Receptor-regulated Interaction of Activator of G-protein Signaling-4 and Gα_i

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 Gα_i-GDP subunits free of Gβγ. 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 Gα_i1 in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Gα_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 Gα_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 α₂-adrenergic receptor-Venus, which were Gα_i-dependent and reduced by agonist, indicating that AGS4-Gα_i complexes are receptor-proximal. These data suggest that AGS4-Gα_i complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.

Introduction

Activators of G-protein signaling (AGS)³ proteins were identified using a yeast-based functional screen of mammalian cDNA libraries for cDNAs that activated G-protein signaling in the absence of a GPCR (1–4). Group II AGS proteins all contain at least one G-protein regulatory (GPR) motif (3, 5) (also termed the GoLoco motif (6)), a 20–25-amino acid motif that binds and stabilizes the GDP-bound conformation of Gα_i/_o/_t and competes with Gβγ for Gα binding (reviewed in Ref. 5). Proteins with multiple GPR motifs can bind to multiple Gα subunits simultaneously, which presents a unique opportunity to act as a scaffold to organize a signaling complex (7, 8).

Functional studies indicate crucial roles for GPR proteins beginning with the original observations in model organisms describing a role for GPR proteins and their interaction with G-proteins in asymmetric cell division (5, 9). Additional functional studies with GPR proteins indicate further functional diversity with roles observed in blood pressure control, fat deposition and energy expenditure, neuronal outgrowth, drug addiction and relapse behavior, autophagy, G-protein-coupled inwardly rectifying potassium channel regulation, and transport of membrane proteins to the cell surface (10–18). These observations indicate crucial functionality of the GPR motif in biological systems and implicate Gα_i-GPR complexes in the regulation of G-protein signaling in unexpected, albeit poorly understood ways. In the context of the group II AGS proteins, which contain multiple GPR motifs, many outstanding questions remain to be addressed. Chief among them is what regulates the formation and disassembly of GPR-Gα_i complexes? Is their interaction with G-protein influenced by GPCR activation or other signals?

AGS4 was identified in the yeast based functional screen for receptor-independent G-protein activators from a human prostate leiomyosarcoma cDNA library (1), and apart from initial reports describing its interaction with Gα_i subunits from cell lysates and purified proteins in vitro, little is known regarding its interaction with Gα_i in the intact cell or how this interaction is regulated. Although AGS3 and AGS5/Leu-Gly-Asn repeat-enriched protein (LGN) both contain seven tetratricopeptide repeats upstream of their four GPR motifs and are broadly expressed in many tissues, AGS4 has a much different domain organization with a proline-rich N terminus followed by three GPR motifs that are somewhat dissimilar as compared with AGS3 and AGS5/LGN (1, 19). AGS4 also appears to be more restricted in its expression to the immune system.⁴ In this study, we report the interaction of AGS4 with Gα_i in the intact cell as determined by bioluminescence resonance energy transfer (BRET) and its regulation by cell surface GPCRs. In addition, the data indicate that AGS4-Gα_i complexes are receptor-proximal as specific BRET signals were observed between AGS4 and the α_2A-adrenergic receptor (α_2A-AR), which were Gα_i-dependent and reduced by an α_2A-AR agonist. These data suggest that AGS4-Gα_i complexes directly couple to a GPCR and may serve as substrates for receptor-induced G-protein activation.

