G Protein-coupled Receptors and Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Both Regulate the Regulator of G Protein Signaling 14 (RGS14)·Gαi1 Complex in Live Cells*

Background: Regulator of G protein signaling 14 (RGS14) is a G protein regulatory (GPR) protein that participates in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Results: RGS14 forms regulated complexes with GPCRs in live cells. Conclusion: RGS14 integrates unconventional and conventional GPCR-dependent G protein signaling pathways. Significance: GPR proteins appear to be at the nexus of divergent G protein signaling pathways. Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gαi/o signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gαi1/3-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gαi1-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gαi/o-dependent complex with a Gi-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α2A-adrenergic receptor (α2A-AR) in a Gαi/o-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α2A-AR complex occurs in the presence of Gαi/o but not Gαs or Gαq. Unexpectedly, RGS14 does not dissociate from Gαi1 in the presence of stimulated α2A-AR, suggesting preservation of RGS14·Gαi1 complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gαi1 complex and the Gαi1-dependent RGS14·α2A-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gαi/o and that these RGS14·Gαi1·GPCR complexes may be substrates for other signaling partners such as Ric-8A.


Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional G␣ i/o signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds G␣ i1/3 -GDP in cells and in vitro.
The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14⅐G␣ i1 -GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a G␣ i/o -dependent complex with a G i -linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the ␣ 2A -adrenergic receptor (␣ 2A -AR) in a G␣ i/o -dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14⅐␣ 2A -AR complex occurs in the presence of G␣ i/o but not G␣ s or G␣ q . Unexpectedly, RGS14 does not dissociate from G␣ i1 in the presence of stimulated ␣ 2A -AR, suggesting preservation of RGS14⅐G␣ i1 complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14⅐G␣ i1 complex and the G␣ i1 -dependent RGS14⅐␣ 2A -AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on G␣ i/o and that these RGS14⅐G␣ i1 ⅐GPCR complexes may be substrates for other signaling partners such as Ric-8A.
Established models of G protein signaling suggest that heterotrimeric G proteins (G␣␤␥ subunits) are linked to specific G protein-coupled receptors (GPCRs), 3 and that these receptors act as guanine nucleotide exchange factors (GEFs) toward the G␣ subunit to promote nucleotide exchange and downstream signaling events (1,2). The regulators of G protein signaling (RGS) proteins act as GTPase accelerating proteins on the activated G␣ subunit, catalyzing GTP hydrolysis to terminate G protein signaling (3)(4)(5).
Recent studies have explored novel unconventional G protein signaling pathways involved with cell division and synaptic signaling/plasticity that can operate independently of GPCRs (6 -13). The hallmark of these unconventional G protein pathways are signaling complexes involving G␣-GDP bound to proteins containing one or more G protein regulatory (GPR) motifs. Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a cytosolic GEF that directly promotes nucleotide exchange on G␣ i , G␣ o , and G␣ q subunits in unconventional G protein signaling (14). Ric-8A also recognizes, binds, and regulates the formation/dissociation of some GPR⅐G␣ i1 -GDP complexes, such as AGS3⅐G␣ i1 -GDP, LGN⅐G␣ i1 -GDP, and RGS14⅐G␣ i1 -GDP (15)(16)(17).
RGS14 is a functionally and structurally complex signaling protein that is most highly expressed in the brain but also present in spleen, thymus, and lymphocytes (18 -21). Within brain, RGS14 is predominately localized in the CA2 subregion of the hippocampus, where it is involved in spatial memory, learning, and synaptic plasticity (22). The unique structure of RGS14, which includes an RGS domain, two Ras/Rap binding domains, and a GPR (also known as GoLoco (23)) motif (20,21) suggests that RGS14 functions in the brain through a variety of signaling mechanisms that may involve both G protein and MAP kinase signaling cascades (24). In addition to possessing GTPase accelerating protein activity toward activated G␣ i/o -GTP subunits, RGS14 also exhibits selective guanine nucleotide dissociation inhibitor activity toward G␣ i1/3 -GDP subunits through direct binding of its GPR motif (18,19,21,(25)(26)(27). In this regard RGS14 shares similarities with the family of Group II activators of G protein signaling (AGS) proteins that are characterized by one or more GPR motifs and mediate unconventional G protein signaling (28 -30). Similar to AGS3 and LGN, which form stable complexes with G␣ i1 -GDP via their GPR motifs (15,16), the RGS14⅐G␣ i1 -GDP signaling complex is a substrate for Ric-8Ainduced dissociation and nucleotide exchange on the resulting free G␣ i1 (17).
