Regulation of the AGS3·G{alpha}i signaling complex by a seven-transmembrane span receptor

Abstract

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind Gα(i) free of Gβγ providing an unusual scaffold for the "G-switch" and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3·Gα(i) signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gα(i1) tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of α(2)-adrenergic receptors or μ-opioid receptors reduced AGS3-RLuc·Gα(i1)-YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by Gβγ sequestration with the carboxyl terminus of GRK2. Gα(i)-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gα(i) and/or Gα(o) indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both Gαβγ and GPR-Gα(i) offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

FIGURE 1.

Interaction between AGS3 and Gα_i as determined by BRET. A, interaction between AGS3-RLuc and Gα_i1-YFP. Left panel, emission spectra for luminescence in HEK-293 cells transfected with pRLuc::AGS3 (10 ng) or pRLuc::AGS3-Q/A (10 ng) and pcDNA3::Gα_i1-YFP (750 ng). Data presented are representative of 5–10 experiments. Right panel, HEK-293 cells were transfected with a fixed amount of pRLuc::AGS3 (10 ng) (n = 9) or pRLuc::AGS3-Q/A (n = 4) and increasing amounts of pcDNA3::Gα_i1-YFP (0–750 ng) and processed for BRET measurements as described under “Experimental Procedures.” B, interaction of AGS3-Venus and Gα_i1-RLuc. HEK-293 cells were transfected with pRLuc::Gα_i1 (50 ng) and increasing amounts of pVenusN1::AGS3 (n = 3) or pVenusN1::AGS3-Q/A (0–500 ng) (n = 3) and processed for BRET measurements as described under “Experimental Procedures.” BRET saturation curves were fitted using a non-linear regression equation assuming a single binding site. Results are expressed as the mean ± S.E. of three independent experiments.

FIGURE 2.

Interaction between AGS3-RLuc and Gα_i1-YFP. A, HEK-293 cells were transfected with a fixed amount of pRLuc::AGS3 (10 ng) and increasing concentrations of pcDNA3::Gα_i1-YFP (0–750 ng) in the absence and presence of pcDNA3::Gβ1 (500 ng) and pcDNA3::Gγ2 (500 ng) (n = 9). Inset, Gβ immunoblot. Each lane contains 50 μg of lysate protein. The fluorescent signal increased by 5–7-fold as the amount of pcDNA3::Gα_i1-YFP increased from 15 to 750 ng. BRET saturation curves were fitted using a non-linear regression equation assuming a single binding site. B, net BRET signal generated from HEK-293 cells transfected with pRLuc::AGS3 and pcDNA3 containing Gα_i1-YFP, Gα_i1-G202T-YFP, or Gα_i1-Q204L-YFP (750 ng) (n = 4). C, net BRET generated from HEK-293 cells expressing Gα_i1-YFP (pcDNA3::Gα_i1-YFP, 750 ng) and wild type AGS3-RLuc (WT, 10 ng) or AGS3-RLuc (10 ng) with single residue mutations in one to four of the GPR motifs (GPR1-Q488A, GPR2-Q541A, GPR3-Q589A, GPR4-Q623A) that disrupt Gα_i binding to the individual GPR motif (n = 5–9). Expression of Gα_i1-YFP was monitored in all experiments by fluorescence measurements (excitation, 485 nm; emission, 535 nm) with no significant difference in Gα_i1-YFP expression among the different experimental groups. The net BRET signal observed with GPR1-Q/A was not significantly different from WT and GPR1–4-Q/A was not significantly different from any of the constructs containing three GPR-Q/A substitutions. A-C, *, p < 0.05 compared with GPR2-Q/A; **, p < 0.05 compared with GPR1,2-Q/A and GPR4-Q/A; #, p < 0.05 compared with GPR1,3-Q/A; ##, p < 0.05 compared with GPR2,4-Q/A; ***, p < 0.05 compared with GPR3,4-Q/A. WT, wild type.

FIGURE 3.

Regulation of AGS3 and Gα_i interaction by the α_2A/D-AR. A, HEK-293 cells were transfected with pRLuc::AGS3 (10 ng) and pcDNA3::Gα_i1-YFP (750 ng) in the presence or absence of pcDNA3::α_2A/D-AR (1 μg). Control (n = 8) and α_2A/D-AR-transfected cells were treated with vehicle or UK-14304 (10 μm) (n = 22) with and without rauwolscine (100 μm, n = 6) for 13 min. Similar experiments were conducted with cells treated with 100 ng/ml of pertussis toxin (n = 6) for 16 h. Cells were then processed for BRET measurements as described under “Experimental Procedures.” *, p < 0.05 compared with basal; **, p < 0.05 compared with UK-14304 treatment. B, HEK-293 cells were transfected with a fixed amount of pRLuc::AGS3 (10 ng) and increasing concentrations of pcDNA3::Gα_i1-YFP (0–750 ng) in the presence of pcDNA3::α_2A/D-AR (1 μg) (n = 8). Cells were treated with vehicle or UK-14304 (10 μm) for 13 min and BRET measurements were performed. C and D, HEK-293 cells were transfected with pRLuc::AGS3 (10 ng) and pcDNA3::Gα_i1-YFP (750 ng) in the presence of pcDNA3::α_2A/D-AR (1 μg). Cells were incubated with different concentrations of UK-14304 (n = 5) (C), with different α₂-AR agonists (10 μm) or the α₂-AR antagonist rauwolscine (100 μm) for 13 min (D), and BRET measurements were performed. D, *, p < 0.05 compared with vehicle (n = 5). A–D, results are expressed as the mean ± S.E. of 4 to 20 experiments.

