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A Key Serine for the GTPase-Activating Protein Function of Regulator of G Protein Signaling Proteins Is Not a General Target for 14-3-3 Interactions

Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom

Received May 23, 2005; accepted September 13, 2005

	Abstract

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Mammalian regulator of G protein signaling (RGS) proteins are highly conserved within the RGS domain. Of amino acids that are universal, a serine residue at the C terminus of this domain has been described as the binding site in RGS7 for 14-3-3 proteins. However, studies with the related RGS3 indicate that the site of interaction is not within the RGS domain. We confirm that the interaction of RGS3 with 14-3-3 and 14-3-3 requires Ser²⁶⁴ and not the RGS domain and show both that mutation of the conserved RGS domain serine, Ser⁴⁹⁶ in RGS3, to either alanine or aspartate does not prevent binding of 14-3-3 proteins and that 14-3-3 proteins do not inhibit GTPase-activating protein (GAP) activity against receptor-activated G_o1. However, mutation of Ser⁴⁹⁶ does directly impair the action of RGS3 as a GAP against receptor-activated G_o1. We mutated the equivalent serine residue in the family B/R4 RGS proteins RGS1 and RGS16. Using two distinct assay formats, conversion to aspartate virtually abolished GAP activity, whereas conversion to alanine decreased potency 20-fold. Neither alteration modulated interactions with 14-3-3 or 14-3-3, but the 14-3-3 proteins did not modulate the GAP activity of the wild-type or mutant RGS proteins. Although interactions between 14-3-3 proteins and many RGS proteins can be observed, this does not involve this conserved serine and does not inherently modify GAP function.

Given these contrasting observations, we have re-examined interactions between 14-3-3 isoforms and both RGS3 and RGS7 and further examined the effects on GAP activity against receptor-activated G_o1 of mutating the highly conserved serine residue in both RGS3 and RGS7 and in a pair of RGS proteins that do not contain the long N-terminal extensions.

	Materials and Methods

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Materials. [-³²P]GTP and [-³²P]ATP were from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Pertussis toxin and PKC were from Sigma Chemical (St. Louis, MO).

Constructs. A fusion protein between the _2A-adrenoceptor and the subunit of the G protein G_o1 that was rendered-insensitive to the ADP-ribosyltransferase activity of pertussis toxin (_2A-AR-Cys³⁵¹IleG_o1) was generated previously (Cavalli et al., 2000).

Human 14-3-3 and , subcloned into pGEX-2T, were obtained from Dr. T. Dubois (University of Edinburgh, Edinburgh, Scotland). The 14-3-3 was used as a PCR template with primers designed to add a BamHI site and an HA-tag (YPYDVPDYA) at the N terminus and an EcoRI site at the C terminus. The fragment was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA). RGS1 (subcloned into pGEX-4T1) and RGS16 (subcloned into pGEX-2TK) were obtained from Dr. A. Meyerdierks (Department of Medical Microbiology, Medizinischer Hochschule, Hannover, Germany) and Dr. C. W. Fong (Institute of Molecular and Cell Biology, Proteos, Singapore), respectively. Serine-to-alanine and serine-to-aspartate mutants of these RGS constructs were generated by a dual overlapping PCR fragment amplification approach to produce mutant fragments of the RGS proteins that were then subcloned into the original constructs with appropriate flanking restriction sites. The _2A-adrenoceptor-RGS16 (_2A-AR-RGS16) fusion construct was a modification of the _2A-AR-RGS4 fusion described previously (Bahia et al., 2003). RGS4 was excised with BamHI and XbaI and replaced with PCR-amplified RGS16 with these sites at the N and C termini, respectively.

Human RGS3 and murine RGS16 were PCR amplified with primers designed to add a KpnI site onto the N-terminal with an XbaI site and a VSV-G tag (YTDIEMNRLGK) onto the C termini. The fragments were subcloned into pcDNA3. N-(RGS3^1–370) and C-terminal (RGS3^371–519) fragments of RGS3 were PCR-amplified with primers designed to add a KpnI site onto the N-terminal and an XbaI site with a VSV-G tag onto the C-terminal of each fragment. Human RGS3 was also PCR-amplified with primers designed to add an EcoRI site onto the N terminus and an XhoI site onto the C terminus. This fragment was then subcloned into pGEX-4T3 to make the GST-RGS3 fusion construct. The fragment was further subcloned into the vector pQE-32 by excising it with EcoRI/XhoI. The EcoRI site was converted to a blunt end and ligated to a blunt-ended BamHI site in pQE-32, whereas the XhoI site was ligated to a SalI site to make a hexahistidine (6xHis)-tagged RGS3 construct. Serine-to-alanine and serine-to-aspartate mutants were also introduced as described above.

Human RGS7 was obtained from the University of Missouri–Rolla cDNA Resource Centre via Dr. A. Tinker (University College London) and was PCR-amplified with primers designed to add an N-terminal BamHI site and a C-terminal SalI site, which was subcloned into pQE-30, or an N-terminal BamHI site and C-terminal NotI site with a VSV-G tag, which was subcloned into pcDNA3. Serine-to-alanine and serine-to-aspartate mutants were also introduced. All subcloning and PCR were carried out using standard techniques and constructs were fully sequenced before use.

