation of the AGS4-Gα_i interaction. A, net BRET signals generated from HEK cells transfected with 2 ng of phRLuc_N3-AGS4 and 750 ng of pcDNA3-Gα_i1-YFP in the presence or absence of 750 ng of pcDNA3-α_2A-AR. Cells were treated with vehicle (−) or the α₂-AR agonist UK14304 (10 μm, +) for 15 min and processed for BRET as described under “Experimental Procedures.” *, p < 0.05. B, BRET saturation signals from HEK cells transfected with a fixed amount (2 ng) of phRLuc_N3-AGS4 and increasing amounts of pcDNA3-Gα_i1-YFP (0–1 μg) in the presence or absence of 750 ng of pcDNA3-α_2A-AR. Cells were treated with vehicle (open boxes) or 10 μm UK14304 (open triangles) for 15 min and processed for BRET measurements. Bottom panel, representative immunoblot for AGS4-Luc (IB: anti-RLuc) and Gα_i1 (IB: anti-Gα_i1) corresponding to the BRET experiment in the upper panel. Each lane contains 50 μg of protein. −, untransfected HEK cells. [³H]RX821002 binding revealed an α_2A-AR density of ∼6.7 pmol/mg. C and D, HEK cells were transfected with 2 ng of phRLuc_N3-AGS4 and 750 ng each of pcDNA3-Gα_i1-YFP and pcDNA3-α_2A-AR. Cells were incubated with increasing amounts of UK14304 (10⁻¹²-10⁻⁴ m) for 15 min (C) or treated with 10 μm UK14304 for 0–30 min (D) and processed for BRET measurements. *, p < 0.05 as compared with 0 time point. E, HEK cells were transfected with 2 ng of phRLuc_N3-AGS4, 750 ng of pcDNA3-Gα_i1-YFP, and 750 ng of either pcDNA3 (no receptor) or pcDNA3-α_2A-AR. Cells were incubated with vehicle or 1 μm UK14304 for 15 min in the absence or presence of 10 μm rauwolscine or 100 ng/ml pertussis toxin (18 h of pretreatment). *, p < 0.05 as compared with vehicle treatment. **, p < 0.05 as compared with UK14304 treatment alone. All data presented are representative of 3–12 experiments, and individual values were the average of triplicate determinations. Error bars in A–E indicate S.E.

FIGURE 3.

AGS4 forms a Gα_i-dependent complex with GPCRs that is regulated by agonist. A, net BRET signals generated from HEK cells transfected with 2 ng of phRLuc_N3-AGS4 and 500 ng of pcDNA3-α_2A-AR-Venus in the presence or absence of 750 ng of pcDNA3-Gα_i1. Cells were treated with vehicle (−), the α₂-AR agonist UK14304 (1 μm), and/or the α₂-AR antagonist rauwolscine (10 μm) for 15 min and processed for BRET as described under “Experimental Procedures.” *, p < 0.05 as compared with vehicle treatment. B, BRET saturation signals from HEK cells transfected with a fixed amount (2 ng) of phRLuc_N3-AGS4 and increasing amounts of pcDNA3-α_2A-AR-Venus (0–1 μg) in the presence or absence of 750 ng of pcDNA3-Gα_i1. Cells were treated with vehicle or 10 μm UK14304 as indicated in the figure for 15 min and processed for BRET measurements. Bottom panel, representative immunoblot of HEK cells (50 μg protein/lane) untransfected (−) or transfected (+) with 250 ng of pcDNA3-α_2A-AR-Venus (IB: Venus), 500 ng of pcDNA3-Gα_i1, and 2 ng of phRLuc_N3-AGS4. C, net BRET signals generated from HEK cells transfected with 2 ng of phRLuc_N3-AGS4, 500 ng of pcDNA3-α_2A-AR-Venus, and 750 ng of pcDNA3-Gα_i3, Gα_i3-Q204L, Gα_i3-G202T, or Gα_s. Cells were treated with vehicle or 10 μm UK14304 as indicated in the figure for 15 min and processed for BRET measurements. *, p < 0.05 as compared with vehicle treatment. Bottom panel, Gα_i3 and Gα_s immunoblot (75 μg of protein lysate per lane). D and E, HEK cells were transfected with 2 ng of phRLuc_N3-AGS4, 500 ng of pcDNA3-α_2A-AR-Venus, and 750 ng of pcDNA3-Gα_i1. Cells were incubated with increasing amounts of UK14304 (10⁻⁸-10⁻⁴ m) for 15 min (D) or treated with 10 μm UK14304 for 0–30 min (E) and processed for BRET measurements. *, p < 0.05 as compared with vehicle treatment (D) or 0 time point (E). F, HEK cells were transfected with 2 ng of phRLuc_N3-AGS4, 500 ng of pcDNA3-α_2A-AR-Venus, and increasing amounts of pcDNA3-Gα_i1 (0–750 ng). Cells were incubated in the presence or absence of 100 ng/ml pertussis toxin (PTX) for 18 h and then treated with vehicle or 10 μm UK14304 for 15 min. *, p < 0.05 as compared with control in each group. **, p < 0.05 as compared with UK14304 treatment in each group. G, HEK cells were transfected with 2 ng of phRLuc_N3-AGS4 and 500 ng of pcDNA3-α_2A-AR-Venus, MOR-YFP, or β₂-AR-Venus in the absence or presence of 750 ng of pcDNA3-Gα_i1 as indicated in the figure. Cells were then treated with 10 μm UK14304, 10 μm [d-Ala², N-MePhe⁴, Gly-ol]-enkephalin (DAMGO), or 10 μm isoproterenol for 15 min as indicated in the figure. *, p < 0.05 as compared with vehicle treatment for each group. All data presented are representative of 4–10 experiments, and individual values were the average of triplicate determinations. Error bars in A–G indicate S.E.