|Molecular Vision 2006;
Received 2 November 2005 | Accepted 9 May 2006 | Published 12 May 2006
Mutation R238E in transducin-α yields a GTPase and effector-deficient, but not dominant-negative, G-protein α-subunit
Brandy Barren, Michael Natochin,
Nikolai O. Artemyev
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA
Correspondence to: Nikolai Artemyev, Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, 52242; Phone: (319) 335-7864; FAX: (319) 335-7330; email: email@example.com
Purpose: Certain forms of inherited and light-induced retinal degenerations are believed to involve excessive phototransduction signaling. A dominant-negative mutant of the visual G-protein, transducin, would represent a major tool in designing potential therapeutical strategies for this group of visual diseases. We thought to further investigate a novel mutant of the transducin-α subunit, R238E, that was recently reported to be a dominant-negative inhibitor of the rhodopsin/transducin/PDE visual system.
Methods: The R238E substitution was introduced into a tranducin-like chimeric Gtα*-subunit. The nucleotide-bound state of the Gtα*R238E mutant was assessed using the trypsin-protection assay. The ability of the Gtα*R238E mutant to interact with Gtβγ, couple to photoexcited rhodopsin (R*), and undergo R*-stimulated guanine nucleotide exchange was examined by a GTPγS binding assay. The GTPase activity of the mutant Gtα* and its interaction with RGS proteins was characterized in the steady-state and single turnover measurements of GTP hydrolysis. A binding assay utilizing the fluorescently-labeled γ-subunit of PDE6 (Pγ) was employed to monitor the effector function of Gtα*R238E.
Results: The Gtα*R238E mutant bound GDP and was capable of the AlF4--induced activational conformational change. The capacity of Gtα*R238E to couple to R* in the presence of Gtβγ was similar to that of Gtα*. However, the mutant GTPase activity was markedly impaired. This defect was further exacerbated by the diminished interactions of Gtα*R238E with the GAP proteins, RGS9 and RGS16. Another consequence of the mutation was the reduction in Gtα*R238E's affinity for Pγ.
Conclusions: Transducin mutant Gtα*R238E exists in a nucleotide-bound state and is fully capable of activational coupling to R*. This mutation results in a significant impairment of Gtα*'s ability to hydrolyze GTP and interact with the inhibitory subunit of PDE6. This phenotype is entirely inconsistent with that of a dominant-negative inhibitor as recently reported.
The visual transduction cascade in rod photoreceptor cells represents a classical model system of G protein-coupled receptor (GPCR) signaling. The cascade is initiated by the interaction of photoexcited rhodopsin (R*) with the visual G protein, transducin (Gt), which causes the exchange of GDP for GTP on the transducin-α subunit (Gtα). GtαGTP is subsequently released to activate the effector enzyme PDE6 by displacing the inhibitory Pγ subunits from the catalytic core PDE6αβ. Activated PDE6 hydrolyzes intracellular cGMP resulting in a closure of cGMP-gated channels and hyperpolarization of the photoreceptor plasma membrane [1,2].
Similar to dominant-negative forms of small monomeric G proteins, dominant-negative mutants of α-subunits of heterotrimeric G-proteins represent effective tools for exploring the G-protein signaling pathways [3,4]. However, the progress in identifying and characterizing dominant-negative Gα subunits has been limited. Three separate mutations in Gsα reducing the protein's affinity for GDP and impairing the ability to bind GTP and assume an active conformation produced an inhibitor of Gs-dependent stimulation of adenylyl cyclase . Another Gsα mutant, S54N, a counterpart of the well-characterized dominant-negative RasS17N, was shown to have a conditional dominant-negative phenotype in suppressing TSH-stimulated cAMP levels in tranfected COS and HEK293 cells [5,6]. Substitutions of the corresponding Ser residues in Goα (S47C) and Giα2 (S48C) also resulted in dominant-negative effects on the G-protein signaling [7,8]. The mechanisms of dominant-negative Gα mutants described to date appear to involve sequestration of Gβγ and/or the Gβγ-dependent sequestration of activated GPCRs [4,6]. Interestingly, native Gtα itself is an excellent, albeit nonselective, dominant-negative inhibitor of G-protein signaling. Owing to the high Gt specificity for R*, transducin is not activated by most other GPCRs so that their signaling is inhibited when Gtα sequesters Gβγ .