Recent evidence suggests that unconventional pathways involving GPR⅐G␣-GDP complexes and conventional pathways involving GPCR⅐G protein complexes may be functionally linked. In particular, the GPR proteins AGS3 and AGS4 appear to interface with GPCRs in a G␣ i -dependent manner (31,32). Compelling evidence also indicates that RGS proteins directly and selectively interact with GPCRs to modulate G protein signaling (for review, see Ref. 33). Given that RGS14 is an RGS protein that interacts with G␣ i/o -GTP but contains a GPR motif that binds G␣ i1/3 -GDP, we examined whether the RGS14⅐G␣ i1 complex can be regulated by a G␣ i/o -linked GPCR.
The non-receptor GEF Ric-8A regulates the RGS14⅐G␣ i1 complex (17) as well as certain GPCR signaling pathways (34, 35). However, it remains unknown whether Ric-8A can modulate GPCR⅐G␣ interactions, especially in the presence of a GPR protein such as RGS14. Therefore, we also studied the effects of Ric-8A on RGS14⅐G␣ i1 ⅐GPCR complex formation and whether RGS14 may be at the interface between conventional and unconventional G protein signaling pathways. Here we report the first evidence that the RGS14⅐G␣ i1 -GDP complex is regulated in concert by both a G␣ i/o -linked GPCR and Ric-8A in live cells. We show that RGS14 forms a stable complex with G␣ i1 via its GPR motif and that this complex is proximal to GPCRs as evidenced by the presence of specific bioluminescence resonance energy transfer (BRET) signals between RGS14 and the ␣ 2A -adrenergic receptor (␣ 2A -AR) in the presence of G␣ i1 . This RGS14⅐␣ 2A -AR complex partially dissociates/rearranges after receptor agonist treatment and is further regulated by Ric-8A. Together, these findings illustrate that RGS14 functions together in both conventional and unconventional G protein signaling and that Ric-8A may recognize and act on GPCR⅐G␣ i ⅐GPR complexes to further regulate G␣ i signaling.

EXPRIMENTAL PROCEDURES
Plasmids and Antibodies-The rat RGS14 cDNA used in this study (GenBank TM accession number U92279) was acquired as described (19). Rat RGS14 was used as a template in PCR reactions using TaKaRa Taq (Fisher) to generate Renilla luciferase (Luc) fusion protein constructs in the phRLucN2 vector graciously provided by Dr. Michel Bouvier (University of Montreal). The following oligonucleotides and restriction enzymes were used in the PCR amplification and subsequent digestion: RGS14 forward primer 5Ј-GCT CTC GAG GCC ACC ATG  CCA GGG AAG CCC AAG CAC-3Ј, XhoI; reverse primer  5Ј-CGC GGT ACC TGG TGG AGC CTC CTG AGA ACC-3Ј,  KpnI. The RGS14-Luc GPR mutant, in which invariant glutamine and arginine residues (Gln 515 and Arg 516 ) were both mutated to alanine, was generated by site-directed mutagenesis using a Stratagene site-directed mutagenesis kit according to the manufacturer's instructions and is referred to as RGS14(GPR-null). Oligonucleotide primers used to create RGS14-Q515A/ R516A-Luc (RGS14(GPR-null)) are as follows: RGS14(GPRnull) forward primer 5Ј-GGG GCC CAT GAC GCC GCC GGA CTT CTT CGC AAA G-3Ј and reverse primer 5Ј-CTT TGC GAA GTC CGG CGG CGT CAT GGG CCC C-3Ј. The RGS14-Luc RGS domain mutant, in which invariant glutamic acid and asparagine (Glu 92 and Asn 93 ) residues were both mutated to alanine, was generated by site-directed mutagenesis using a Stratagene kit and is referred to as RGS14(RGS-null). Oligonucleotide primers used to create RGS14-E92A/N93A-Luc (RGS14(RGS-null)) are as follows: RGS14(RGS-null) forward primer 5Ј-AAG GAA TTC AGC GCC GCC GCC GTA ACT TTC TGG CAA GC-3Ј and reverse primer 5Ј-GCT TGC CAG AAA GTT ACG GCG GCG GCG CTG AAT TCC TT-3Ј). The RGS14-Luc RGS/GPR double mutant referred to as RGS14(RGS/GPR-null) was generated by using RGS14(RGSnull) as a template and RGS14(GPR-null) primers in site-directed mutagenesis. In all cases, the plasmids were sequenced to confirm the fidelity of the PCR.
