Structural Determinants of G-protein α Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2)

Abstract

“Regulator of G-protein signaling” (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of Gα subunits (known as GTPase-accelerating protein or “GAP” activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Gα_q subunits. As many vasoconstrictive hormones signal via G_q heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for Gα_q over Gα_i/o substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Gα_i-directed binding and GAP activities without perturbing its association with Gα_q. Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of Gα_i at 2.8-Å resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Gα complexes highlighted the roles of these residues in wild type RGS2 that weaken Gα_i subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective Gα_q GAP that modulates cardiovascular function.

G protein-coupled receptors (GPCRs)⁴ form an interface between extracellular and intracellular physiology, as they convert hormonal signals into changes in intracellular metabolism and ultimately cell phenotype and function (1–3). GPCRs are coupled to their underlying second messenger systems by heterotrimeric guanine nucleotide-binding protein (“G-proteins”) composed of three subunits: Gα, Gβ, and Gγ. Four general classes of Gα subunits have been defined based on functional couplings (in the GTP-bound state) to various effector proteins. G_s subfamily Gα subunits are stimulatory to membrane-bound adenylyl cyclases that generate the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP); conversely, G_i subfamily Gα subunits are generally inhibitory to adenylyl cyclases (4). G_12/13 subfamily Gα subunits activate the small G-protein RhoA through stimulation of the GEF subfamily of RGS proteins, namely p115-RhoGEF, LARG, and PDZ-RhoGEF (5). G_q subfamily Gα subunits are potent activators of phospholipase-Cβ enzymes that generate the second messengers diacylglycerol and inositol triphosphate (6); more recently, two additional Gα_q effector proteins have been described: the receptor kinase GRK2 and the RhoA nucleotide exchange factor p63RhoGEF (7, 8).

The duration of GPCR signaling is controlled by the time Gα remains bound to GTP before its hydrolysis to GDP. RGS proteins are key modulators of GPCR signaling by virtue of their ability to accelerate the intrinsic GTP hydrolysis activity of Gα subunits (reviewed in Refs. 9 and 10). RGS2/G0S8, one of the first mammalian RGS proteins identified (11) and member of the R4-subfamily (10), has a critical role in the maintenance of normostatic blood pressure both in mouse models (12, 13) and in humans (14, 15); additionally, Rgs2-deficient mice exhibit impaired aggression and increased anxiety (16, 17), behavioral phenotypes with potential human clinical correlates (18, 19).

Although many RGS proteins are promiscuous and thus act on multiple Gα substrates in vitro (e.g. Ref. 20), RGS2 exhibits exquisite specificity for Gα_q in biochemical binding assays and single turnover GTPase acceleration assays (20, 21). Consistent with this in vitro selectivity,⁵ mice deficient in RGS2 uniquely exhibit constitutive hypertension and prolonged responses to vasoconstrictors, as would be expected upon loss of a potent negative regulator of Gα_q that mediates signaling from various vasoconstrictive hormones such as angiotensin II, endothelin, thrombin, norepinephrine, and vasopressin (22). In addition, RGS2-deficient mice respond to sustained pressure overload with an accelerated time course of maladaptive cardiac remodeling (23), a pathophysiological response that evokes myocardial hypertrophy known to be critically dependent on Gα_q signaling (24, 25).

To gain insight into the structural basis of the unique Gα substrate selectivity exhibited by RGS2, a series of point mutants in RGS2 were evaluated that enable this protein to bind and accelerate GTP hydrolysis by Gα_i; we subsequently delineated the structural determinants of the Gα_i/mutant RGS2 interaction using x-ray crystallography. Three key positions, first identified by Heximer and colleagues (21) and highlighted in our structural studies as key determinants of RGS2 substrate selection, were also found to be conserved throughout the evolution of the RGS2 protein in a manner suggestive of specialization toward cardiovascular signaling modulation.

EXPERIMENTAL PROCEDURES

Chemicals and Assay Materials

Unless otherwise noted, all chemicals were the highest grade available from Sigma or Fisher Scientific (Pittsburgh, PA).