FIGURE 4.

Regulation of AGS3 and Gα_i interaction by G-protein-coupled receptors and second messengers. A, HEK-293 cells were transfected with pRLuc::AGS3 (10 ng) and pcDNA3::Gα_i1-YFP (750 ng) in the presence of pcDNA3 (1 μg) (Control), pcDNA3::α_2B-AR (1 μg) (n = 3), pcDNA3::μ-opioid receptor (1 μg) (n = 4), or pcDNA3::M3-MR (1.5 μg) (n = 3). Cells were incubated with agonist for 13 min prior to addition of coelenterazine H and processed for BRET measurements as described under “Experimental Procedures.” *, p < 0.05 compared with receptor-transfected cells without agonist treatment (Basal). B, HEK-293 cells were transfected with pRLuc::AGS3 (10 ng), pcDNA3::Gα_i-YFP (750 ng), and pcDNA3::μ-opioid receptor (1 μg) (n = 4). Cells were incubated with increasing concentrations of DAMGO for 13 min prior to addition of coelenterazine H, and the cells were processed for BRET measurements as described under “Experimental Procedures.” C, HEK-293 cells were transfected with pRLuc::AGS3 (10 ng) and pcDNA3::Gα_i1-YFP (750 ng) and treated with forskolin (10 μm) or ionomycin (1 μm) for 5 min (n = 4) to increase intracellular cAMP and calcium, respectively. Cells were then processed for BRET measurements as described under “Experimental Procedures.” Results are expressed as the mean ± S.E. of four independent experiments. D, HEK-293 cells were transfected with pRLuc::AGS3-SH (1 ng) or pRLuc::AGS3-MYR-SH (2 ng), pcDNA3::Gα_i1-YFP (750 ng), and pcDNA3:: α_2A/D-AR (1 μg). Cells were treated with vehicle or UK-14304 (10 μm) (n = 7) for 13 min and processed for BRET measurements. Results are expressed as the mean ± S.E. *, p < 0.05 compared with its vehicle treated.

FIGURE 5.

Influence of agonist incubation time on the regulation of AGS3-RLuc·Gα_i1-YFP BRET and reversal of the agonist-induced reduction in AGS3-RLuc·Gα_i1-YFP BRET. HEK-293 cells were processed for BRET assays as described in the legend to Fig. 3. In A, agonist and antagonist were added at the same time. B, reversal of agonist-mediated effects on AGS3-RLuc·Gα_i1-YFP BRET by rauwolscine. BRET measurements were obtained twice from three sets of tubes. One set of tubes contained UK-14304 (1 μm). In a second set of tubes, UK-14304 (1 μm) and rauwolscine (10 μm) were added at the same time. In a third set of tubes, samples were first incubated with UK-14304 (1 μm) for 2 min and the first BRET measurement was performed and then rauwolscine (10 μm final) was added and the incubation continued for 1 min before the second BRET measurement. *, p < 0.05 compared with UK-14304 (n = 4). **, p < 0.05 compared with the initial BRET measurement for UK-14304 alone after a 2-min incubation with agonist (n = 4).

FIGURE 6.

Influence of GRK2-CT and RGS4 on basal and receptor-mediated regulation of AGS3-RLuc·Gα_i1-YFP BRET. A, HEK-293 cells were transfected with a fixed amount of pRLuc::AGS3 (10 ng) and increasing concentrations of pcDNA3::Gα_i1-YFP (0–750 ng) in the presence and absence of pcDNA3::RGS4-C2S (500 ng) or pcDNA3::GRK2-CT (500 ng) (n = 5). Cells were processed for BRET measurements as described under “Experimental Procedures.” B, HEK-293 cells were transfected with pRLuc::AGS3 (10 ng), pcDNA3::α_2A/D-AR (750 ng), and increasing concentrations of pcDNA3::Gα_i1-YFP (0–750 ng) in the presence and absence of pcDNA3::RGS4-C2S (500 ng) or pcDNA3::GRK2-CT (500 ng) (n = 5). Cells were treated with vehicle or UK-14304 (10 μm) (n = 5) for 13 min and processed for BRET measurements. *, p < 0.05 compared with control. C, HEK-293 cells were transfected with pRLuc::AGS3 (10 ng), pcDNA3::α_2A/D-AR (750 ng), and pcDNA3::Gα_i1-YFP (750 ng) or pcDNA3::Gα_i1-YFP-G184S (750 ng) in the presence and absence of pcDNA3::GRK2-CT (500 ng), pcDNA3::RGS4-C2S (500 ng), or pcDNA3::RGS4-C2S-N128A (500 ng). Cells were treated with vehicle or UK-14304 (10 μm) for 13 min and processed for BRET measurements. Results are expressed as percent inhibition of net BRET by agonist. Results are expressed as the mean ± S.E. of 3–5 independent experiments. *, p < 0.05 compared with control. #, p < 0.05 compared with the pcDNA3::RGS4-C2S transfected group. Right panel, GRK2 and RGS4 immunoblot. Each lane contains 50 μg of lysate protein.