BRET-BRET experiments were performed as previously described (31,32). Briefly, HEK293 cells were transiently transfected with BRET donor and acceptor plasmids using polyethyleneimine. Forty-eight hours after transfection, the culture medium was removed, and cells were washed once with PBS and harvested with Tyrode's solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.37 mM NaH 2 PO 4 , 24 mM NaHCO 3 , 10 mM HEPES, and 0.1% glucose (w/v), pH 7.4). Each group of cells was distributed into gray 96-well OptiPlates (PerkinElmer Life Sciences) in triplicate, with each well containing 1 ϫ 10 5 cells. The acceptor (YFP/Venus-tagged) protein expression levels were evaluated by measuring total fluorescence using the TriStar LB 941 plate reader (Berthold Technologies) with excitation and emission filters at 485 and 535 nm, respectively. Data were analyzed using the MikroWin 2000 program. After fluorescence measurement, coelenterazine H (Nanolight Technology; 5 M final concentration) was added and luminescencedetected in the 480 Ϯ 20 and 530 Ϯ 20 nm windows for donor (Luc) and acceptor (YFP/Venus), respectively, by the TriStar LB 941 plate reader. BRET signals were determined by calculating the ratio of the light intensity emitted by the YFP/Venus divided by the light intensity emitted by Luc. Net BRET values were corrected by subtracting the background BRET signal detected from the expression of the donor fusion protein (Luc) alone. Agonists used were UK14304 (Sigma) and isoproterenol (Sigma). Immunoblots were performed as described previously (39).
Immunofluorescence and Confocal Imaging-Transfected HEK293 cells were treated with either vehicle or 10 M UK14304 diluted in serum-free DMEM for 5 min at 37°C. Cells were then fixed at room temperature for 15 min in buffer containing 3.7% paraformaldehyde diluted in PBS. Cells were washed in PBS and incubated for 8 min with 0.4% Triton X-100 diluted in PBS. Cells were then blocked for 1 h at room temperature in PBS containing 10% goat serum and 3% bovine serum albumin. Next, cells were incubated in this same buffer with a 1:1000 dilution of rabbit anti-FLAG and/or mouse anti-G␣ i1 antibodies at room temperature for 2 h. Cells were washed with PBS (3ϫ) and incubated with 1:300 dilutions of Alexa 546 goat anti-rabbit and/or Alexa 633 goat anti-mouse secondary antibodies at room temperature for 1 h. Cells were washed with PBS again (3ϫ) and mounted with ProLong Gold Antifade Reagent (Invitrogen). Confocal images were taken using a 63ϫ oil immersion objective from a LSM510 laser scanning microscope with AxioObserver Stand (Zeiss). Images were processed using the ZEN 2009 Light Edition software and Adobe Photoshop 7.0 (Adobe Systems).

RGS14 Interacts
Selectively with G␣ i1 through Its GPR Motif-RGS14 has two distinct G␣-binding domains. The RGS domain binds activated G␣ i/o subunits (18,19,21), whereas the GPR motif binds inactive G␣ i1 and G␣ i3 (19,26,27,40). That RGS14 is recruited from the cytosol to the plasma membrane and colocalizes with wild-type G␣ i1 (Fig. 1A, supplemental Fig. S1, and Refs. 17 and 27) suggests that RGS14 forms a stable complex with G␣ i1 at the plasma membrane, which we sought to quantitatively measure using BRET. We therefore measured the strength and selectivity of a BRET signal between RGS14-Luc and various YFP-tagged G␣ subunits (36,(41)(42)(43) (Fig. 1B). Of note, the YFP tag was inserted into the loop joining the ␣B and ␣C helices of each G␣ (36,41,43), preserving nucleotide binding and hydrolysis properties similar to the wild-type protein (36). Transfection of HEK cells with increasing amounts of G␣-YFP plasmid and a fixed amount (5 ng) of RGS14-Luc plasmid showed a robust, saturable BRET signal in the presence of G␣ i1 -YFP, whereas no BRET signal was observed between RGS14-Luc paired with either G␣ s -YFP or G␣ q -YFP (Fig. 1B). This BRET signal saturation is indicative of a specific interaction between RGS14 and G␣ i1 (44).
To further show BRET signal selectivity for RGS14-Luc interactions with G␣ i1 -YFP, we performed a competition assay in cells co-expressing untagged G␣ subunits ( Fig. 1C) to determine which G␣ subunits could displace G␣ i1 -YFP from RGS14-Luc and disrupt the BRET signal. As expected, the previously reported RGS14 binding partners G␣ i1 and G␣ i3 each disrupted the RGS14/G␣ i1 BRET signal, indicative of competition with G␣ i1 -YFP for RGS14 binding. By contrast, G␣ i2 , G␣ o , G␣ s , and G␣ q did not disrupt G␣ i1 -YFP binding to RGS14. This selectivity for G␣ i1 and G␣ i3 binding is entirely consistent with earlier reports showing RGS14 binding to only G␣ i1 and G␣ i3 but not other G␣ through its GPR motif, further validating our BRET system (18,19,21,26,27,40).