Protein Expression and Purification

Using ligation-independent cloning, DNA encoding human RGS2 (Lys⁷¹–His²⁰⁹), fused to either hexahistidine alone (His₆) or to His₆-tagged enhanced yellow fluorescent protein (YFP), was hybridized into a Novagen (San Diego, CA) pET vector-based prokaryotic expression construct as previously described (26, 27). Point mutations corresponding to Cys¹⁰⁶ to serine (C106S), Asn¹⁸⁴ to aspartate (N184D), Arg¹⁸⁸ to glutamate (R188E), and Glu¹⁹¹ to lysine (E191K) were made using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA). For expression of hexahistidine- and His₆-YFP fusion RGS2 constructs, BL21(DE3) Escherichia coli were grown to an A_{600 nm} of 0.7–0.8 at 37 °C before induction with 0.5 mm isopropyl β-d-thiogalactopyranoside. After culturing for 14–16 h at 20 °C, cells were pelleted by centrifugation and frozen at −80 °C. Bacterial pellets were then resuspended in N1 buffer (50 mm HEPES, pH 8.0, 400 mm NaCl, 30 mm imidazole, 5% (w/v) glycerol) and lysis of bacterial slurry was performed using an Emulsiflex (Avestin, Ottawa, Canada) according to the manufacturer's instructions. Cellular lysates were clarified by centrifugation at 100,000 × g for 30 min at 4 °C. The supernatant was applied to a Ni²⁺ chelating fast protein liquid chromatography column (FF HisTrap; GE Healthcare, Piscataway, NJ), washed with 7 column volumes of N1 buffer then 3 column volumes of N1 buffer containing an additional 30 mm imidazole before elution of recombinant RGS2 protein with N1 buffer containing 300 mm imidazole. His₆-tagged RGS2 protein was cleaved with tobacco etch virus protease overnight at 4 °C and dialyzed into N1 buffer containing 5 mm dithiothreitol. To separate residual His₆-RGS2 from untagged, cleaved RGS2, the protein was passed over a second Ni²⁺-chelating fast protein liquid chromatography column. The flow-through was pooled, concentrated to final volume of ∼5 ml, and resolved using a calibrated 150-ml size exclusion column (Sephacryl S200; GE Healthcare) using S200 buffer (10 mm HEPES pH 8.0, 300 mm NaCl, 5 mm dithiothreitol, 5% (w/v) glycerol). Fractions containing monodisperse protein were then pooled and concentrated to ∼500 μm, as determined by A_{280 nm} measurements upon denaturation in 8 m guanidine hydrochloride. Concentration was calculated based on the predicted extinction coefficient (ProtParam, Swiss Institute for Bioinformatics). Additional details regarding protein purification for crystallography can be found online at the SGC Oxford website. Human RGS16 constructs, C-terminal biotinylated Gα_i1, N-terminal deleted (ΔN30) Gα_i1, CFP-Gα_i1, and Gα_i3 were purified exactly as previously described (20, 28, 29).

Single Turnover GTPase Assays

Single turnover [γ-³²P]GTP hydrolysis assays were conducted using recombinant Gα_i1 and various concentrations of RGS proteins as previously described (20). Briefly, 100 nm Gα_i1 in reaction buffer (50 mm Tris pH 7.5, 0.05% C₁₂E₁₀, 1 mm dithiothreitol, 10 mm EDTA, 100 mm NaCl, and 5 μg/ml bovine serum albumin) was incubated for 10 min at 30 °C with 1 × 10⁶ cpm (2 nm) of [γ-³²P]GTP (specific activity of 6500 dpm/Ci). The reaction was then chilled on ice for 5 min prior to the addition of 10 mm MgCl₂ and 100 μm unlabeled GTPγS (final concentration) with or without added RGS protein. Reactions were kept on ice and 100-μl aliquots were taken at the indicated time points, quenched in 900 μl of charcoal slurry, and centrifuged before 600-μl aliquots of supernatant were counted via liquid scintillation.

Surface Plasmon Resonance

Optical detection of protein-protein interactions by surface plasmon resonance (SPR) was performed using a Biacore 3000 (GE Healthcare) exactly as previously described (20, 29, 30).

Förster Resonance Energy Transfer (FRET)-based Binding Assays

Förster resonance energy transfer was used to measure binding between Gα_i1 and the triple point mutant RGS2 (C106S,N184D,E191K) as previously described (26, 28). In brief, FRET between recombinant Gα_i1-CFP and YFP-RGS2(C106S,N184D,E191K) proteins was measured using a SpectraMax Gemini fluorescence reader (Molecular Devices, Sunnyvale, CA) using an excitation wavelength of 433 nm (455 nm cutoff) and emission scans from 470 to 535 nm at 2-nm intervals.

Structure Determination

Purified Gα and RGS2(C106S, N184D,E191K) proteins were mixed at a molar ratio of 1:1.5 and incubated at 4 °C for 20 min. The sample was passed through an S200 gel filtration column pre-equilibrated with 25 mm HEPES, pH 7.5, 150 mm NaCl, 5% glycerol, 2 mm dithiothreitol, 100 μm AlCl₃, 20 mm sodium fluoride, and 100 μm GDP. Protein fractions that eluted as a complex were identified using SDS-PAGE and the fractions were pooled and concentrated to 23 mg/ml prior to crystallization condition screens using a 150-nl drop volume with an TTP Labtech Mosquito nanoliter liquid-handling system. The crystal of the RGS2(C106S,N184D,E191K)-Gα_i3 complex used for data collection was crystallized by vapor diffusion in sitting drops of 400 nl of protein and 200 nl of reservoir solution containing 0.1 m HEPES, pH 7.5, and 2 m ammonium sulfate (TTP Labtech Mosquito).