A potential significance of a dominant-negative Gtα mutant is underlined by the fact that a subset of inherited and light-induced retinal degenerations, as well as various forms of stationary night blindness, are caused by excessive phototransduction signaling [10-12]. Mutations in the genes encoding arrestin and rhodopsin kinase cause prolonged R* signaling activity and lead to an Oguchi form of human congenital night blindness [13-16]. Mutations in rod opsin T94I, A292E, and G90D constitutively activate transducin in the absence of 11-cis-retinal and also cause congenital stationary night blindness in humans [10,17]. Mutation K296E disrupting the attachment of 11-cis-retinal to opsin is associated with autosomal dominant retinitis pigmentosa . The mutant opsin is constitutively active in vitro, but appears to be inactivated by phosphorylation and bound arrestin in vivo . Defects in 11-cis-retinal biosynthesis due to mutations in RPE65 are linked to Leber congenital amaurosis, a severe early-onset form of retinal degeneration [11,19]. A constitutive activation of transducin by the chromophore-free opsin triggers this visual disorder . A dominant-negative mutant of Gtα capable of sequestering R* and blocking activation of native Gt may become a very useful instrument in probing the mechanisms of visual dysfunctions. Furthermore, it may become a part of therapeutical strategy for more severe forms of retinal degeneration due to excessive phototransduction signaling. However, no Gtα mutants capable of blocking the phototransduction cascade have been described until recently. A new study indicated a unique dominant-negative phenotype of the R238E substituted transducin-like chimeric αT* subunit . The mutant was reported to be incapable of R*-dependent αGDP-GTP exchange and interaction with PDE6. The uniqueness of the stated phenotype was that the mutant exists in the nucleotide-free state, and can effectively block R*-induced activation of αT* independently of sequestration of Gβγ. In this study, we sought to further explore the mechanism of the R238E mutant and its interactions with the key phototransduction molecules, R*, RGS9, and PDE6. The Gtα-like Gtα-Giα1 chimeric protein Gtα*  was used as the template to construct the Gtα*R238E mutant. An effector competent Gtα* was generated based on Chi8  by introducing two mutations (H244N247) [22,24]. The αT* protein was produced by making analogous mutations to Chi6 [23,25]. Gtα* is more similar to transducin-α than αT* due to the fact that it contains switch III from native transducin-α. Surprisingly, while we detected specific impairments in the mutant GTPase activity and interactions with RGS proteins and PDE6, Gtα*R238E was fully competent of binding guanine nucleotides and activational coupling to R*.
Guanosine 5'-[γ-35S]thiotriphosphate triethylammonium salt (GTPγS; 1100 Ci/mmol) and guanosine 5'-[γ-32P]triphosphate triethylammonium salt (about 5000 Ci/mmol) were from Amersham Biosciences (Piscataway, NJ). GTP and GDP were from Sigma-Aldrich (St. Louis, MO). Restriction enzymes were from New England Biolabs (Ipswich, MA), Inc. Cloned Pfu DNA polymerase was from Stratagene (La Jolla, CA). DH5α and BL21(DE3) bacterial strains were from Invitrogen (Carlsbad, CA). His-Bind resin was from Novagen (La Jolla, CA). Trypsin treated with L-(tosylamido-2-phenyl)-ethylchloromethylketone was purchased from Worthington Biochemical Corp. (Lakewood, NJ). Primers were synthesized by IDT, Inc. (Coralville, IA). Bovine (WL Lawson Co., Lincoln, NE) outer rod segment (ROS) membranes and urea-washed ROS membranes (uROS) were prepared as before [26,27]. Gtβγ was purified as described . The γ subunit of rod PDE6 (Pγ) labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (BC, Molecular Probes, Eugene, OR) was obtained and purified . The RGS-domain of RGS9 (RGS9d, amino acids 284-461) was expressed and purified as previously described [22,30]. Cloning, expression, and purification of GST-RGS16 were performed as previously described . Purified proteins were stored in 50% glycerol at -20 °C or without glycerol at -80 °C.