RGS14 Forms a Complex with the ␣ 2A -Adrenergic Receptor in a G␣ i/o -dependent Manner-The GPR proteins AGS3 and AGS4 form G␣ i -dependent complexes with GPCRs that are regulated by receptor activation (31,32). Therefore, we sought to investigate whether the RGS14⅐G␣ i1 complex can also be regulated by GPCRs in cells. Subcellular localization data showed that although RGS14 remained predominately cytosolic in the presence of co-expressed ␣ 2A -AR, it was recruited to the plasma membrane in the presence of both overexpressed ␣ 2A -AR and G␣ i1 in the absence of agonist (Fig. 3, left panel). This suggests formation of an RGS14⅐G␣ i1 ⅐␣ 2A -AR complex at the plasma membrane. Although RGS14 and G␣ i1 remained at the plasma membrane, the ␣ 2A -AR internalized in the presence of agonist UK14304 (Fig. 3, right panel).
To further examine the regulatory effects of GPCRs on RGS14⅐G␣ i1 complexes, we analyzed the BRET signals between RGS14-Luc and Venus-tagged ␣ 2A -AR or ␤ 2 -AR (Fig. 4). As expected, little to no detectable BRET signal was observed between RGS14 and the G s -linked ␤ 2 -AR in the absence or presence of both G␣ i1 and the receptor agonist isoproterenol FIGURE 1. RGS14 selectively interacts with G␣ i1 and G␣ i3 in the basal state of live cells as observed by BRET. A, FLAG-RGS14 and G␣ i1 -Glu-Glu (G␣ i1 -EE) plasmids were transfected into HEK cells alone and in combination. Cells were fixed, subjected to immunocytochemistry, and analyzed using confocal microscopy with a 63ϫ objective as described under "Experimental Procedures." Images are representative of cells observed in three separate experiments. Scale bars represent 10 m. B, top, a diagram shows the principle of BRET using the RGS14-Luc/G␣ i1 -YFP pair. Non-radiative emission from the Luc tag excites the YFP if the donor/acceptor pairs are Ͻ100 Å, which then emits at 535 nm. Bottom, HEK cells were transfected with 5 ng RGS14-Luc plasmid alone or in combination with 10, 50, 100, 250, or 500 ng of either G␣ i1 -YFP, G␣ s -YFP, or G␣ q -YFP plasmid. BRET signals (luminescence measured: donor, 480 Ϯ 20 nm; acceptor, 530 Ϯ 20 nm) were measured, and net BRET was calculated by first calculating the 530 Ϯ 20/480 Ϯ 20 nm ratio and then subtracting the background BRET signal determined from cells transfected with the RGS14-Luc plasmid alone. C, top panel, HEK cells were transfected with 5 ng of RGS14-Luc and 250 ng of G␣ i1 -YFP plasmids alone (con) or in combination with 1 g of untagged G␣ i1 , G␣ i2 , G␣ i3 , G␣ o , G␣ s , or G␣ q plasmid. Net BRET signals are shown between RGS14-Luc and G␣ i1 -YFP. Bottom panel, shown is a representative immunoblot of the different untagged G␣ subunits used in the BRET experiment. All BRET graphs are representative of at least three separate experiments. (Fig. 4A). Very low specific BRET signals were observed between RGS14 and ␣ 2A -AR both in the absence and presence of receptor agonist UK14304 (Fig. 4B, filled circles and open  circles, respectively). However, a 3-fold increase in BRET signal was observed between ␣ 2A -AR and RGS14 in the presence of co-expressed G␣ i1 (Fig. 4B, filled triangles). This signal was reduced by ϳ50% in the presence of UK14304 (Fig. 4B, open  triangles). This agonist-induced reduction in BRET correlates with the lack of co-localization between RGS14 and the ␣ 2A -AR after agonist stimulation (Fig. 3, right panel). Furthermore, ago-   nist-induced dissociation of the RGS14⅐␣ 2A -AR complex was completely blocked by pretreatment with pertussis toxin (PTX) (Fig. 4B, right panel). The very low BRET signals observed between RGS14 and the ␤ 2 -AR in the presence of G␣ i1 (Fig. 4A) illustrate that the BRET signals observed between RGS14 and the ␣ 2A -AR are indeed specific and are not simply the result of "bystander BRET," i.e. RGS14 localizing at the plasma membrane with G␣ i1 and randomly interacting with the receptor.