FIGURE 7.

Gα_i-dependent and agonist-sensitive interaction between AGS3 and the α₂-adrenergic receptor. A, HEK-293 cells were transfected with a fixed amount of pRLuc::AGS3 (10 ng) and increasing amounts of α_2A-AR-Venus plasmid (0–750 ng) in the absence or presence of pcDNA3::Gα_i1 (1 μg). Cells were incubated with vehicle or UK-14304 (10 μm) and BRET assays were performed (n = 5) as described under “Experimental Procedures.” Cells were exposed to agonist for 2 min. B, HEK-293 cells were transfected with pRLuc::AGS3 (10 ng) and α_2A-AR-Venus plasmid (750 ng) with increasing amounts of pcDNA3::Gα_i1 (0–1 μg). One group of cells was incubated with pertussis toxin (100 ng/ml) for 16 h before the experiment. Control cells (n = 3) and pertussis toxin-pretreated cells (n = 3) were incubated with UK-14304 (10 μm) and BRET assays were performed. Cells were exposed to agonist for 2 min. Results are expressed as the mean ± S.E. *, p < 0.05 compared with vehicle for each amount of pcDNA3::Gα_i1. **, p < 0.05 compared with UK-14304 treated for each amount of pcDNA3::Gα_i1. C, net BRET signal generated from HEK-293 cells transfected with pRLuc::AGS3 (10 ng), α_2A-AR-Venus (250 ng), and pcDNA3::Gα_i3, pcDNA3::Gα_i3-G202T, or pcDNA3::Gα_i3-Q204L (1 μg) (n = 5). Inset, Gα_i3 immunoblot. Each lane contains 50 μg of lysate protein. D, reversal of agonist-mediated effects on Gα_i-dependent AGS3-RLuc·α_2A-AR-Venus BRET by rauwolscine. Cells were transfected with pRLuc::AGS3 (10 ng), 250 ng of α_2A-AR-Venus plasmid, and 1 μg of pcDNA3::Gα_i1. BRET measurements were obtained twice from three sets of tubes. One set of tubes contained UK-14304 (1 μm). In a second set of tubes, UK-14304 (1 μm) and rauwolscine (10 μm) were added at the same time. In a third set of tubes, samples were first incubated with UK-14304 (1 μm) for 2 min and the first BRET measurement was performed and then rauwolscine (10 μm final) was added and the incubation continued for 1 min before the second BRET measurement. *, p < 0.05 compared with UK-14304 (n = 4). **, p < 0.05 compared with the initial BRET measurement for UK-14304 alone after a 2-min incubation with agonist (n = 4).

FIGURE 8.

AGS3-RLuc interaction with G-protein-coupled receptors. HEK-293 cells were transfected with pRLuc::AGS3-Q/A (10 ng) and α_2A-AR-Venus plasmid (250 ng) or pRLuc::AGS3 (10 ng) and α_2A-AR-Venus plasmid (50 ng), pEYFP-N1::α_2B-AR (750 ng), pEYFP-N1::μ-OR (750 ng), or β₂-AR-Venus plasmid (1 μg) in the presence and absence of pcDNA3::Gα_i1 (750 ng) (n = 4). pcDNA3::Gα_i1 (750 ng)-transfected cells were also incubated with agonist for 2 min (UK-14304, 10 μm for α_2A-AR-Venus and α_2B-AR-YFP; DAMGO, 10 μm for μ-OR-YFP; isoproterenol, 100 μm for β₂-AR-Venus). BRET assays were performed as described under “Experimental Procedures.” *, **, and ***, p < 0.05 compared with the corresponding vehicle control (n = 4).

FIGURE 9.

Subcellular distribution of AGS3-RLuc and Gα_i1-YFP. HEK-293 cells were transfected with pRLuc::AGS3 (10 ng), pcDNA3::Gα_i1-YFP (750 ng), and/or pcDNA3::α_2A/D-AR (1 μg). Cells were incubated with vehicle or UK-14304 (10 μm) and lysed to obtain membrane and cytosol fractions. Luminescence (A) and fluorescence (B) were measured in samples containing 50 μg of protein as described under “Experimental Procedures.” Results are expressed as the mean ± S.E. of three independent experiments. * and **, p < 0.05 compared with corresponding vehicle control. RLU, relative luminescence units. RFU, relative fluorescent units (excitation, 485 nm; emission, 535 nm).