Mutagenesis and expression of Gtα*R238E
The Gtα*R238E mutant was obtained using the pHis6-Gtα* vector for expression of the transducin-like chimera, Gtα* , as a template in the PCR amplifications.
Reverse primer GCT TTC ATG CAT TTC GTT CAC TTC GTC ATC CTC AAC carrying the mutation R238E (red color) and a unique NsiI restriction site (blue color) was paired with the N-terminus Gtα* primer ATC ACG CCA TGG GGG CTG GGG CCA GC carrying a NcoI restriction site (red color). The NcoI/NsiI-digested PCR product (about 700 bp) was ligated to the pHis6-Gtα* vector that was digested with the same enzymes. The construct was sequenced at the University of Iowa DNA Core Facility. Gtα* and Gtα*R238E proteins were expressed and purified .
Gtα* and Gtα*R238E (2 μg) were incubated for 10 min at 25 °C in 20 mM HEPES buffer (pH 8.0) containing 100 mM NaCl, 2 mM MgSO4 (buffer A) and 50 μM GDP. Where indicated, 10 mM NaF and 50 μM AlCl3 were included in the buffer. To obtain GTPγS-bound Gtα* and Gtα*R238E, 2 μg of each protein were preincubated with bleached uROS (20 nM rhodopsin) and Gtβγ (0.2 μg) in the presence of 50 μM GTPγS for 1 h at 25 °C. Digestions with trypsin (10 μg/ml) were performed for 10 min at 25 °C and stopped by the addition of sodium dodecyl sulfate (SDS) sample buffer and immediate heat treatment (100 °C, 5 min) . Proteolytic fragments were analyzed by SDS-gel electrophoresis followed by Coomassie Blue staining.
GTPγS binding assay
Gtα* and Gtα *R238E (1 μM) alone, or mixed with 2 μM Gtβγ and uROS membranes (50 nM, 1 μM or 5 μM rhodopsin) were incubated for 2 min at 25 °C in the presence of light. Binding reactions were started with the addition of 20 μM or 200 nM [35S]GTPγS (1 μCi). Aliquots of 20 μl were withdrawn at the indicated times, mixed with 1 ml ice-cold 20 mM Tris-HCl (pH 8.0) buffer containing 130 mM NaCl, 2 mM MgSO4, and 1 mM GTP, passed through Whatman cellulose nitrate filters (0.45 μm), and washed three times with 3 ml of the same buffer without GTP . The filters were dissolved in 5 ml of a xylene-based 3a70B counting cocktail (RPI Corp., Mt. Prospect, IL) and [35S]GTPγS was measured in a liquid scintillation counter. The kapp values for the binding reactions were calculated as the slopes of the linear fits.
GTPase activity assays
Single-turnover GTPase activity measurements were carried out in suspensions of uROS membranes (5 μM rhodopsin) reconstituted with Gtα* or Gtα*R238E (1 μM each) and Gtβγ (2 μM) in buffer A [32,33]. Where indicated, RGS9d (3 μM) or GST-RGS16 (0.5 μM) were added. After incubation for 5 min at 25 °C, 200 nM [γ-32P]GTP (0.5 μCi) was added to the mixtures to initiate the reaction. GTPase hydrolysis was quenched at the indicated times by mixing 20 μl aliquots with 100 μl of 7% (v/v) perchloric acid. Nucleotides were precipitated with 700 μl of 10% (w/v) charcoal suspension in phosphate-buffered saline, and free [32Pi] was measured by liquid scintillation counting. GTPase rate constants were calculated by fitting the experimental data to an exponential function: %GTP hydrolyzed=100(1-e-kt), where kcat is the rate constant for GTP hydrolysis.
Steady state GTPase reactions were performed in the presence of Gtα* or Gtα*R238E (1 μM), Gtβγ (2 μM), and the absence or presence of varying concentrations of rhodopsin (uROS) . Proteins were mixed with 20 μM [γ-32P]GTP (0.5 μCi) in 100 μl of buffer A to start the reaction. Aliquots (20 μl) were withdrawn at the indicated times and transferred to 100 μl of 7% (v/v) perchloric acid. Nucleotides were precipitated with 700 μl of 10% (w/v) charcoal suspension in phosphate-buffered saline, and free [32Pi] was measured by liquid scintillation counting. Results were fit with linear regression.