Ric-8A and GPCR Regulation of the RGS14⅐G␣ i1 Complex
The interaction between RGS14 and the ␣ 2A -AR was dependent on the presence of G␣ i/o family members (Fig. 4C). Specific BRET signals were observed between RGS14 and the ␣ 2A -AR in the presence of G␣ i1 , G␣ i2 , G␣ i3 , and G␣ o , with lower signals observed in the presence of G␣ s and G␣ q . The agonist-mediated dissociation of the RGS14⅐␣ 2A -AR complex was observed in the presence of all four G␣ i/o family members tested but not G␣ s or G␣ q (Fig. 4C).
To determine which domains of RGS14 are important for associating with the ␣ 2A -AR, we performed BRET experiments using the RGS14 constructs with mutations in the RGS domain and GPR motif as described in Fig. 2B (Fig. 4D). BRET signals observed between either RGS14-WT or RGS14(RGS-null) and the ␣ 2A -AR in the presence of co-expressed G␣ i1 were compa- rable, with similar reductions in response to receptor agonist UK14304. This suggests that the RGS domain of RGS14 is not required for the formation of the G␣ i1 -dependent complex with the ␣ 2A -AR. In contrast, the BRET signals observed between the ␣ 2A -AR and RGS14(GPR-null) in the presence of G␣ i1 were reduced by ϳ50% in the absence of agonist compared with RGS14-WT, indicating that the GPR motif is critical to forming a complex with the ␣ 2A -AR in the presence of G␣ i1 . Together, these results indicate that RGS14 forms a complex with the ␣ 2A -AR in the presence of a G␣ i/o protein and that the GPR motif is critical in promoting the formation of this complex (see supplemental Fig. S2A).
The RGS14⅐G␣ i1 Complex Remains Intact after ␣ 2A -AR Stimulation-Because the presence of G␣ i1 promotes the formation of an RGS14⅐␣ 2A -AR complex that is regulated by agonist, we examined the effects of ␣ 2A -AR stimulation on the RGS14⅐G␣ i1 complex (Fig. 5). To test this, we measured the BRET signal between RGS14-Luc and G␣ i1 -YFP in the presence of untagged ␣ 2A -AR. The RGS14⅐G␣ i1 complex remains intact in the presence of the ␣ 2A -AR regardless of receptor stimulation (Fig. 5A). This is in marked contrast to the decrease in BRET signal observed between AGS4-Luc and G␣ i1 -YFP in the presence of stimulated ␣ 2A -AR (Fig. 5A and Ref. 32). Together, these findings suggest that the ␣ 2A -AR dissociates from RGS14 after agonist stimulation but that the dissociated RGS14 remains in complex with G␣ i1 (supplemental Fig. S2B). This portrays a novel mechanism of GPR⅐G␣ i complex function with GPCRs that may be unique to RGS14 compared with other Group II AGS proteins.
The GPR motif is still critical for RGS14 interactions with G␣ i1 in the presence of the ␣ 2A -AR (Fig. 5B), as Ͼ80% reductions in BRET signals were observed between G␣ i1 and both RGS14(GPR-null) and RGS14(RGS/GPR-null) regardless of the presence of receptor. This indicates that even the presence of a GPCR cannot facilitate RGS14 interactions with G␣ i1 in the absence of a functional GPR motif.
Ric-8A Promotes Dissociation of the RGS14⅐G␣ i1 Complex-Because we observed Ric-8A regulation of RGS14⅐G␣ i1 complexes in vitro (17), we sought to quantitatively measure Ric-8A-mediated dissociation of RGS14⅐G␣ i1 complexes in live cells using BRET (Fig. 6A). As expected (17), increasing Ric-8A protein levels induced a decrease in BRET between RGS14-Luc and G␣ i1 -YFP (Fig. 6C). Ric-8A-induced reductions in RGS14/ G␣ i1 BRET were inhibited by pertussis toxin (ϩPTX) (Fig. 6C), which blocks Ric-8A binding and GEF activity toward G␣ i subunits (49). Expression of Ric-8A also induces an increase in G␣ i1 -YFP protein expression levels (Fig. 6B), which is consistent with recent evidence showing that Ric-8A is important for the functional expression and stability of G␣ subunits (50). Interestingly, the effect of Ric-8A on G␣ i1 -YFP expression levels was not blocked by pertussis toxin pretreatment, suggesting that the effect of Ric-8A on G␣ i expression is independent from its GEF activity.
We next studied the effects of Ric-8A on RGS14⅐G␣ i1 complexes in the presence of the ␣ 2A -AR (Fig. 7A). In the absence of Ric-8A, RGS14⅐G␣ i1 complexes remained intact after receptor stimulation as before (see Fig. 5A). In the absence of receptor agonist, Ric-8A promoted a decrease in the RGS14/G␣ i1 BRET signal. In the presence of agonist, Ric-8A induced an even greater decrease in the BRET signal (Fig. 7A). These findings suggest that Ric-8A can recognize and act on RGS14⅐G␣ i1 complexes in the presence of GPCRs and even more so in the presence of activated receptors.