After cryoprotection in a solution of 2 m ammonium sulfate, 0.1 m HEPES, pH 7.5, and 20% (w/v) d-glucose, crystals were flash cooled in liquid nitrogen. A complete data set was collected at 100 K on a Rigaku/MSC FR-E rotating anode x-ray generator equipped with an R-AXIS HTC image plate detector. Diffraction images were evaluated with MOSFLM (31), and data were scaled using SCALA (32). The crystal belonged to the space group P3₂21 with unit cell dimensions a = 114.54 Å, b = 114.54 Å, and c = 99.33 Å. A molecular replacement solution was found in this space group using PHASER (33) with the RGS10/Gα_i3 complex (PDB code 2IHB) as the search model. The RGS2 coordinates from PDB code 2AF0 were superimposed onto the RGS10 coordinates of the RGS10/Gα_i3 positioned complex and rigid body refinement into the electron density was performed using REFMAC5 (34). Difference density in the GDP binding site was modeled using the higher resolution structure of Gα_i3 in the RGS8/Gα_i3 complex (PDB code 2ODE) with one molecule of GDP, a tetrafluoroaluminate ion, and a magnesium ion coordinated by two additional water molecules. Several rounds of manual rebuilding in COOT (35) and restrained refinement with REFMAC5 (34), using Translation/Libration/Screw (TLS) groups calculated with TLSMD (36), resulted in the final structural model described in Table S1. Coordinates of the RGS2(C106S,N184D,E191K)-Gα_i3 complex were deposited in the Protein Data Bank with entry code of 2V4Z.

Cellular cAMP Signaling Assays

HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) in 6-well dishes with 4 μg of total DNA including pGloSensor^TM-20F cAMP biosensor plasmid (Promega Corp., Madison WI), dopamine D2 receptor, and empty vector, HA-RGS2(WT), or HA- RGS2(C106S,N184D,E191K). The RGS2 expression vectors encoded solely the RGS domain (amino acid Lys⁷¹–His²⁰⁹; with an N-terminal HA epitope tag) to avoid the influence of non-RGS domain regions on adenylyl cyclase function (e.g. Ref. 37). Twenty-four hours post-transfection, cells were re-plated on poly-d-lysine-treated, clear-bottom, white 96-well plates at a density of 60,000 cells/well. Forty-eight hours post-transfection, culture medium was aspirated and cells were washed once with assay medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum (without phenol), 15 mm HEPES, pH 7.4) before being incubated for 2 h with 40 μl/well of equilibration medium (assay medium with 4% GloSensor^TM substrate). After 2 h, 6.6 μl of 6× final concentration of quinpirole (diluted in 10 μm forskolin-containing assay medium) was added to each well and allowed to incubate for 10 min before GloSensor emission was read on a MicroBeta Plate Counter (PerkinElmer). Before plotting, luminescence counts were normalized to 100% maximal response for each condition to account for variability in GloSensor expression, transfection efficiency, and the exact number of cells per well.

RESULTS AND DISCUSSION

Evaluating Point Mutations to RGS2 That Facilitate Interaction with Gα_i1

RGS2 is the only member of the R4-subfamily known to bind specifically to Gα_q and not to Gα_i/o heterotrimeric G-protein subunits in vitro (20, 21). Three amino acids within RGS2 were identified by Heximer and colleagues (21) as potential selectivity determinants in studies of Gα_o-directed GAP activity by RGS domain chimera derived from RGS2 and RGS4 sequences: namely, cysteine 106, asparagine 184, and glutamate 191. In the present study, we mutated these three amino acids to the highly conserved corresponding amino acids in R4-subfamily members (Cys¹⁰⁶ to serine, Asn¹⁸⁴ to aspartate, and Glu¹⁹¹ to lysine; supplementary Fig. S1) to identify their respective contributions to Gα substrate specificity.

RGS2 proteins containing these point mutations, either singly, in tandem, or all three together, were expressed in E. coli and purified to homogeneity (Fig. S2). SPR spectroscopy was used (e.g. Fig. 1) to assess if any individual mutation, or combination of point mutations, was capable of changing the selectivity of RGS2. All mutants retained wild type binding toward Gα_q (e.g. Fig. 1B). Single mutations to RGS2 (C106S, N184D, or E191K) did not enhance binding to Gα_i1 and only minimal enhancements to binding were observed with the C106S,N184D, C106S,N191D, and E191K,N184D double mutants (e.g. Fig. 1A); in contrast, the triple mutant RGS2 exhibited a dramatic increase in Gα_i1 binding versus wild type RGS2. Although the magnitude of binding of the RGS2 double mutants was significantly less than that observed with the triple mutant, binding isotherms were nonetheless generated for all double mutants along with the triple mutant by injecting increasing concentrations of RGS2 protein over the Gα_i1·GDP·AlF₄⁻ surface. Using equilibrium binding analyses (Fig. 2), dissociation constants (K_D values) for the RGS2/Gα_i1·GDP·AlF₄⁻ interaction were estimated to be ≥5.3, ≥8.6, and ≥21.1 μm, for C106S,N184D, E191K,N184D, and C106S,E191K, respectively, whereas the K_D value was determined to be 1.25 μm for the RGS2(C106S,N184D,E191K) triple mutant. Dissociation constants derived for the RGS2 double mutants are likely underestimated given an inability to attain saturating concentrations of these particular RGS2 analytes and thereby attain maximal binding (B_max).

FIGURE 1.

Gα_i1 and Gα_q selectivity of wild type RGS2 versus RGS2 point mutants profiled by SPR. Gα_i1-biotin (left) or His₆-Gα_q (right) were immobilized on sensor surfaces for binding analyses of the indicated RGS2 protein analytes (3 μm final concentration). Analyte injections were performed at a flow rate of 20 μl/min for 600 s (start time = 0 s) over surfaces of Gα subunits in the inactive state (GDP-bound; dashed lines) or in the transition state for GTP hydrolysis (i.e. GDP·AlF₄⁻-bound; solid lines). Legend in panel A also applies to sensorgrams of panel B.