EXPERIMENTAL PROCEDURES

Materials

Polyethyleneimine (PEI) (25 kDa molecular mass, linear form), was obtained from Polysciences, Inc. Benzyl-coelenterazine was obtained from NanoLight Technology (Pinetop, AZ). UK14304, rauwolscine HCl, and pertussis toxin were purchased from Sigma. Materials for cell culture were purchased from Invitrogen. Gray 96-well Optiplates were obtained from PerkinElmer Life Sciences. Anti-Gα_i1 and anti-Renilla luciferase were purchased from Millipore (Billerica, MA). Anti-Gα_i3 and Gα_s were kindly provided by Dr. Thomas Gettys (Pennington Biomedical Research Center, Baton Rouge, LA). AGS4 GPR mutants, Gα_i1-YFP-G202T, Gα_i1-YFP-Q204L, and Gα_i1-YFP-N149I were generated by site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA). Gα_i3, Gα_i3-G202T, and Gα_i3-Q204L cDNAs were obtained from the cDNA Resource Center (University of Missouri, Columbus, MO). pcDNA3.1-Gα_i1-YFP was generated by Dr. Gibson (20) and provided by Gregory G. Tall (University of Rochester School of Medicine and Dentistry). Construction of expression vectors for α_2A-AR, β₂-AR, and μ-opioid receptor (MOR) were previously described (21–23). MOR-YFP plasmid was kindly provided by Dr. Lakshmi A. Devi (Mount Sinai Medical Center, New York, NY). All other reagents and materials were obtained as described elsewhere (22, 24).

Cell Culture and Transfection

HEK-293 cells were maintained in Dulbecco's minimal essential medium (high glucose, without phenol red) containing 5% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells were grown in the presence of 5% CO₂ at 37 °C in a humidified incubator. For transfection, 8 × 10⁵ cells/well were seeded on 6-well plates and cultured overnight at 37 °C. BRET donor and acceptor plasmids were used for transfection with PEI (1 mg/ml in distilled H₂O) at a DNA:PEI ratio of 1:4. PEI and plasmid DNA were diluted in separate tubes with 100 μl of serum-free medium. DNA and PEI solutions were vortexed at maximum speed for 3–5 s and incubated for 15 min at room temperature prior to addition to the cells. Cells were incubated for 48 h prior to collection for experiments. Cell lysates and immunoblotting were performed as described previously (24).

BRET

Initial experiments were performed to optimize the BRET system for AGS4-Gα_i1 interactions and to ensure the specificity of observed signals. All studies involved saturation BRET analysis, altering donor/acceptor ratios and/or time course analysis. Forty-eight hours after transfection, the cells were washed once with phosphate-buffered saline and harvested with Tyrode's solution (140 mm NaCl, 5 mm KCl, 1 mm MgCl₂, 1 mm CaCl₂, 0.37 mm NaH₂PO₄, 24 mm NaHCO₃, 10 mm HEPES, pH 7.4, and 0.1% glucose (w/v)). Cells were distributed in triplicate at 1 × 10⁵ cells/well into gray 96-well plates. Fluorescence and luminescence signals were measured with a TriStar LB 941 plate reader (Berthold Technologies, Oak Ridge, TN). Total fluorescence (excitation, 485 nm; emission, 535 nm) was first measured to quantify acceptor expression. The luciferase substrate coelenterazine H (5 μm final concentration) was then added, and luminescence was measured (donor, 480 ± 20 nm; acceptor, 530 ± 20 nm). Net BRET values were determined by first calculating the 530 ± 20:480 ± 20 nm ratio and then subtracting the background BRET signal determined from cells transfected with the Renilla luciferase (RLuc) expression vector phRLuc_N3 alone. Spectral measurements were conducted with the protocol described above using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA). BRET saturation curves and statistical analyses were measured using GraphPad Prism (GraphPad Software, San Diego, CA). Data were analyzed by analysis of variance with significant differences between groups determined by Tukey's post-hoc test.

RESULTS AND DISCUSSION

Key questions in the field and for AGS4 in particular are what regulates the formation and disassembly of AGS4-Gα_i complexes and is the AGS4-G-protein interaction influenced by GPCR activation or other signals? As an initial approach to address these questions, we used BRET with contingent binding proteins tagged with RLuc or yellow fluorescent protein (YFP). The enzymatic oxidation by luciferase of substrates such as coelenterazine and subsequent non-radiative emission can excite YFP if the two proteins are in close proximity (< 100 Å). The strength of the BRET signal for two proteins fused with RLuc and YFP, respectively, depends upon distance between the fluorophores, their relative orientations, and the relative expression levels of donor (RLuc) and acceptor (YFP). Specific interactions exhibit saturation of BRET signals using a constant amount of the luciferase donor and increasing amounts of the YFP acceptor (25). AGS4 was tagged with either YFP or RLuc, and YFP or RLuc was inserted into the loop connecting helices αB and αC of the helical domain in Gα_i1, which confers nucleotide binding and hydrolysis properties similar to the untagged protein (20, 22).