Ric-8A Potentiates Dissociation of the RGS14⅐ ␣ 2A -AR Complex Caused by Receptor Agonist-Because Ric-8A induced dissociation of G␣ i1 from RGS14 in the presence of the ␣ 2A -AR, we next investigated the effect of Ric-8A on the RGS14⅐␣ 2A -AR complex in the presence of G␣ i1 (Fig. 7B). Ric-8A had little  NOVEMBER 4, 2011 • VOLUME 286 • NUMBER 44 effect on the RGS14⅐␣ 2A -AR complex in the presence of co-expressed G␣ i1 in the absence of agonist. However, BRET signals between RGS14 and the ␣ 2A -AR in the presence of G␣ i1 and receptor agonist were further reduced by ϳ25% in the presence of Ric-8A (red lines) compared with the absence of Ric-8A (black lines) (Fig. 7B). These findings suggest that Ric-8A acts to facilitate dissociation of RGS14 from activated ␣ 2A -AR in the presence of G␣ i1 (see supplemental Fig. S2C).

DISCUSSION
RGS14 is unusual among RGS protein family members in that it possesses two distinct G␣ binding domains; that is, an  RGS domain that accelerates GTP hydrolysis on activated G␣ i/o subunits (18,19,21) and a GPR motif that forms a tight complex with inactive G␣ i1/3 subunits (17,19,(25)(26)(27). RGS14 also belongs to a second family of signaling proteins, the Group II AGS proteins, which are characterized by the presence of one or more GPR motifs that mediate newly appreciated "unconventional" G protein signaling events (28,29). Recent studies of AGS3 and AGS4 demonstrate that these GPR domain-containing proteins interact with G␣ i to form complexes with G␣ i/olinked GPCRs in cells (31,32). Our results with RGS14 support those findings but also highlight some important differences that will be discussed. Overall, our findings indicate the following: 1) RGS14 selectively interacts with G␣ i1/3 in live cells through its GPR motif, 2) RGS14 forms a G␣ i/o -dependent complex with the G i/o -linked ␣ 2A -AR in live cells, 3) RGS14 dissociates from the ␣ 2A -AR after agonist treatment but remains bound to G␣ i1 , 4) Ric-8A potentiates agonist-stimulated dissociation of the RGS14⅐␣ 2A -AR complex, and 5) Ric-8A induces dissociation of G␣ i1 and ␣ 2A -AR from RGS14, having a greater effect in the presence of stimulated ␣ 2A -AR. Taken together, these findings suggest that RGS14 integrates both unconventional Ric-8A/G protein signaling and conventional GPCR/G protein signaling. A summary and interpretation of these findings is shown in Fig. 8.
RGS14 Selectively Interacts with Inactive G␣ i1/3 in Live Cells through Its GPR Motif-Our BRET analysis and confocal imaging indicate that the interaction of RGS14 with inactive G␣ i1/3 occurs at the plasma membrane of live cells ( Fig. 1 and supple-mental Fig S1). Consistent with previous studies (18,19,26,27,40), the capacity of both G␣ i1 and G␣ i3 (but not G␣ i2 , G␣ o , G␣ s , or G␣ q ) to disrupt the BRET between RGS14-Luc and G␣ i1 -YFP indicates that the observed BRET signal is specific for interactions between RGS14 and G␣ i1/3 (Fig. 1C).
To clarify which RGS14 domains are involved in the RGS14⅐G␣ i1 interaction, we measured the BRET signal between mutant forms of RGS14-Luc and G␣ i1 -YFP that specifically blocked RGS and/or GPR motif functions (Fig. 2). These studies show that the majority of the observed RGS14⅐G␣ i1 interaction is conferred by the GPR motif of RGS14 interacting with G␣ i1 . The fact that the BRET signal was never completely abolished in the presence of the RGS14 and G␣ i1 double mutants that ablate G␣ binding to both the GPR and RGS domains (Fig. 2, B and C) is consistent with the existence of a third G protein binding site on RGS14, as has been postulated (51).