FIGURE 2.

Quantitation of RGS2 binding to Gα_i1. SPR was performed as described in the legend to Fig. 1, with the concentration of the RGS2 analyte titrated from 1 nm to 50 μm. Sensorgrams were subsequently used in equilibrium saturation binding analyses to determine RGS2/Gα_i1 interaction binding affinities. Dissociation constants (K_D values) were estimated to be ≥21.1 (95% CI, 11.6–30.7 μm), ≥5.3 (95% CI 3.1–7.5 μm), and ≥8.6 (95% CI 5.4–11.9 μm) for the double mutants RGS2(C106S,E191K), RGS2(C106S,N184D), RGS2(N184D,E191K), respectively, and determined to be 1.25 (95% CI, 1.0–1.6 μm) for the triple mutant RGS2(C106S,N184D,E191K). A K_D value for the wild type RGS2/Gα_i1 interaction could not be estimated because saturation was not obtained at concentrations tested.

To determine whether the enhanced affinity of the RGS2 triple mutant was the result of improvements to a canonical RGS domain/Gα interaction interface, a highly conserved, surface-exposed arginine within this canonical interface (Arg¹⁸⁸ in the αVIII helix; Fig. S1) was mutated to glutamic acid. As has been shown for other RGS proteins (38), this single charge-reversal point mutation (R188E) on the Gα-binding surface of the RGS2 triple mutant abolished binding to Gα_i1·GDP·AlF₄⁻ (Fig. 2B, bottom panel).

To quantify any difference in the ability of the RGS2(C106S,N184D,E191K) triple mutant to bind Gα_q, increasing concentrations of wild type RGS2 and RGS2 triple mutant proteins were separately injected over an immobilized Gα_q·GDP·AlF₄⁻ surface (Fig. 3). Dissociation constants were determined to be 55 nm (95% confidence interval (CI) of 23–87 nm) and 17 nm (95% CI, 9–27 nm) for wild type RGS2- and RGS2(C106S,N184D,E191K)-bound Gα_q, respectively.

FIGURE 3.

Quantification of RGS2 binding to Gα_q. SPR was performed as described in the legend to Fig. 1, using an immobilized His₆-Gα_q·GDP·AlF₄ surface and RGS2 analyte concentrations from 0.5 to 1000 nm. Using equilibrium saturation binding analyses, RGS2/Gα_q dissociation constants were determined to be 55 nm (95% CI, 23.4–86.9 nm) for wild type RGS2 and 17 nm (95% CI, 8.7–27.0 nm) for the RGS2(C106S,N184D,E191K) triple mutant.

To confirm these SPR-derived results with an orthogonal technique of assessing the RGS domain/Gα interaction, FRET measurements were performed using a YFP-RGS2 (C106S,N184D,E191K)/Gα_i1-CFP pair, similar to the RGS4/Gα_i1 interaction FRET assay we have previously described (28). In the presence of GDP, aluminum tetrafluoride, and Mg²⁺ (“AMF”), binding between RGS protein and the Gα subunit is observed as an increase in YFP emission and decrease in CFP emission; in the presence of GDP alone, no binding is observed as expected (28, 39) and so the ratio of YFP to CFP emission remains low. The relative affinities of wild type RGS2, RGS16, and RGS2 triple mutant were assessed by using this FRET binding assay in a competitive manner: unlabeled RGS protein was added in increasing amounts to a fixed concentration of YFP-RGS2(C106S,N184D,E191K) and Gα_i1-CFP proteins. As expected, only unlabeled RGS2(C106S,N184D,E191K) and RGS16 proteins were able to inhibit the binding of the RGS2(C106S,N184D,E191K)/Gα_i1 FRET pair (Fig. 4), with observed IC₅₀ values of 526 nm (95% CI, 236–1171 nm) and 115 nm (78–168 nm), respectively. At no concentration tested was wild type RGS2 able to inhibit binding of the RGS2(C106S,N184D,E191K)/Gα_i1 FRET pair (Fig. 4B), consistent with the lack of affinity between wild type RGS2 and Gα_i subunits seen in our present SPR analyses and previously published studies (20, 21).

FIGURE 4.

Competition FRET assays of the Gα_i1-CFP/YFP-RGS2(triple) interaction. A, the fusion proteins YFP-RGS2(C106S,N184D,E191K) and Gα_i1-CFP interact in the presence of GDP and AlF₄·Mg²⁺ (“AMF”) but not in the presence of GDP alone. This interaction can be inhibited by the addition of unlabeled RGS2(C106S,N184D,E191K) “triple” mutant protein (IC₅₀ value of 526 nm; 95% CI, 236–1171 nm), but not by the addition of buffer alone. B, the addition of unlabeled wild type RGS2 to the Gα_i1-CFP/YFP-RGS2(triple mutant) FRET pair does not result in a decrease in FRET; however, the addition of RGS16 (known to have affinity for Gα_i1 (20) competitively inhibits binding (IC₅₀ value of 115 nm; 95% CI, 78–168 nm).