Analysis of different AGS4 and Gα_i BRET donor/acceptor pair combinations revealed that the AGS4-RLuc-Gα_i1-YFP (YFP at position 122) BRET pair yielded the strongest BRET signal and the highest signal-to-noise ratio (data not shown). The higher BRET signals detected by C-terminal tagged AGS4-RLuc and Gα_i1-YFP likely reflect the ability of the donor AGS4-RLuc to simultaneously bind more than one acceptor Gα_i1-YFP due to the presence of three GPR motifs in AGS4. Robust, specific, and saturable BRET signals were observed between AGS4-RLuc and Gα_i1-YFP (Fig. 1, A and B). BRET signals were not observed in cells expressing AGS4-Q/A, which contains Gln-Ala mutations in each of the three GPR motifs in AGS4, rendering it unable to bind Gα_i (26) (Fig. 1, A and B), thus confirming the specificity of the BRET signal. AGS4-RLuc-Gα_i1-YFP BRET signals were also decreased following co-expression of Gβγ subunits, consistent with the ability of Gβγ subunits to compete with GPR motifs for Gα binding (8, 27) (Fig. 1B). AGS4-RLuc-Gα_i-YFP BRET signals were markedly reduced upon introduction of the Q204L mutation in Gα_i-YFP, which alters GTP hydrolysis consistent with the known preference of GPR motifs for GDP-bound Gα_i subunits. The N149I mutation, which renders Gα_i incapable of binding GPR motifs (28), also predictably reduced the AGS4-Gα_i BRET signal (Fig. 1C). Interestingly, treatment of cells with pertussis toxin, which ADP-ribosylates a cysteine residue in Gα_i/_o subunits four amino acids from the C terminus and renders Gα incapable of being activated by GPCRs, had no effect AGS4-RLuc-Gα_i-YFP BRET signals (Fig. 1C).

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.

Truncations of AGS4 revealed that the AGS4-RLuc-Gα_i1-YFP BRET signals require the AGS4 C-terminal GPR domain (AGS4-CT-Leu⁵⁷–Cys¹⁶⁰) (Fig. 1D). However, although no BRET signals were observed between the AGS4 N terminus (AGS4-NT-Met¹–Ser⁵⁶) and Gα_i-YFP, the reduction in the overall magnitude of the BRET signals of AGS4-CT-RLuc as compared with full-length AGS4-RLuc suggests that the N terminus of AGS4 may influence the ability of AGS4 to interact with Gα_i, consistent with the idea that residues outside of the core GPR motif may influence the GPR-Gα_i interaction (1, 19, 26, 29).

As an initial approach to define the relative influence of each of the GPR motifs in AGS4 on Gα_i binding, we tested AGS4-RLuc constructs with the Q/A substitution in each of the AGS4-GPR motifs (Q80A, Q122A, and Q151A) in AGS4-RLuc-Gα_i-YFP BRET experiments (Fig. 1E). These data are interesting in a number of aspects. First, individually mutated AGS4-GPR motifs show a progressive decrease in Gα_i1-YFP BRET signals with GPRIII-Q/A having the most significant effect. In addition, AGS4-RLuc constructs with mutations in GPRIII in combination with either GPRI (GPRI,III-Q/A) or GPRII (GPRII–III-Q/A) have significantly lower BRET signals with Gα_i1-YFP than AGS4-Q/A mutations in the first two GPR motifs (GPRI–II-Q/A). Taken together these data suggest that GPR-III is important for Gα_i binding in cells. Alternatively, the decreased BRET signals generated in GPR-III Q/A mutants may reflect the proximity of the C-terminal RLuc tag to GPR-III and increased resonance energy transfer to Gα_i-YFP. Secondly, Q/A mutations in both GPR-I and GPR-III of AGS4 (GPRI,III) still yield significant Gα_i-YFP BRET signals with GPR-II as the sole GPR motif, suggesting that in the intact cell, GPR-II does indeed contribute to Gα_i binding consistent with earlier observations (1). This is in contrast to a previous report in which a glutathione S-transferase fusion protein containing AGS4-GPRII was essentially inactive as a guanine nucleotide dissociation inhibitor for Gα_i1 unless Ala¹²¹ was changed to Asp (19).