Fluorescence-binding measurements were performed on an F-2500 Fluorescence Spectrophotometer (Hitachi; Tokyo, Japan) in 0.5 ml of buffer A at 25 °C. Fluorescence of PγBC (15 nM) was measured with an excitation of 445 nm and emission of 495 nm in the presence of increasing concentrations of the GDP-, AlF4--, or GTPγS-bound Gtα* or Gtα*R238E . The AlF4-- or GTPγS-bound proteins were obtained by preincubation of 10 mM NaF and 50 μM AlCl3 for 10 min, or 50 μM GTPγS in the presence of bleached uROS membranes (100 nM rhodopsin) and Gtβγ (100 nM) for 1 h, followed by centrifugation for 15 min at 13,000x g. The Kd values were calculated by fitting the data to the equation: F/Fo=1+[(F/Fo)max-1]X/(Kd+X).
Fo is the basal fluorescence of PγBC, F is the fluorescence after the addition of Gtα* or Gtα*R238E, (F/Fo)max is the maximal relative increase of fluorescence, and X is the concentration of free Gtα* or Gtα*R238E. The X value is determined as [Gtα*]total-[Gtα*]bound=[Gtα*]total-[PγBC](F/Fo)/(Fmax-Fo); where [PγBC] is the concentration of PγBC in the assay (15 nM).
Protein concentrations  were measured using IgG as a standard. GraphPad Prizm (version 4) was used to fit the experimental data and plot the graphs. The kapp, kcat, and Kd values are expressed as mean±SE for three independent experiments.
Expression and nucleotide-bound state of GtαR238E
Gtα* is readily expressed in E. coli and has been utilized in the past to generate mutations of Gtα residues [22,32]. After purification using His-Bind resin, the Gtα*R238E mutant was expressed about 5 fold less (about 1 mg/l of culture) compared to Gtα* expression. After HPLC purification using a MonoQ column, Gtα*R238E was >95% pure and yielded about 0.5 mg/l of culture. To assess the folding and the nucleotide-binding state of Gtα*R238E, a trypsin-protection assay was performed. This assay takes advantage of the protection of the switch II region from tryptic cleavage in activated (AlF4- or GTPγS-bound) Gtα, resulting in a band of about 34 kDa. The trypsin-protection test of Gtα*R238E resembled Gtα* (Figure 1). The band of about 20 kDa was observed for Gtα*R238E in the presence of GDP, indicating that the mutant binds GDP. Activation by both AlF4- and GTPγS resulted in the quantitive appearance of 34 kDa bands, demonstrating protection of the switch II region. The protection in the presence of AlF4- proves that Gtα*R238E contains bound GDP, since AlF4- mimicks the γ-phosphate of GTP .
Rhodopsin activation of Gtα*R238E
The ability of R* to activate Gtα* and Gtα*R238E was measured by a GTPγS binding assay in the absence and presence of purified Gtβγ and uROS. In the absence of Gtβγ and uROS, both Gtα* and Gtα*R238E displayed low basal rates of GTPγS binding rates (Figure 2). With Gtβγ and uROS (50 nM rhodopsin) present, the GTPγS binding rates of both Gtα* and Gtα*R238E increased dramatically (0.078 min-1 and 0.088 min-1, respectively, Figure 2). The rates of GTPγS binding of Gtα* and Gtα*R238E further accelerated to the same level of about 2 min-1 using higher concentrations of uROS (1 μM rhodopsin, not shown). The similarity of the rates indicates that mutant Gtα*R238E interacts with Gtβγ, couples to rhodopsin, and undergoes rhodopsin-stimulated GDP to GTPγS exchange.