Determinants of RGS2 GAP Activity on Gα_i1 in Vitro

Using SPR and FRET, we demonstrated that all three point mutations were required to facilitate high affinity binding of RGS2 to Gα_i1. To determine whether this enhanced binding affected the ability of RGS2 to accelerate GTP hydrolysis by Gα_i1, we performed single turnover GTPase assays with both wild type and triple mutant RGS2 proteins (Fig. 5). At no concentration tested was wild type RGS2 capable of increasing GTP hydrolysis over the intrinsic GTP hydrolysis rate of Gα_i1 (Fig. 5A). In contrast, a substoichiometric amount of RGS16 (a known Gα_i1 GAP; Ref. 40) was able to accelerate Gα_i1 GTPase activity; complete hydrolysis of bound GTP was observed in less than 15 s at 0 °C. Unlike wild type RGS2, the RGS2(C106S,N184D,E191K) triple mutant was able to increase the rate of Gα_i1 GTP hydrolysis in a dose-dependent manner (Fig. 5B); however, adding the R188E mutation to the triple mutant resulted in a complete loss in GAP activity, consistent with the loss of Gα_i1 binding observed in SPR and FRET assays. To further confirm that the mechanism of action of the RGS2(C106S,N184D,E191K) triple mutant in increasing GTP hydrolysis by Gα_i1 was related to a canonical RGS domain/Gα interaction and not the inadvertant addition of a contaminating GTPase, we assessed the effects of both RGS2(C106S,N184D,E191K) and RGS16 proteins on an RGS-insensitive Gα_i1 point mutant: specifically, G183S in the Gα switch I region (41). Neither RGS2(C106S,N184D,E191K) nor RGS16 proteins were able to increase the intrinsic rate of GTP hydrolysis exhibited by this RGS-insensitive Gα_i1 (Fig. 5, C and D).

FIGURE 5.

The triple mutant RGS2(C106S,N184D,E191K), but not wild type RGS2, accelerates the GTP hydrolysis rate of Gα_i1. A, increasing concentrations of wild type RGS2 (as indicated) are unable to accelerate the GTP hydrolysis of 200 nm Gα_i1. Intrinsic GTP hydrolysis by isolated Gα_i1 (k_obs) was measured at 0.0075 s⁻¹ (95% CI 0.0055–0.010 s⁻¹), whereas k_obs values of 0.0076 (0.0055–0.0097), 0.0066 (0.0054–0.0078), and 0.0086 (0.0069–0.010) s⁻¹ were observed upon the addition of 50, 2500, or 5000 nm wild type RGS2, respectively. RGS16 is a potent GAP for Gα_i subunits (e.g. Ref. 20) and, at substoichiometic concentrations (50 nm), was found to accelerate GTP hydrolysis by Gα_i1: k_obs of at least 0.18 s⁻¹ (an underestimate as the measurement is limited by sampling time constraints). B, the triple mutant RGS2(C106S,N184D,E191K) was observed to accelerate GTP hydrolysis by 200 nm Gα_i1 in a dose-dependent manner: k_obs values of 0.0075 (0.0055–0.0095), 0.0079 (0.0068–0.0089), and 0.028 (0.023–0.032) s⁻¹ were observed upon the addition of 0, 10, and 50 nm RGS2(triple) protein, respectively. Higher concentrations of RGS2(triple) protein (200, 500, 1000, and 5000 nm) led to GTPase rates of at least 0.1–0.2 s⁻¹ (again underestimated due to sampling time constraints). The triple mutant also containing a fourth, loss-of-function point mutation (namely, RGS2(C106S,N184D,E191K,R188E)) was unable to accelerate GTP hydrolysis by Gα_i1: with a k_obs value of 0.0076 (0.0066–0.0086) s⁻¹. C, the single point mutation to Gα_i1 (glycine 183 to serine, G183S (41)) renders Gα_i1 insensitive to the GAP activity of RGS proteins. The intrinsic hydrolysis rate of the Gα_i1(G183S) mutant was determined to be 0.0053 (0.0037–0.0069) s⁻¹. Upon addition of 200, 3000, or 5000 nm of the RGS2(C106S,N184D,E191K) triple mutant, the k_obs was found to be 0.0036 (0.0026–0.0046), 0.0042 (0.0060–0.0078), and 0.0025 (0.00017–0.0048) s⁻¹, respectively; the k_obs for GTP hydrolysis after addition of 200 nm RGS16 was observed to be 0.0064 (0.0052–0.0076) s⁻¹. D, the k_obs values are plotted versus concentration of RGS protein to demonstrate the dose-dependent increase in GAP activity upon the addition of RGS2(C106S,N184D,E191K) protein to wild type Gα_i1, but not the RGS-insensitive Gα_i1(G183S) mutant.