RGS14 Selectively Interacts with the ␣ 2A -AR Receptor in a G␣ i/o -dependent Manner-Because RGS14 interacts with G␣ i/o family members, we examined whether RGS14 can be regulated by a G i/o -linked GPCR, specifically the ␣ 2A -AR. RGS14, G␣ i1 , and the ␣ 2A -AR co-localized at the plasma membrane when all three proteins were expressed together in cells (Fig. 3, left panel), consistent with the possibility that a ternary protein complex forms at the plasma membrane. After treatment with the ␣ 2A -AR agonist UK14304, RGS14 and G␣ i1 remained at the plasma membrane, whereas the ␣ 2A -AR partially internalized (Fig. 3, right panel), suggesting that the ternary complex dissociates. This hypothesis was supported in our BRET experiments. Co-expression of G␣ i1 resulted in an approximate 3-fold increase in RGS14/␣ 2A -AR BRET compared with RGS14 and ␣ 2A -AR alone (Fig. 4B). The G␣ i1 -dependent RGS14/␣ 2A -AR BRET signal was reduced ϳ50% after receptor activation by agonist, and this agonist effect was blocked by pertussis toxin pretreatment (Fig. 4B, right panel). This implies that functional coupling of the ␣ 2A -AR to G␣ i1 disrupts the RGS14⅐␣ 2A -AR complex. It is possible that the interacting sites between GPCR⅐G␣ i are different between the inactive and active states, the latter being sensitive to PTX. This is suggested by previous work on the phenomenon of guanine nucleotide-sensitive agonist binding to GPCRs and more recent work demonstrating preformed complexes of GPCRs and G proteins (52,53).
As expected, RGS14 interaction with the ␣ 2A -AR is dependent on the presence of G␣ i/o as G␣ q and G␣ s failed to elicit a robust RGS14/␣ 2A -AR BRET signal. Somewhat unexpectedly, RGS14⅐␣ 2A -AR association is promoted indiscriminately by the presence of any G␣ i/o family member (G␣ i1 , G␣ i2 , G␣ i3 , and G␣ o ) (Fig. 4C). This is surprising given that the RGS14⅐␣ 2A -AR interaction was highly dependent on the GPR motif (Fig. 4D), which only interacts with G␣ i1 and G␣ i3 in the absence of receptor. One possible explanation may be that RGS14 recognizes a receptor if the receptor is bound to any G␣ i/o protein, reflecting the promiscuity of RGS14 GTPase accelerating protein activity toward activated G␣ i/o subunits. In this regard, RGS14 is similar to RGS2. In the absence of receptor, RGS2 acts specifically on G␣ q (54). However, RGS2 is capable of interacting with G␣ i in the presence of a G i/o -linked GPCR (55), albeit with 30-fold lower affinity than for G␣ q (56). We note that . We propose that two pools of G␣ i exist in cells. Top, one pool localizes with GPCRs and G␤␥/ GPR proteins at the plasma membrane to participate in conventional GPCRdependent G protein signaling. In the resting state (left) of our model, a GPCR⅐G␣ i ⅐RGS14 complex forms and remains intact. Ric-8A has little effect on this complex in the absence of stimulation. Upon receptor stimulation (right), the RGS14⅐G␣ i complex dissociates from the GPCR, where it can be further acted upon by Ric-8A. Bottom, the second G␣ i pool forms complexes with GPR proteins at the plasma membrane in the absence of a GPCR to participate in unconventional GPCR-independent signaling. According to our findings, RGS14 forms a complex with G␣ i through its GPR motif. Ric-8A can recognize this RGS14⅐G␣ i complex, catalyze GTP exchange on G␣ i , and induce dissociation of the complex.
RGS14 complexes with receptor are dependent on both the G protein and the receptor because the G s -linked ␤ 2 -AR failed to interact with RGS14 in the presence of G␣ i1 (Fig. 4A).
The GPR motif interaction with G␣ i1 is important in promoting formation of the RGS14⅐␣ 2A -AR complex (Fig. 4D). The RGS14/␣ 2A -AR BRET signal was greatly reduced in the presence of RGS14(GPR-null) compared with RGS14-WT, indicating that G␣ i1 has a reduced capacity to bring RGS14 and the ␣ 2A -AR in close proximity when it cannot bind the GPR motif. Even when G␣ i1 could no longer bind either the RGS domain or GPR motif, there was still a slight BRET signal between RGS14(RGS/GPR-null) and the ␣ 2A -AR. Several possibilities exist to explain these results; 1) there may be another (undefined) G␣ i1 binding site on RGS14 (51), 2) RGS14 may be bound to G␣ i1 at a distinct site on the extreme C terminus of G␣ i1 (17), or 3) an unknown binding partner/scaffold may facilitate an RGS14⅐␣ 2A -AR interaction.