Cell Culture, Transient Transfection, and Cell Lysates. HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) newborn calf serum and 2 mM glutamine. Before transfection, cells were seeded onto 10-cm dishes at 50 to 70% confluence. Constructs were transfected into the cells using the Lipofectamine reagent (Invitrogen) according to the manufacturer's protocol. Forty-eight hours after transfection, the medium was removed, and the cells washed twice with 1x PBS (120 mM NaCl, 25 mM KCl, 10 mM Na₂HPO₄, and 3 mM KH₂PO₄, pH 7.4). Cells intended for membrane preparation were scraped into 5 ml of 1x PBS, pelleted, and stored at -80°C. Cells for lysates were harvested with 1 ml of lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 50 mM NaF, 15 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1 mM EDTA, and 1 complete protease inhibitor tablet; Roche Diagnostics, Indianapolis, IN) and incubated, rotating, at 4°C for 1 h. Lysed cells were centrifuged at 12,000g for 10 min at 4°C, and the supernatant was transferred to fresh tubes. Protein concentration was determined by BCA assay, and the lysates were stored at -80°C.

Membrane Preparation. Cells were resuspended in 1x 10 mM Tris-HCl and 0.1 mM EDTA, pH 7.5, and disrupted with a Teflon-on-glass homogenizer for 2 min before being passed through a 25-gauge needle 20 times. Unbroken cells were removed by centrifugation at 500g for 5 min at 4°C. The supernatant fraction was centrifuged at 90,000g for 30 min at 4°C to recover the membranes, which were resuspended in 1x 10 mM Tris-HCl and 0.1 mM EDTA, pH 7.5, and the protein concentration was determined by BCA assay before being stored at -80°C.

Purification of 6xHis-Tagged Proteins. pQE-30/32 plasmids (QIAGEN, Valencia, CA) containing full-length cDNAs of RGS3 or RGS7 fused to the 6xHis tag were transformed into Escherichia coli strain BL21 DE3. LB medium (400 ml) containing 100 µg/ml ampicillin was inoculated with a 10-ml starter culture of the transformed cells and were grown to an optical density at 600 nm of 0.5. Expression of the 6xHis-tagged protein was induced by the addition of 1 mM isopropyl-, D-thiogalactopyranoside for 5 h before harvesting the cells by centrifugation at 6000g for 15 min at 4°C. Pellets were resuspended in denaturing lysis buffer (containing 6 M urea), and the 6xHis-tagged protein was purified using Ni-NTA agarose (QIAGEN) according to the manufacturer's instructions. The eluted proteins were analyzed by SDS-PAGE and BCA protein assay before being dialysed against three changes of 1x PBS containing 5% glycerol at 4°C over 2 days, before storage at -80°C.

Purification of GST-Fusion Proteins. pGEX plasmids (GE Healthcare) containing the appropriate cDNA fused to glutathione S-transferase were transformed and grown up as described in the previous section. Cell pellets were lysed by resuspending in 20 ml of 1x PBS (with protease inhibitors) containing 0.5 mg/ml lysozyme followed by incubation at 4°C with rotation for 1 h. The resuspended cells were sonicated for 4 x 30 s each on ice, with 30 s for cooling between bursts. Dithiothreitol was added to a final concentration of 5 mM and Triton X-100 (as a 10% stock) to a final concentration of 1%. The lysates were then incubated at 4°C with rotation for 1 h, and the insoluble material was removed by centrifugation at 12,000g for 15 min at 4°C. One milliliter of a suspension of washed (three times with 10 volumes of 1x PBS with protease inhibitors) glutathione Sepharose was added to each cleared lysate and incubated at 4°C overnight with rotation. The lysates were spun at 500g for 5 min at 4°C, and the supernatant was removed from the pellet of glutathione Sepharose beads. The beads were washed with 3 x 10 ml of 1x PBS with protease inhibitors, and the GST fusion proteins were eluted with 5 x 1.5 ml of glutathione solution (10 mM concentration of reduced glutathione in 50 mM Tris-HCl, pH 8.0). The eluted proteins were analyzed by SDS-PAGE and BCA protein assay before being dialyzed against three changes of 1x PBS containing 5% glycerol at 4°C over 2 days before storage at -80°C.

PKC Phosphorylation of Recombinant RGS Protein. Recombinant RGS protein (5–10 µg) was incubated for 30 min at 37°C in a total volume of 100 µl with 20 mM HEPES, pH 7.4, 10 mM MgCl₂, 0.1 mM CaCl₂, 100 µM ATP, 20 µg/ml diacylglycerol, 100 µg/ml phosphatydylserine, 0.03% Triton X-100, and 0.5 U of PKC (or equivalent volume enzyme dilution buffer). Reactions were then placed on ice until added to pull-down assays. Phosphorylation was monitored by including 5 µCi of [-³²P]ATP, followed by SDS-PAGE and detection by autoradiography.