Determinants of RGS2 Activity on G_i-coupled GPCR Signaling in Cells

To validate in a cellular context the change in Gα specificity exhibited in vitro by the RGS2(C106S,N184D,E191K) triple mutant, we used an intracellular cAMP biosensor to measure G_i heterotrimer-mediated inhibition of forskolin-stimulated cAMP production in HEK293T expressing the G_i-coupled D2 dopamine receptor along with either wild type RGS2 or the RGS2(C106S,N184D,E191K) mutant. Upon treatment of transfected cells with forskolin, a robust increase in luminescence was observed from the cAMP sensor, reflecting direct activation of adenylyl cyclase by forskolin (4); upon administration of the dopamine D2/D3-receptor selective agonist, quinpirole, dose-dependent inhibition of this cAMP production was observed. Wild type RGS2 had no effect on the IC₅₀ of quinpirole (Fig. 6). However, cellular expression of the RGS2(C106S,N184D,E191K) triple mutant resulted in a significantly higher IC₅₀ for quinpirole (762 versus 18 nm for empty vector; Fig. 6), indicating that the gain of Gα_i-directed activity is readily apparent in a cellular context as well as in vitro for the RGS2 triple mutant.

FIGURE 6.

The triple mutant RGS2(C106S,N184D,E191K), but not wild type RGS2, inhibits dopamine D2-receptor influence on forskolin-stimulated cAMP production. HEK293T cells were transiently co-transfected with expression vectors for the GloSensor cAMP biosensor and the G_i-coupled dopamine D2-receptor with empty vector, wild type RGS2, or the RGS2(triple) mutant. Inhibition of forskolin-stimulated cAMP production was determined after activation of the D2 receptor with various concentrations of quinpirole as indicated. The IC₅₀ (95% CI) for quinpirole was determined to be 18 (12–26), 14 (9–22), and 762 (498–1170) nm in the presence of empty vector, wild type RGS2, and the triple mutant, respectively. Inset, post-transfection cell lysates were immunoblotted with anti-HA epitope tag antibody to confirm the equivalent overexpression of HA-RGS2 and HA-RGS2(C106S,N184D,E191K) proteins.

Structural Determinants of RGS2 Interaction with Gα Subunits

To determine the structural basis for the Gα selectivity of RGS2, we used x-ray crystallography to obtain a structural model of the RGS2 triple mutant bound to a Gα_i subunit. A diffraction pattern data set was collected on a single crystal containing a complex between the RGS2(C106S,N184D,E191K) triple mutant and Gα_i3·GDP·AlF₄⁻ and was refined to 2.8-Å resolution (supplemental Table S1). The resultant structural model revealed canonical RGS domain/Gα interactions (20, 42), specifically, contacts between the flexible switch regions of Gα_i3 and the nine α-helical bundle formed by the RGS2 triple mutant (Fig. 7).

FIGURE 7.

Overall structural features of the RGS2(C106S,N184D,E191K)-Gα_i3·GDP·AlF₄⁻ complex. A, the tertiary structure of Gα_i3 is composed of a Ras-like domain (red) and an all α-helical domain (blue) and is present in a transition-state mimetic form bound to a molecule of GDP (magenta) and tetrafluoroaluminate (AlF₄⁻) ion (gray/blue sticks). The three critical switch regions of Gα (numbered Sw I to Sw III) are colored cyan. All three switch regions are engaged by the RGS2 RGS domain (yellow-green). Panel B represents the same structural model as in panel A, but rotated to highlight contacts made by residues serine 106, aspartate 184, and lysine 191 of the RGS2(C106S,N184D,E191K) triple mutant. This same orientation of the complex is presented in Fig. 8B.

One of the three mutation sites within the RGS2 triple mutant, aspartate 184, is observed to form a double salt bridge (Fig. 8A and Fig. S3) with the neighboring arginine 188, the latter being an αVIII residue completely conserved among all other R4-subfamily RGS domains (Fig. S1). Asparagine 184 of wild type RGS2, located between helix αVII and αVIII, is an aspartic acid in all other R4-subfamily RGS domains (Fig. S1). The additional terminal oxygen present in the aspartate side chain (and missing in asparagine) normally allows two salt bridges to be formed (Fig. 8A) with the conserved αVIII helix arginine residue (e.g. Arg¹⁷⁰ of RGS16, Arg¹⁸⁸ of RGS2). These salt bridges are not consistently observed in all unliganded RGS domain structures (20); however, this double salt bridge is present in all R4-subfamily RGS domains complexed with Gα_i/o subunits (Table S2), suggesting that their formation is important for making the RGS domain competent to bind Gα_i/o subunits. The importance of this Arg-Asn side chain interaction is supported by the loss of Gα_i binding and Gα_i-directed GAP activity when this αVIII helix arginine is mutated to glutamate (Figs. 2 and 5). The significance of this intramolecular interaction is further supported by observations that mutating the analogous αVIII helix arginine in RGS4 (Arg¹⁶⁷) and RGS12 (Arg⁸²¹) results in loss of Gα_i/o binding and Gα_i/o-directed GAP activity (38, 43, 44). Although Arg¹⁸⁸ of RGS2 does not make any critical contacts with Gα_i3 per se, it has a critical role in orienting Asp¹⁸⁴ (Fig. 8B) to form a conserved hydrogen bond with the main chain amide of a threonine residue in the Gα switch I region (Thr¹⁸² of Gα_i (20, 42); Thr¹⁸³ of Gα_o (45)). In the structure of wild type, uncomplexed RGS2 (PDB code 2AF0; Ref. 20), asparagine at this position (Asn¹⁸⁴) forms only a single hydrogen bond with terminal amine of Arg¹⁸⁸ and, rotated in this manner, the side chain cannot at the same time form a hydrogen bond with the Thr¹⁸² backbone (Fig. 8A and Table S2).