RGS14 Remains Bound to G␣ i1 after Dissociating from the ␣ 2A -AR-Although RGS14 dissociated from the ␣ 2A -AR after agonist treatment in the presence of co-expressed G␣ i1 (Fig. 4), it remained in complex with G␣ i1 via the GPR motif (Fig. 5). This finding is unexpected and differs from previous observations that show AGS3 and AGS4 dissociating from G␣ i after receptor activation (Fig. 5A and Refs. 31 and 32)). Our result suggests that RGS14 and G␣ i1 remain bound after receptor activation. This result is reminiscent of other findings showing that, in contrast to established models of G protein signaling (1), G␤␥ may not necessarily always dissociate from G␣. In some cases G␤␥ may rearrange relative to G␣-GTP after receptor activation (53), although in others G␤␥ does appear to dissociate (Refs. 57-59 and references therein). Irrespective of the mechanism involved, our findings represent a novel mechanism of action for GPCR⅐G␣⅐RGS complexes, where the active conformation of the ␣ 2A -AR favors release of an RGS14⅐G␣ i1 complex that may then be able to function as a signaling complex on its own or with other binding partners (such as potential MAP kinase signaling partners (24)). This complex may be regulated and function independently of the GPCR.
Ric-8A Is a Key Regulator of the GPCR⅐G␣ i1 ⅐RGS14 Complex-Although Ric-8A has been shown to influence GPCR signaling (34, 35, 60), little is known mechanistically about if or how Ric-8A may directly interact with and regulate GPCR⅐G protein complexes. We recently demonstrated that Ric-8A induces dissociation of RGS14 from G␣ i1 in vitro (17). In this study we sought to quantitatively measure the dissociative effects of Ric-8A on RGS14⅐G␣ i complexes in live cells using BRET (Fig.  6). Pertussis toxin blocked Ric-8A-mediated dissociation of the RGS14⅐G␣ i1 complex (Fig. 6, C and D), consistent with recent reports showing that pertussis toxin inhibits Ric-8A GEF activity on G␣ i1 and that Ric-8A binds to G␣ i1 at a region overlapping with the pertussis toxin binding site (17,49). In the absence of pertussis toxin, Ric-8A facilitated RGS14⅐G␣ i1 complex dissociation (Fig. 6, C and D). Ric-8A also induced dissociation of the RGS14⅐G␣ i1 complex in the presence of the ␣ 2A -AR, even in the absence of ␣ 2A -AR stimulation (Fig. 7A). This may be explained by Ric-8A effects on G␣ i1 expression levels. Because Ric-8A overexpression also induced an increase in G␣ i1 expression (Fig. 6B), it may be that there is an overabun-dance of G␣ i1 that is free to bind RGS14. The number of RGS14⅐G␣ i1 complexes may, therefore, outnumber the number of ␣ 2A -ARs, resulting in free RGS14⅐G␣ i1 complexes on which Ric-8A may act in the absence of receptor activation.
Ric-8A did not induce dissociation of the RGS14⅐␣ 2A -AR complex in the absence of receptor stimulation (Fig. 7B). This is in contrast to its effects on the RGS14⅐G␣ i1 complex in the presence of unstimulated receptor. It is possible that Ric-8A facilitates dissociation of RGS14⅐G␣ i1 complexes that are not associated with receptors, accounting for the decrease in RGS14/G␣ i1 BRET seen in the presence of unstimulated receptor (Fig. 7A). In a cellular signaling context, Ric-8A may function similarly to the Arr4 protein in yeast that serves a feedforward facilitating role in pheromone receptor-G protein signaling mating responses (61). Consistent with this idea is that Ric-8A potentiates taste-receptor signaling by a potential feed-forward mechanism (34).
Taken together, these studies show that RGS14 can associate with a GPCR⅐G␣ i/o complex in a regulated fashion and that Ric-8A is a regulatory partner in this process. Although Ric-8A potentiated dissociation of RGS14⅐G␣ i1 complexes from the ␣ 2A -AR in both the absence and presence of receptor stimulation, it had no effect on dissociating the RGS14⅐␣ 2A -AR complex itself in the absence of stimulation. We postulate that two pools of RGS14⅐G␣ i1 complexes may exist (Fig. 8). One subset resides at membranes (plasma and others?) in the absence of a GPCR, and the other directly complexes to a cell surface receptor. Ric-8A acts differently on the RGS14⅐G␣ i1 complex depending on whether or not the complex is coupled to a GPCR. In the absence of a GPCR (Fig. 8, bottom), Ric-8A can recognize and induce dissociation of the RGS14⅐G␣ i1 complex. When the RGS14⅐G␣ i1 complex is associated with a GPCR (Fig.  8, top), Ric-8A may not affect RGS14⅐G␣ i1 complexes unless the receptor is activated. In this case Ric-8A induces dissociation of G␣ i1 from RGS14 and subsequently RGS14 from receptor.
Our findings demonstrate that RGS14 functions in a unique mechanism to integrate both conventional GPCR⅐G protein signaling and unconventional GPCR-independent G protein signaling. These results highlight newly appreciated roles of GPR proteins at the interface of G protein signaling pathways, making them significant targets in the study of non-canonical G protein regulation and function.