Pull-Down Experiments. Aliquots (100–400 µl) of HEK293 cell lysate (dependent on protein expression from the construct) were mixed with 200 µl of dialyzed GST fusion protein (0.5–2 µM) and 40 µl of washed glutathione Sepharose slurry. Lysis buffer was added to give a constant final volume. After overnight incubation at 4°C with rotation, the beads were centrifuged at 500g for 5 min at 4°C, and the supernatant was removed. The beads were then washed with 4 x 1-ml aliquots each of lysis buffer, and 40 µl each of SDS-PAGE sample buffer was added. The proteins were allowed to elute for 15 min at room temperature and were then heated to 65°C for 5 min before analysis by SDS-PAGE. Experiments with 6xHis-tagged proteins were essentially similar, but Ni-NTA agarose was used in place of glutathione Sepharose.

High-Affinity GTPase Assays. High-affinity GTPase assays were carried out as described by Hoffmann et al. (2001) and Ward and Milligan (2004), using the procedure adapted to a 96-well microtiter plate format. In the case of assays requiring the addition of GST-RGS, GST-14-3-3, or 6xHis-RGS proteins, these were combined with the membranes and allowed to incubate on ice for 30 min before the addition to the assay mix. The volumes and concentrations of the components added to the assay were adjusted to allow for a constant reaction volume of 100 µl to be maintained in all cases.

	Results

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An HA-tagged version of the isoform of 14-3-3 was expressed in HEK293 cells, and after SDS-PAGE analysis of cell lysates, it was detected with the anti-HA antibody 12CA5 predominantly as a 33-kDa polypeptide (Fig. 1A). This polypeptide interacted with, and was pulled down by, a 6xHis-tagged form of the regulator of G protein signaling, RGS3 (Fig. 1B). All mammalian RGS proteins contain a serine residue within a conserved Asp-Ser-Tyr-Xaa-Arg motif at the C-terminal end of the RGS domain that defines this protein family. Previous work (Benzing et al., 2000) has indicated that this serine is central for the binding of 14-3-3 proteins to both RGS3 and RGS7. Because 14-3-3 binding routinely requires a phosphorylated serine residue (Mackintosh, 2004) we altered this serine (Ser⁴⁹⁶ in RGS3) to either alanine or aspartic acid. Neither of these modifications altered the ability of 14-3-3 to interact with 6xHis RGS3 (Fig. 1B). To examine the role of the RGS box of RGS3 in interactions with 14-3-3, RGS3 was split into the RGS domain and C terminus, consisting of amino acids 371 to 519, and the N-terminal domain consisting of amino acids 1 to 370 (Fig. 2). The VSV-G epitope tag sequence was added to the C terminus of each of these fragments and to full-length RGS3 to facilitate detection. After expression in HEK293 cells, full-length RGS3-VSV-G migrated through SDS-PAGE predominantly as a 75-kDa polypeptide with a small amount of a higher molecular mass species that might represent an SDS-PAGE-resistant dimer. The C-terminal fragment migrated predominantly as a 23-kDa species with lower levels of a 46-kDa species, whereas in SDS-PAGE, the N-terminal fragment was predominantly a 52-kDa species with evidence of some capacity to dimerize (Fig. 2). Full-length RGS3-VSV-G was pulled down by GST fusions of both 14-3-3 and 14-3-3 (Fig. 3A). Again, this was not prevented by the introduction of Ser⁴⁹⁶Ala or Ser⁴⁹⁶Asp mutations into RGS3 (Fig. 3A). It is interesting that although the VSV-G-tagged C-terminal 23-kDa RGS domain fragment of RGS3 did not interact with either 14-3-3 or 14-3-3, no matter the identity of the amino acid at position 496, the VSV-G-tagged N-terminal 52-kDa fragment of RGS3 did interact with both 14-3-3 isoforms (Fig. 3B). Recent studies have suggested a role for RGS3Ser²⁶⁴ within the N-terminal domain and outside the RGS domain in interactions with 14-3-3 isoforms (Niu et al., 2002). In agreement with this, the mutation of Ser²⁶⁴ to either alanine or aspartic acid did eliminate interactions with 14-3-3 and 14-3-3 (Fig. 3C).

View larger version (41K): [in this window] [in a new window]   Fig. 1. Interaction of 14-3-3 with RGS3. A, an HA-14-3-3 construct expressed in HEK293 cells was subjected to SDS-PAGE and immunoblotted with anti-HA antibody. B, lysates of HEK293 cells expressing the HA-14-3-3 construct were used in pull-down experiments using 6xHis-tagged forms of RGS3: no RGS3 (1), wild-type RGS3 (2), Ser496Asp RGS3 (3), and Ser496AlaRGS3 (4). Samples were resolved by SDS-PAGE and immunoblotted to detect HA-14-3-3.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Production of N- and C-terminal fragments of RGS3. A, RGS3 was split to generate N-terminal 1 to 370 and C-terminal 371 to 519 (containing the RGS domain sequence) fragments. Each of these was C-terminally tagged with the VSV-G epitope sequence to assist detection. Two serine residues that were mutated in the studies are highlighted. B, Full-length and the N- and C-terminal RGS3-VSV-G constructs were expressed in HEK293 cells, resolved by SDS-PAGE, and detected with anti-VSV-G antibody.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Interactions between 14-3-3 isoforms and RGS3 are not defined by the RGS domain. A, Western blots of pull-down experiments using lysates of HEK293 cells expressing RGS3-VSV-G, Ser496Asp RGS3-VSV-G, or Ser496Ala RGS3-VSV-G with 1 µM GST-tagged 14-3-3 or 14-3-3 were probed with anti-VSV-G antibody. B, equivalent pull downs from lysates of HEK293 cells expressing the RGS3-VSV-G N- and C-terminal (incorporating Ser496Asp and Ser496Ala mutations) fragments with GST-tagged 14-3-3 or 14-3-3 were probed with anti-VSV-G antibody. C, equivalent pull downs from lysates of HEK293 cells expressing wild-type RGS3-VSV-G and Ser264Asp and Ser264Ala mutants of RGS3-VSV-G. Input levels of the forms of RGS3 are shown as lysates.