FIGURE 8.

Particular Gα selectivity determinants inferred from the structural model of the triple mutant RGS2(C106S,N184D,E191K) bound to Gα_i3. A, illustration of the αVII–αVIII region of the RGS domain to highlight the intramolecular interaction between the highly conserved αVIII helix arginine (Arg¹⁸⁸ of RGS2) and position 184 (asparagine in wild type RGS2 and aspartate in the triple mutant). RGS2(C106S,N184D,E191K) triple mutant (yellow-green; PDB code 2V4Z), unliganded wild type RGS2 (gray; PDB code 2AF0), and the Gα_i1-bound RGS16 (dark green; PDB code 2IK8) were aligned by sequence and then structure (Cα atoms) using the Align command with default align settings of MacPyMOL (DeLano Scientific, Palo Alto, CA), resulting in root mean square deviations of 0.92 and 0.80 Å, respectively. The conserved Arg¹⁸⁸ makes salt bridges with the terminal oxygens of the Asp¹⁸⁴ side chain in the RGS2(C106S,N184D,E191K) mutant and the analogous asparate side chain in RGS16; however, only one contact can be made between Arg¹⁸⁸ and the Asn¹⁸⁴ side chain of wild type RGS2. Loss of the second salt bridge creates a torsion in the wild type RGS2 Asp¹⁸⁴ residue, resulting of the loss of the stabilizing hydrogen bond to Thr¹⁸² in switch I of the Gα subunit. B, critical contacts between the three mutated positions of RGS2(C106S,N184D,E191K) (yellow-green) and its Gα binding partner (Ras-like domain in red; all-helical domain in blue; switch regions in cyan; bound GDP in magenta). The modeled terminal atoms of the Lys¹⁹¹ side chain (spheres) within RGS2(C106S,N184D,E191K) are in close enough proximity to make a hydrogen bond with Glu⁶⁵ of the Gα all-helical domain. Asp¹⁸⁴ makes two hydrogen bonds with Arg¹⁸⁸ and an additional bond with the backbone amine of the peptide bond connecting Thr¹⁸¹ and Thr¹⁸², both located within switch I of Gα. Ser¹⁰⁶ of the RGS2 triple mutant is tightly packed with the backbone carbonyl and γ-hydroxyl of Gα Thr¹⁸², both being less than 3.9 Å from β-carbon of Ser¹⁰⁶. Additionally, the Gα switch II residue Lys²¹⁰ is 3.8 Å from the Ser¹⁰⁶ α-carbon.

The aspartate substitution at position Asn¹⁸⁴ is critical to allow binding of RGS2 to Gα_i; however, this single substitution alone is not sufficient to engender robust Gα_i binding (Fig. 1). Ser¹⁰⁶ is completely conserved among all R4-subfamily RGS domains except RGS2, in which this position is a cysteine residue (Fig. S1). Mutating Cys¹⁰⁶ to serine was also necessary to obtain high affinity binding to Gα_i subunits (Figs. 1 and 2); whereas the Ser¹⁰⁶ side chain was not observed in the structural model to make any critical contacts with Gα_i3, this residue is tightly packed among other residues (Fig. 8B). The structure of the RGS2(C106S,N184D,E191K)-Gα_i3 complex reveals that the β-carbon of Ser¹⁰⁶ is closely juxtaposed with the backbone carbonyl and γ-hydroxyl of Thr¹⁸² within switch I of Gα_i3; additionally, the α-carbon of Ser¹⁰⁶ is 3.8 Å from the terminal amine of Lys²¹⁰ within switch II of Gα_i3. In conjunction with the SPR binding data, the observed tight packing of Ser¹⁰⁶ within the RGS2(C106S,N184D,E191K)-Gα_i3 complex suggests that the Cys¹⁰⁶ residue of wild type RGS2 prevents high affinity binding to Gα_i subunits by steric blockade of interactions with switch I and switch II of the Gα subunit.

Although amino acid positions 106 and 184 are completely conserved among all R4-subfamily RGS domains except RGS2, the specific amino acid at position 191 is conserved only in its basic character, being either a lysine or an arginine in all R4-subfamily RGS domains (Fig. S1). In wild type RGS2, this position is instead an acidic residue (glutamate 191). In the structural data derived from the RGS2(C106S,N184D,E191K)-Gα_i3 complex, electron density was present only for the α-, β-, and γ-carbons of the mutated Lys¹⁹¹; however, the final ordered carbon atom was found to be only 5.1 Å from the hydroxyl oxygen of Glu⁶⁵ in the αA helix of the Gα_i3 all-helical domain. Electron density was present to fit the Cα, Cβ, Cγ, and Cδ atoms of the Lys¹⁹¹ residue (Fig. S3). The Cϵ and terminal amine were modeled by superimposing a Lys over those parts of the carbon atom chain that could be placed with electron density, revealing that this basic side chain would be less than 3.0 Å from the hydroxyl oxygen of Gα_i3 Glu⁶⁵ and thus within hydrogen bonding distance. It is possible that the high salt concentration necessary for crystallization screened the electrostatic contribution of this interaction away, resulting in a partially disordered side chain. In wild type RGS2, this salt bridge would be lacking and this position instead would create electrostatic repulsion between RGS2 Glu¹⁹¹ and the all-helical domain of Gα_i3. The importance of all-helical domain contacts to RGS protein selectivity for Gα substrates has been previously speculated for the retinal-specific proteins RGS9-1 and Gα-transducin (46); our present finding with RGS2 provides one of the first structural insights into these interactions. These RGS domain/all-helical domain interactions, whereas typically underappreciated when considering the structural determinants of the RGS protein/Gα interaction interface (e.g. Refs. 42 and cf. 20), may provide a unique point of interdiction to exploit with selective RGS protein inhibitors.