GTPase activity of Gtα*R238E and its interaction with RGS
To study the effect of R238E on the GTPase activity of Gtα*R238E, steady state GTPase assays were performed in the absence or presence of Gtβγ and uROS containing 1 μM R*. Raising the concentration of R* above 1 μM did not increase the rate of GTP hydrolysis, suggesting that under these conditions GDP-GTP exchange on Gtα* is not a rate-limiting step (not shown). Without uROS present, both Gtα* and Gtα*R238E demonstrated negligible levels of GTPase activity (not shown). In the presence of uROS, the rate of GTP hydrolysis by Gtα* was 1.21 mol Pi/mol Gtα* min (Figure 3). The GTPase activity of Gtα*R238E using the same concentration of uROS was about 7.5 fold lower than that of Gtα* (Figure 3).
To confirm that the lower GTPase activity of Gtα*R238E in the steady state experiments was due to the reduced catalytic rate of GTP hydrolysis, a single turnover GTPase assay ([GTP] <[Gtα*βγ]) was utilized next. The catalytic rate of GTP hydrolysis for Gtα*R238E (0.003 s-1) is about 7 fold slower than that Gtα* (0.023 s-1; Figure 4A,B), thus indicating that the mutation caused a defect in GTPase activity. The rate values determined under single turnover conditions are similar to the steady-state activities of Gtα* (0.020 s-1) and Gtα*R238E (0.0027 s-1) observed in the presence of 1 μM R*. Control experiments showed that under the conditions in Figure 4A, the GTPγS-binding rates for Gtα* (0.16 s-1) and Gtα*R238E (0.14 s-1) were similarly high and significantly exceeded the measured GTPase rates (Figure 4C).
Next, we studied the effects of two GAP proteins, RGS9d, and RGS16, on the GTPase activity of Gtα*R238E. RGS9 is a natural GAP for transducin in photoreceptor cells , whereas RGS16 is a potent GAP for transducin in vitro . As expected, the addition of the RGS domain of RGS9 or RGS16 increased the GTP hydrolysis rate of Gtα* (about 4 fold and about 6.5 fold, respectively; Figure 4A). The GTPase rate in the presence of RGS16 (0.15 s-1) became comparable to the GTPγS-binding rate in Figure 4C and may thus be limited by the binding reaction under these conditions. In contrast, the addition of RGS9d or RGS16 only slightly increased the GTPase rate of Gtα*R238E (about 1.3 fold and 2 fold, respectively; Figure 4B). This implies that Gtα*R238 is defective in its ability to interact with the RGS proteins.
Gtα*R238E interaction with its effector Pγ
Activated Gtα*GTP binds to the Pγ subunit of the effector enzyme PDE6, thus allowing cGMP hydrolysis by the rod PDE6βγ subunits. The fluorescence-binding assay was utilized to determine the affinity of interaction between Gtα* or Gtα*R238E and fluorescently labeled Pγ, PγBC . The affinity of Gtα*GDP for PγBC was only about 1.5 fold higher than that of the GDP-bound Gtα*R238E (Figure 5). However, Gtα*GDP displayed a significantly greater increase in affinity for PγBC upon activation with AlF4- than Gtα*R238E (Figure 5), suggesting that the mutation leads to an effector impairment of Gtα* (Figure 5). Similar results were obtained for the GTPγS-bound forms of Gtα* and Gtα*R238E (not shown).
A significance of developing a dominant-negative mutant of Gtα for understanding the mechanisms of visual disorders linked to rhodopsin/transducin mediated signaling is evident. However, no such mutant has been described prior to a recent study of the αT*R238E mutant with the substitution of the conserved Arg residue within the switch III region of chimeric transducin-α . The reported phenotype of αT*R238E is unique and unusual in that it exists as a stable protein in a nucleotide-free state, binds to R* without being activated, fails to interact with PDE6, and blocks the R*-induced activation of αT* independently of Gβγ sequestration . These properties of αT*R238E would suggest that the R238E mutant of transducin-α might be an ideal candidate for an effective dominant-negative inhibitor of phototransduction. We set out to further investigate the mechanism of the R238E mutant. Another goal of our study was to compare the R238E mutation to another substitution at this position, R238Q, made in Gtα*, a close analog of αT*. We previously examined Gtα*R238Q in order to understand the mechanism of a corresponding gain-of-function R243Q mutant in C. elegans Gqα . Although Gln and Glu are conservative residues, the properties of Gtα*R238Q appeared to be dramatically different from the stated phenotype of αT*R238E [21,32]. The analysis of Gtα*R238E, done by using a set of assays similar to those utilized previously in characterization of Gtα*R238Q, might have provided additional clues to the mechanism of the R238E mutation.