We have constructed previously a fusion protein (_2A-AR-Cys³⁵¹IleG_o1) between the _2A-adrenoceptor and the subunit of the G protein G_o1 that has been rendered insensitive to the ADP-ribosyltransferase activity of pertussis toxin by altering the reactive cysteine residue to isoleucine (Cavalli et al., 2000). When expressed in HEK293 cells that were treated with pertussis toxin (25 ng/ml for 24 h) to produce ADP-ribosylation and inactivation of endogenously expressed members of the family of pertussis toxin-sensitive G_i/G_o G proteins, this fusion protein generated a large stimulation of high-affinity GTPase activity upon the addition of _2A-adrenoceptor agonists such as the natural ligand adrenaline (Fig. 4). This reflected an increase in the V_max of GTPase activity without an alteration in the apparent K_m value for GTP (Fig. 4A). The addition of recombinantly expressed 6xHis-RGS3 to membranes of pertussis toxin-treated HEK293 cells expressing _2A-AR-Cys³⁵¹IleG_o1 did not modify basal GTPase activity but resulted in a large elevation of adrenaline-stimulated high-affinity GTPase activity (Fig. 4Bi) that was not modified by the coaddition of GST-14-3-3 (Fig. 4Bi). Likewise, the presence of GST-14-3-3 did not modulate the stimulatory GAP effects of either 6xHis-Ser⁴⁹⁶Ala RGS3 or 6xHis-Ser⁴⁹⁶Asp RGS3 (Fig. 4B, ii and iii). However, both Ser⁴⁹⁶Ala RGS3 and Ser⁴⁹⁶Asp RGS3 were significantly less able to enhance the effects of adrenaline on the GTPase activity of _2A-AR-Cys³⁵¹IleG_o1 than wild-type RGS3 (Fig. 4B). These studies suggested a direct role of Ser⁴⁹⁶ in RGS3 in the GAP activity of this RGS rather than an indirect role involving interactions with 14-3-3 isoforms. To demonstrate that the 6xHis-RGS3 and GST-14-3-3 recombinant proteins were able to interact when both were present in the GTPase assay, pull-down experiments were carried out as shown in (Fig. 4C). The 6xHis-RGS3 was able to pull down GST-14-3-3, and this interaction was unaffected by treatment with the PKC isoform. PKC, however, was able to cause phosphorylation of the 6xHis-RGS3, but Ser⁴⁹⁶ was not a key site for phosphorylation because the extent of incorporation of ³²P into 6xHis-RGS3 was unaffected by the Ser⁴⁹⁶Asp mutation (Fig. 4D). To further explore the role of Ser⁴⁹⁶, we added different amounts of purified 6xHis wild-type RGS3, Ser⁴⁹⁶Ala RGS3, and Ser⁴⁹⁶Asp RGS3 to membranes of pertussis toxin-treated HEK293 cells expressing _2A-AR-Cys³⁵¹IleG_o1 and examined the effects on high-affinity GTPase activity stimulated by a maximally effective concentration (10 µM) of adrenaline. Each of the forms of RGS3 enhanced adrenaline-stimulated GTPase activity in a concentration-dependent manner, but wild-type RGS3 was clearly more effective than equimolar amounts of either Ser⁴⁹⁶Ala RGS3 or Ser⁴⁹⁶Asp RGS3 (Fig. 4E).