Unique Determinants of RGS2 Gα_q Selectivity Are Conserved among Species with Cardiovascular Systems

Current knowledge of Gα selectivity suggests that R4-subfamily members, as well as proteins from the more ancestral RZ-subfamily (e.g. RGS17, -19, and -20), can act as GAPs for both Gα_i and Gα_q subunits (20, 47), with the R4-protein RGS2 particularly attuned to Gα_q over Gα_i. Given its unique Gα selectivity and its specialized role in cardiovascular signal transduction, RGS2 is likely to have arisen from the R4-subfamily in response to the development of cardiovascular structures and function.

In evolutionary terms, Gα_q emerged as the harbinger of a distinct and recognizable Gα subfamily in fungi, and Gα_q subunits are present in all metazoans including sponges (48, 49). Although RZ-subfamily RGS proteins are represented within the genomes of nematodes and arthropods (50), a distinct R4-subfamily does not appear until the evolution of urochordates. The genome of the urochordate Ciona intestinalis (sea squirt) encodes at least two RGS proteins (Fig. 7), an ortholog of the ancestral RZ-subfamily progenitor found in nematodes and arthropods, as well as a newly divergent R4-subfamily member (but not an RGS2 ortholog per se). With specialized tissues such as a notochord, digestive tract, single chamber heart, and gonads, C. intestinalis is commonly considered an excellent modern representative of the precursor to higher vertebrates (51, 52). Agnatha (jawless fish) such as the sea lamprey Petromyzon marinus are considered the most primitive extant members of early vertebrates (53) and represent the first vertebrate to exhibit cardiac innervation (54). Although the P. marinus cardiovascular system is more advanced than the open system of C. intestinalis, it is still considered primitive in that it lacks an elastin-reinforced vasculature (55), coronary circulation, and a pericardial-contained fourth chamber (conus or bulbus arteriosus) to dampen systolic oscillations in blood pressure (54). Similar to C. intestinalis, the genome of P. marinus encodes at least two RGS proteins, the ancestral RZ member and a single R4 member (Fig. 9); however, no RGS2-like protein has yet been identified in this species.

FIGURE 9.

Emergence of the specialized R4-subfamily member RGS2 and evolutionary conservation of its three Gα_q-selectivity residues. Full-length, protein open reading frame sequences for RGS2, RGS4, and RGS20 orthologs were obtained from the genomes of the indicated multicellular organisms and aligned using T-Coffee version 1.37 (61). Sequence alignments were manually adjusted using SEAVIEW version 3.2 (62) prior to the generation of a Markov-chain Monte Carlo-based phylogeny using MrBayes version 3.1.2 (Refs. 63 and 64); the dendrogram was visualized using Njplot version 2.3 (65).

As chordates evolved into the Gnathostomata (jawed vertebrates), the cardiovascular system rapidly developed coronary vessels, inhibitory vagal innervation, excitatory adrenergic innervation, and responses to prostaglandins, nitric oxide, and endothelin (56). This advance is marked in Danio rerio by the addition of multiple R4 proteins, specifically including a Gα_q-specific RGS2 protein (Fig. 9). This unique member of the R4-subfamily, with cysteine, asparagine, and aspartate at the three key specificity positions, is highly conserved in the extant representatives of all subsequent evolutionary steps: amphibians (e.g. Xenopus laevis and Xenopus tropicalis), avians (e.g. Gallus gallus) and mammals (Fig. 9); the three defining residues are seen to be unique among all R4-subfamily members within a given species (e.g. human R4 paralogs aligned in Fig. S1). Only amphibians (X. laevis and X. tropicalis) do not contain all three RGS2-defining amino acids (Fig. 9): whereas the RGS2 signature residue asparagine is present at position 184, serine (not cysteine) is present at position 106, and a neutral glutamine (not glutamate) is present at position 191. (Note that the latter glutamine is not seen in RGS2, RGS4, nor RGS20 paralogs.) Even though the conservation is not absolute in the amphibians, we have shown that asparagine in position 184 is sufficient on its own to significantly reduce Gα_i affinity (i.e. ∼20-fold; compare K_D of >21 μm for the C106S,E191K RGS2 double mutant versus K_D of 1.25 μm for the C106S,N184D,E191K triple mutant in Fig. 2). In conclusion, the conservation of these three key residue positions suggests that RGS2 has indeed evolved from the R4-subfamily to be a specialized Gα_q GAP for the modern cardiovascular system by acquiring particular residues at one or more of three key positions that have been highlighted in our mutagenesis/crystallography studies.