Unexpectedly, our analysis yielded a Gtα*R238E phenotype strongly contradicting that of a dominant-negative inhibitor. The trypsin-protection assay (Figure 1) shows that Gtα*R238E binds GDP and is fully capable of undergoing an activational conformational change induced by AlF4- or GDP-GTP exchange. A reduced sensitivity of αT*R238E to trypsin in the absence of AlF4- was previously reported, suggesting a partially activated state of the mutant . Our data do not confirm this observation. The proteolytic cleavage patterns of Gtα* and Gtα*R238E are essentially identical, demonstrating similar GDP-bound states for the two proteins. Furthermore, the R238E mutation had no effect on the capacity of Gtα* reconstituted with Gβγ to couple to R*. The GTPγS-binding assay revealed equivalent activation rates for Gtα* and Gtα*R238E. The ability of Gtα*R238E to hydrolyze GTP was tested in single turnover and steady-state GTPase measurements. The later assay utilized sufficiently high concentrations of R*, such that the GDP-GTP exchange is not a rate limiting step. Both assays uncovered a 7-8 fold reduction in the rate of GTP hydrolysis by Gtα*R238E. This impairment was augmented by the diminished capacity of the GAP proteins, RGS9d and RGS16, to stimulate the GTPase activity of the mutant. A second notable deficiency of Gtα*R238E is its reduced affinity for the effector molecule. However, this deficit did not result from the inability of Gtα*R238E to undergo activational conformational change because, a clear increase in the mutant's affinity for Pγ was observed for the activated forms of Gtα*R238E.
The GTPase and effector impairments of Gtα*R238E are generally similar to the phenotype of the previously characterized R238Q and R238A mutants . The effects of the R238E mutation on GTP hydrolysis and its potentiation by RGS can be readily rationalized on the basis of the crystal structures of Gtα [38-41]. GtαR238 forms a salt bridge with E39 [38,39], which in the transitional state for GTP hydrolysis makes a water-mediated hydrogen bond with a major catalytic and RGS contact residue Q200 [40,41]. A decreased effector competence of R238E might be caused by subtle conformational changes resulting from the loss of the R238-E39 bridge and the contact between R238 and Q143 in the Gtα helical domain . Another possibility is that the R238E substitution interferes with the interaction between the α3 helix of Gtα and Pγ [24,41]. Theoretically, because of reduced effector function, Gtα*R238E could have been a weak inhibitor of phototranduction by competing with Gtα for R*. However, the high catalytic efficiency of R* would require that Gtα*R238E be present in massive excess to Gtα and R* for any significant inhibition to occur. In addition, the GTPase impairment of GtαR238E increases the lifetime of its activated form and makes any potential competition with Gtα for R* even less realistic.
It is unlikely that the minor sequence variations between αT* and Gtα* can account for the drastic difference in the phenotypes of their R238E mutants. αT* and Gtα* have four substitutions: three conservative substitutions (L228M, E234D, and M236V) and only one nonconservative (A231V). The R238E mutant's parent proteins, Chi6 and Chi8, are very similar in all respects [23,24]. Our examination of the crystal structures of Gtα [38-41] does not provide an apparent explanation as to why the R238E mutants αT* and Gtα* might have such strikingly dissimilar properties. However, if the R238E mutation yields a dominant-negative phenotype only in the context of αT*, but not in the context of a more transducin-like Gtα*, this mutant cannot be considered as a dominant-negative mutant of transducin and would not be a practical tool for studies of visual disorders related to excessive signaling. Nevertheless, advances toward the generation of dominant-negative mutants from other classes of Gα subunits indicate the achievability of dominant-negative Gtα mutants in future.
This work was supported by National Eye Institute Grant R01EY012682.
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