View larger version (30K): [in this window] [in a new window]   Fig. 4. Effects on RGS3 on adrenaline-stimulated GTPase activity of a 2-AR-Cys351IleGo1 fusion protein. A, high-affinity GTPase assays were performed at varying concentrations of GTP on membranes prepared from HEK293 cells expressing 2-AR-Cys351IleGo1 (closed symbols, basal activity; open symbols, stimulated with 10 µM adrenaline). Data are presented as an Eadie-Hofstee transformation. In the experiment shown, Vmax values were 18.6 and 100.5 pmol/mg/min for basal and stimulated activity, respectively. Corresponding Km values were 321 and 367 nM, respectively. B, high-affinity GTPase activity measured at 0.5 µM GTP of membranes prepared from cells expressing an 2-AR-Cys351IleGo1 fusion construct plus added 6xHis-RGS3 (i), 6xHis-Ser496Asp RGS3 (ii), and 6xHis-Ser496AlaRGS3 (iii) (each at 0.5 µM). 1, basal; 2, stimulated with 10 µM adrenaline; 3, basal + RGS; 4, 10 µM adrenaline + RGS; 5, 10 µM adrenaline + RGS + GST-14-3-3. GST-14-3-3 was present at 1 µM. C, Western blot of a pull-down experiment between GST-14-3-3 and 6xHis-RGS3 or 6xHis-RGS3 phosphorylated by PKC [6His-RGS3(P)]. The 6xHis tags were pulled down with Ni-NTA agarose, and the blot was probed with anti-GST antibody. D, autoradiograph of a PKC phosphorylation experiment using 6xHis-RGS3 or 6xHis-Ser496Asp RGS3 as substrates. Reactions were initiated by the addition of enzyme dilution buffer (-) or diluted PKC (+) and were performed in the presence of 5 µCi [-32P]ATP to allow for the identification of the phosphorylated bands. E, GTPase activity measured at 0.5 µM GTP of membranes as in B with varying amounts of 6xHis-tagged RGS3 (), Ser496Asp RGS3 (), or Ser496Ala RGS3 (). Stimulation was done with 10 µM adrenaline.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Alteration of Ser173 in RGS1 and Ser166 in RGS16: effects on GAP activity. Adrenaline (10 µM)-stimulated high-affinity GTPase activity was measured at 0.5 µM GTP in membranes prepared from cells expressing the 2A-AR-Cys351IleGo1 fusion construct. Samples contained differing concentrations of GST-wild-type RGS1 (), GST-Ser173AspRGS1 (), and GST-Ser173Ala RGS1 () (A); and GST-wild-type RGS16 (), GST-Ser166Asp RGS16 (), and GST-Ser166Ala RGS16 () (B).

As with RGS3, the addition of GST-14-3-3 along with the GST-RGS16 proteins did not modify the ability of GST-RGS16 to function as a GAP, and this was true whether measuring the high-level GAP activity of GST wild-type RGS16 or the much lesser effect of Ser¹⁶⁶AspRGS16, in which we reasoned that the aspartic acid might act as a phosphoserine mimetic (Fig. 6), and this was true when the molar ratio of GST-RGS16 to GST-14-3-3 was varied between 1:1 and 1:10 (data not shown). Despite this and the lack of interactions between GST-14-3-3 and the C-terminal domain of RGS3-VSV-G containing the RGS box, interactions could be observed between HA-14-3-3 and GST wild-type RGS16 in pull-down studies (Fig. 7). Again, however, this was unchanged by the alteration of Ser¹⁶⁶ of RGS16 to either alanine or aspartic acid (Fig. 7). Previous studies on RGS16 have suggested an important role for Ser⁵³ in function (Chen et al., 2001). We thus also tested whether alteration of this serine would modify interactions with HA-14-3-3. However, alteration of Ser⁵³ to either alanine or glutamic acid did not inhibit the interaction and, indeed, seemed to improve the interaction as monitored in pull-down studies (Fig. 7).

View larger version (17K): [in this window] [in a new window]   Fig. 6. 14-3-3 does not inhibit the GAP effect of RGS16. Adrenaline (10 µM)-stimulated high-affinity GTPase activity was measured at 0.5 µM GTP in membranes prepared from cells expressing the 2A-AR-Cys351IleGo1 fusion. The effect of GST-14-3-3 on wild-type and Ser166Asp RGS16-stimulated activity was assessed at a 1:1 molar ratio (2 µM each) of the RGS and 14-3-3.

View larger version (49K): [in this window] [in a new window]   Fig. 7. 14-3-3 interacts with RGS16: pull-down studies. Lysates of HEK293 cells expressing HA-14-3-3 or vector-transfected controls were incubated with 0.5 µM GST fusions of wild-type RGS16 or point mutations at Ser53 or Ser166. GST pull downs were resolved by SDS-PAGE, and the presence of 14-3-3 was monitored using an anti-HA antibody.