We then sought to determine the influence of GPCR activation on the AGS4-Gα_i interaction. Co-expression of the α_2A-adrenergic receptor had no effect on AGS4-RLuc-Gα_i-YFP BRET signal generation (Fig. 2A), and BRET signals were detected at levels of Gα_i1-YFP that were similar to the endogenous level of Gα_i1 detected by immunoblotting (Fig. 2B, lower panel). However, when the α₂-AR agonist UK14304 was added, an ∼30% reduction in AGS4-RLuc-Gα_i-YFP BRET signal was observed (Fig. 2, A and B). This agonist-mediated effect was dose-dependent (Fig. 2C), occurred within 2 min of agonist treatment, and persisted for up to 30 min (Fig. 2D). The UK14304-mediated reduction in AGS4-RLuc-Gα_i1-YFP BRET was blocked by the α₂-AR antagonist rauwolscine (Fig. 2E). Similar reductions in AGS4-RLuc-Gα_i1-YFP BRET were observed upon co-expression of CXCR4 and the mu-opioid receptor (MOR) after treatment with CXCL12 and [d-Ala², N-MePhe⁴, Gly-ol]-enkephalin, respectively (data not shown). Similar reductions in BRET signals were observed between the GPR protein AGS3 fused to Renilla luciferase and Gα_i1-YFP in cells expressing Gα_i-coupled GPCRs upon treatment with agonist.⁵

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.

The effect of receptor activation on the AGS4-Gα_i interaction led us to ask whether AGS4-Gα_i complexes were actually substrates for receptor-stimulated activation of Gα_i at the cell surface. We first examined the subcellular distribution of AGS4-YFP and Gα_i to determine whether their localization might overlap with that of a receptor present at the cell surface. Although AGS4 is primarily localized to the cytosol (supplemental Fig. S1) (1), co-expression with Gα_i resulted in a dramatic recruitment of AGS4-GFP to the cell cortex (supplemental Fig. S1), suggesting that AGS4-Gα_i complexes are at least within the same subcellular compartment as GPCRs, i.e. at the cell surface. Gα_i-mediated recruitment of AGS4-GFP to the cell cortex was not observed for AGS4-Q/A-GFP, nor in the context of the GTP hydrolysis-deficient Gα_i3-Q204L variant (supplemental Fig. S1). As AGS4-RLuc-Gα_i-YFP BRET signals were dramatically reduced or absent in the context of the Gα_i-Q204L and AGS4-Q/A variants (Fig. 1), the co-localization of wild-type AGS4 and Gα_i suggest that their co-localization at the cell surface likely results from their interaction. We then measured agonist-induced changes in AGS4-RLuc and Gα_i1-YFP distribution in crude membrane and cytosol fractions (supplemental Fig. S2). Receptor activation decreased the amount of AGS4-RLuc in the membrane fraction with a concomitant increase in the cytosol fraction as measured by both luminescence and immunoblotting (supplemental Fig. S2A), whereas Gα_i1-YFP distribution was unaltered (supplemental Fig. S2B). These data suggest that AGS4 and Gα_i physically dissociate after receptor activation, with AGS4 moving into the cytosol and Gα_i remaining at the plasma membrane.