As an alterative to adding recombinantly expressed forms of RGS proteins to membranes of HEK293 cells expressing the _2A-AR-Cys³⁵¹IleG_o1 target, we generated a series of fusion proteins between the _2A-adrenoceptor and various wild-type and mutant forms of the RGS proteins (Bahia et al., 2003; Ward and Milligan, 2004). These were then coexpressed in HEK293 cells along with the pertussis toxin-insensitive Cys³⁵¹IleG_o1 and membranes prepared from pertussis toxin-treated cells. When examining membranes coexpressing _2A-AR-RGS16 and Cys³⁵¹IleG_o1, as in membranes expressing _2A-AR-Cys³⁵¹IleG_o1 (Fig. 3), the addition of adrenaline resulted in a large increase in high-affinity GTPase activity. However, when GTPase activity was analyzed at a wide range of GTP concentrations, the characteristics of GTP hydrolysis were very distinct from those in membranes expressing only ₂-AR-Cys³⁵¹IleG_o1. Now, in addition to the large increase in GTPase V_max, a substantial increase in the measured K_m value for GTP was recorded (Fig. 8A). As discussed previously (Cavalli et al., 2000; Hoffmann et al., 2001; Cladman and Chidiac, 2002), these are exactly the characteristics expected when agonist stimulation of G protein GTPase activity is enhanced by the action of an RGS and proves the functionality of the RGS fused to the _2A-adrenoceptor in this construct as a GAP for Cys³⁵¹IleG_o1 (Bahia et al., 2003; Ward and Milligan, 2004). This effect required coexpression of the G protein. When membranes prepared from pertussis toxin-treated HEK293 cells expressing only _2A-AR-RGS16 were used to measure high-affinity GTPase activity, no effect of adrenaline could be observed (Fig. 8B). To extend this work, we also generated _2A-AR-Ser¹⁶⁶AlaRGS16 and _2A-AR-Ser¹⁶⁶AspRGS16 fusions. When _2A-AR-Ser¹⁶⁶AlaRGS16 was coexpressed with Cys³⁵¹IleG_o1, the addition of adrenaline generated data equivalent to _2A-AR-RGS16 in that both GTPase V_max and the measured K_m values for GTP increased greatly (Fig. 9A). In total contrast, when Cys³⁵¹IleG_o1 was coexpressed with _2A-AR-Ser¹⁶⁶AspRGS16, adrenaline was able to stimulate GTPase V_max only weakly, and there was no increase in the measured K_m value for GTP (Fig. 9B), confirming the virtual lack of GAP activity of Ser¹⁶⁶AspRGS16 noted earlier for the recombinant version of this protein. Expression levels of the _2A-AR-RGS16 fusions varied between 12 and 15 pmol/mg protein in individual transfections (for the actual values, see figure legends 8 and 9). As expected from the above, the addition of recombinant 14-3-3 to membranes coexpressing _2A-AR-RGS16 and Cys³⁵¹IleG_o1 was without effect on either basal or adrenaline-stimulated GTPase activity (Fig. 10A) and this was also the case for the limited effect of adrenaline in membranes coexpressing _2A-AR-Ser¹⁶⁶AspRGS16 and Cys³⁵¹IleG_o1 (Fig. 10B).

View larger version (15K): [in this window] [in a new window]   Fig. 8. An 2A-adrenoceptor-RGS16 fusion protein acts as both a GEF and a GAP for Cys351IleGo1. Basal () and adrenaline (10 µM)-stimulated () high-affinity GTPase assays was measured at varying concentrations of GTP in membranes of pertussis toxin-treated HEK293 cells expressing 2A-AR-RGS16 with (A) or without (B) coexpressed Cys351IleGo1. The inset in A represents an Eadie-Hofstee transformation of the data to allow the visual inspection of the alteration in the apparent Km value for GTP. The Vmax and Km values calculated by linear regression were: for A, basal activity, Vmax = 23.8 ± 1.7 pmol/mg/min, and Km = 331 ± 43 nM; adrenaline-stimulated, Vmax = 116.2 ± 10.2 pmol/mg/min, and Km = 712 ± 94 nM; for B, basal activity, Vmax = 8.6 ± 0.9 pmol/mg/min, and Km = 306 ± 60 nM; adrenaline-stimulated, Vmax = 9.6 ± 0.8 pmol/mg/min, and Km = 386 ± 60 nM. Expression levels of the fusion constructs in these membranes were determined by ligand binding studies using [3H]RS-79948-197 (Ward and Milligan, 2004) 2A-AR-RGS16/Cys351IleGo1, 11.9 pmol/mg (A) or 2A-AR-RGS16, 15.0 pmol/mg (B).

View larger version (19K): [in this window] [in a new window]   Fig. 9. Altering Ser166 in RGS16: the effects on GAP activity against Cys351IleGo1. Basal () and adrenaline (10 µM)-stimulated () high-affinity GTPase assays was measured at varying concentrations of GTP in membranes of pertussis toxin-treated HEK293 cells expressing Cys351IleGo1 and either 2A-AR-Ser166AlaRGS16 (A) or 2A-AR-Ser166AspRGS16 (B). The insets represent Eadie-Hofstee transformations of the data to allow visual inspection of any alteration in the apparent Km for GTP. The Vmax and Km values calculated by linear regression were: for A, basal activity, Vmax = 21.2 ± 1.3 pmol/mg/min, and Km = 343 ± 37 nM; adrenaline-stimulated, Vmax = 156.5 ± 11.2 pmol/mg/min, and Km = 1140 ± 111 nM; for B, basal activity, Vmax = 27.5 ± 1.9 pmol/mg/min, and Km = 323 ± 40 nM; adrenaline-stimulated, Vmax = 34.4 ± 2.0 pmol/mg/min, and Km = 277 ± 30 nM. Expression levels of the fusion constructs in these membranes were determined as in Fig. 8: 2A-AR-Ser166AlaRGS16/Cys351IleGo1, 15.7 pmol/mg (A), and 2A-AR-Ser166AspRGS16/Cys351IleGo1, 14.3 pmol/mg (B).

View larger version (16K): [in this window] [in a new window]   Fig. 10. 14-3-3 does not regulate the GAP activity of 2A-AR-RGS16. Basal and adrenaline (10 µM)-stimulated high-affinity GTPase assays were performed at 0.5 µM GTP in membranes prepared from HEK293 cells coexpressing Go1C351I with either 2A-AR-wild-type RGS16 (top) or 2A-AR-Ser166AspRGS16 (bottom). GST-14-3-3 was present at 2.6 µM.

Given previous reports of an effect of 14-3-3 isoforms on the GAP activity of RGS7 (Benzing et al., 2000, 2002), we explored the effects of each of wild type, Ser³⁷⁹Ala and Ser³⁷⁹Asp RGS7, on basal and adrenaline-stimulated GTPase activity of the _2A-AR-Cys³⁵¹IleG_o1 fusion protein. Mirroring other results, wild-type RGS7 and Ser³⁷⁹Ala RGS7 produced a large enhancement of this activity that was unaffected by the coaddition of either 14-3-3 or 14-3-3 (Fig. 11). Ser³⁷⁹Asp RGS7 was far less effective as a GAP for adrenaline-activated _2A-AR-Cys³⁵¹IleG_o1 (Fig. 11).

View larger version (16K): [in this window] [in a new window]   Fig. 11. RGS7 is a GAP for Cys351IleGo1, but this is unaffected by 14-3-3. Basal (1 and 3) and adrenaline (10 µM)-stimulated (2, 4–6) high-affinity GTPase assays were performed at 0.5 µM GTP in membranes prepared from HEK293 cells expressing the 2A-AR-Cys351IleGo1 fusion protein. Assays also contained forms of 6xHis-RGS7 (0.4 µM)(3–6) and GST-14-3-3 (5) or GST-14-3-3 (6) (each 0.5 µM). Top, 6xHis-wild-type RGS7; middle, 6xHis-Ser379Asp RGS7; bottom, 6xHis-Ser379Ala RGS7.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the current studies, we demonstrate and quantify the effects of mutating a totally conserved serine residue in the RGS domain of mammalian RGS proteins. On the basis of the atomic level structure of the RGS domain of RGS4 complexed with G_i1 (Tesmer et al., 1997), this serine (RGS4Ser¹⁶⁴) is in direct contact with the G protein and is part of a contact interface at the junction of helices 7 and 8 of the RGS domain. The effect of mutating this residue on GAP activity was assessed in two distinct assays. First, akin to other studies, wild-type or mutated RGS proteins were produced recombinantly in bacteria, and after purification and quantification, these proteins were added to membranes of HEK293 cells expressing a _2A-AR-Cys³⁵¹IleG_o1 fusion protein. The addition of adrenaline to membranes expressing the fusion protein resulted in a substantial stimulation of high-affinity GTPase activity, reflecting the activation of the receptor-associated G protein, and the addition of the purified RGS proteins markedly increased this agonist-induced activity as anticipated for an active RGS GAP. For RGS1 and RGS16, mutation of the serine residue to aspartic acid almost eliminated GAP activity, whereas mutation to alanine reduced the potency of the proteins as RGS GAPs some 20-fold. The serine-to-aspartic acid alterations could be related to aspartic acid acting as a phosphoserine mimetic, and if so, then phosphorylation of this serine would be anticipated to be highly detrimental to GAP activity. Of course, bacterially expressed proteins are not modified post-translationally in this way; thus, the second assay used the coexpression of proteins in HEK293 cells. When heterologously expressed in mammalian cells, substantial amounts of RGS proteins are not effectively targeted to the plasma membrane (Chidiac and Roy, 2003). Likewise, the mutation of proteins frequently alters steady-state expression levels. To limit both of these potential issues, we generated fusion proteins in which RGS16, as a model, was fused to the C-terminal tail of the _2A-adrenoceptor, akin to the _2A-AR-Cys³⁵¹IleG_o1 construct, and as we have done previously for RGS4 (Bahia et al., 2003). The equivalent fusions incorporating Ser¹⁶⁶AlaRGS16 and Ser¹⁶⁶AspRGS16 were also produced and expressed in HEK293 cells with or without Cys³⁵¹IleG_o1, which was anticipated to act as a target for both elements of the receptor RGS fusions, the _2A-adrenoceptor as a guanine nucleotide exchange factor (GEF), and the RGS16 as a GAP. Analysis of agonist-stimulated GTP hydrolysis at different concentrations of GTP confirmed the functionality of both the receptor and wild-type RGS16 in this fusion because both GTPase V_max and apparent K_m for GTP increased greatly (Bahia et al., 2003; Ward and Milligan, 2004). As with the recombinant RGS studies, the receptor element of the _2A-AR-Ser¹⁶⁶AspRGS16 fusion was able to act as a GEF for Cys³⁵¹IleG_o1 because adrenaline-stimulated high-affinity GTPase activity was present. However, again in this assay, Ser¹⁶⁶AspRGS16 GAP activity was negligible. These studies confirmed a key role for Ser¹⁶⁶ in RGS16, and in this case, if Ser¹⁶⁶Asp is a phosphoserine mimetic, then Ser¹⁶⁶ is not constitutively phosphorylated to a significant extent, despite the protein being expressed in mammalian cells. It was noted that the RGS1 Ser¹⁷³Ala mutant displayed a higher V_max value than the wild-type RGS1. Although a definitive explanation for this cannot be provided, on the basis of the X-ray structure of RGS4 complexed with G_i1 (Tesmer et al., 1997), this may be caused by the pivotal position that this residue occupies. Although the RGS serine-to-alanine mutants made in the course of this study all displayed reduced potency, unlike the serine-to-aspartic acid mutants, at sufficiently high concentrations, they each display maximal activity at least as good as the wild-type RGS. In the case of RGS1, the mutation may therefore modify the contact between the RGS and G to improve efficacy.