We then sought to determine whether AGS4 and Gα_i were actually forming a complex with receptors. Under basal conditions, specific BRET signals were not observed between AGS4-RLuc and α_2A-AR-Venus (Fig. 3, A and B). However, when co-expressed with Gα_i, dramatic and specific BRET signals were observed (Fig. 3, A and B).⁶ We did not observe significant alterations in either the basal or the agonist-induced changes in BRET observed between AGS4-RLuc and α_2A-AR-Venus when either Gα_i1 or Gα_i3 was used (data not shown). BRET signals were not observed between AGS4-RLuc and α_2A-AR-Venus in the context of the Gα_i-Q204L or Gα_i-G202T mutations or Gα_s (Fig. 3C), nor were they observed with the AGS4-Q/A-RLuc variant, which cannot bind Gα_i (data not shown), indicating that the AGS4-Gα_i interaction is required for the BRET signals observed between AGS4-RLuc and α_2A-AR-Venus. In addition, the Gα_i-dependent AGS4-RLuc-α_2A-AR-Venus BRET signals were reduced by ∼40–50% upon treatment with the α₂-AR agonist UK14304 (Fig. 3, A and B). As was observed for the agonist-regulated BRET signals between AGS4-RLuc and Gα_i1-YFP (Fig. 2), the reductions in Gα_i-dependent BRET signals between AGS4-RLuc and α_2A-AR-Venus exhibited were dose-dependent and occurred on a similar timescale (Fig. 3, D and E). The agonist-mediated reduction in AGS4-RLuc-α_2A-AR-Venus BRET was blocked by the antagonist rauwolscine (Fig. 3A) and by pertussis toxin pretreatment (Fig. 3F). Similar Gα_i-dependent and agonist-induced reductions in BRET signals were also observed between AGS4-RLuc and MOR-YFP but were not observed with AGS4-RLuc and β₂-AR-Venus, which is primarily a Gα_s-coupled receptor (Fig. 3G).

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.

In the case of AGS4-RLuc-Gα_i-YFP BRET signals, receptor activation resulted in a reduction in BRET that could result either due to dissociation of AGS4-RLuc-Giα-YFP complexes or from a conformational rearrangement of the complex. The observation that receptor activation leads to a redistribution of AGS4 from the plasma membrane to the cytosol supports the first possibility. The reduction in agonist-modulated AGS4-RLuc-Gα_i-YFP BRET signal suggests that the agonist-regulated effect either occurs in a spatially restricted manner (e.g. at the plasma membrane) or is the result of second messengers. It is also possible that the agonist-induced reductions in BRET may arise from subunit exchange between the Gα_i-YFP complexed with AGS4-RLuc and wild-type, endogenous (i.e. untagged) Gα_i from Gαβγ heterotrimers or that “free” Gβγ released from activated Gαβγ heterotrimers competes with AGS4-RLuc for Gα_i-YFP binding. Regarding the latter scenario, although data indicate that increased expression of Gβγ subunits does indeed reduce AGS4-RLuc-Gα_i-YFP BRET (Fig. 1B), this is likely due to an overall reduction in the amount of AGS4-RLuc-Gα_i-YFP complexes being formed in the presence of excess Gβγ rather than the release of free Gβγ subunits occurring via receptor activation.

The data therefore suggest that AGS4-Gα_i complexes are directly coupled to and are regulated by a GPCR. The agonist-induced reduction in Gα_i-dependent BRET signals between AGS4-RLuc and α_2A-AR-Venus (Fig. 3) and the agonist-induced reductions in AGS4-RLuc-Gα_i-YFP (Fig. 2) are consistent with nucleotide exchange occurring on Gα_i while complexed with AGS4-RLuc and suggest that once bound to GTP, the Gα_i subunit dissociates from AGS4-RLuc, resulting in the decrease in BRET signals in both cases. The pertussis toxin blockade of these agonist-regulated BRET signals suggests that the mechanism of activation of an AGS4-Gα_i complex is similar to that of receptor-mediated activation of Gαβγ heterotrimers and that GPCRs provide one mode of regulation of AGS4-Gα_i complexes. As AGS4 and other proteins containing more than one GPR motif can bind multiple Gα_i subunits simultaneously (7, 8, 19), these data have far reaching implications for G-protein signal processing and may provide cells with additional flexibility to modulate signal efficiency, specificity, duration, and location in ways previously unappreciated.