A limitation of the studies using RGS3 and RGS7 is that they are relatively large proteins, and thus levels of expression of recombinant forms of these two polypeptides and the various mutants were insufficient to generate enough protein to perform full concentration-response curves to measure their potency as GAPs against Cys³⁵¹IleG_o1. It was clear, however, that RGS3 was significantly less potent as a GAP in such assays than either RGS1 or RGS16. We have reported previously differences in the capacities of RGS1, RGS16, and RGS-G-interacting protein to enhance _2A-adrenoreceptor agonist-stimulated GTPase activity of G_o1 (Hoffmann et al., 2001), and in a similar vein, Hooks et al. (2003) recently noted that RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of G_i family G proteins with differential selectivity and maximal activity, whereas Tovey and Willars (2004) have noted that RGS2, RGS3, and RGS4 differentially regulate signaling via the M₃ muscarinic acetylcholine receptor. This is not inherently surprising, and although many RGS proteins are coexpressed (Mittmann et al., 2002), because there are selective expression patterns of RGS family members (Cho et al., 2003) and regulation of their levels by a range of cellular challenges (Grant et al., 2000; Taymans et al., 2003; Cho et al., 2004; Riddle et al., 2005), RGS proteins may be potential targets for therapeutic intervention (Zhong and Neubig, 2001; Cho et al., 2004; Riddle et al., 2005).

Recent studies have shown certain RGS proteins, including RGS3 and RGS7, to interact with 14-3-3 proteins, and at least for RGS7, this interaction has been reported to inhibit GAP function (Benzing et al., 2000). It is interesting that the amino acid reported to be key to this interaction is the serine equivalent to Ser¹⁶⁶ in RGS16. If interactions of an RGS with a 14-3-3 protein and a G protein subunit were both to require this serine, and hence, to be mutually exclusive, this would provide a potential explanation for the reported 14-3-3 protein inhibition of RGS GAP activity. Furthermore, because interactions with 14-3-3 proteins generally require a phosphorylated amino acid, then a serine is an obvious potential target. We confirmed in pull-down studies that RGS3 can interact with both 14-3-3 and 14-3-3. The work of Niu et al. (2002) indicates that the interaction of RGS3 with 14-3-3 is mediated by a residue within the N-terminal extension of the RGS3 and outside of the RGS box. This residue, Ser²⁶⁴, was identified by yeast two-hybrid screening, followed by deletion and point mutational analysis. In agreement with this study, Niu et al. (2002) found that Ser²⁶⁴Ala abolished binding of 14-3-3 to RGS3.

14-3-3 did not alter the GAP activity of RGS3 against the _2A-AR-Cys³⁵¹IleG_o1 fusion protein, and this was true also for the Ser⁴⁹⁶ RGS3 mutants, even when the molar ratio of 14-3-3 to RGS proteins was increased to 10:1, although such interactions are expected to be stoichiometric. Although pull-down studies did indicate a degree of interaction between the 14-3-3 and both RGS16 and RGS1, there was no effect of the coaddition of 14-3-3 to assays that measured the GAP activity of RGS16.

In conclusion, although a central role of this fully conserved serine residue for the GAP function of RGS proteins is now abundantly clear, it seems that the reported effect of 14-3-3 proteins on RGS GAP activity is not general, and that as concluded previously for RGS3, this conserved serine is not the key amino acid for RGS/14-3-3 interactions. Examination of the atomic level structure of RGS4 complexed with G_i1 suggests that the effect of mutation of this conserved serine is to modulate interactions with both Thr¹⁸³ of G_o1 and intermolecular contacts with a functionally important and conserved asparagine in the RGS box. Given the key role of Thr¹⁸³ in the GTPase activity of G_o1, effects on GTPase activity and, hence, RGS GAP activity by modification of this serine, are easily explained at a molecular level and do not require interactions with 14-3-3.

	Footnotes

 
These studies were supported by the Wellcome Trust.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.015073.

ABBREVIATIONS: RGS, regulator of G protein signaling; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; PCR, polymerase chain reaction; HA, hemagglutinin; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Ni-NTA, nickel-nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; PKC, protein kinase C; 6xHis, hexahistidine; RS-79948-197, (8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine.

Address correspondence to: Dr. Graeme Milligan, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail: g.milligan{at}bio.gla.ac.uk

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