©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Effector Site That Stimulates G-protein GTPase in Photoreceptors (*)

Vladlen Z. Slepak (1), Nikolai O. Artemyev (2)(§), Yun Zhu (3), Charles L. Dumke (3), Leah Sabacan (2), John Sondek (5)(¶), Heidi E. Hamm (2), M. Deric Bownds (3) (4), Vadim Y. Arshavsky (3)(**)

From the (1)Division of Biology, California Institute of Technology, Pasadena, California 91125, (2)Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60680, (3)Laboratory of Molecular Biology, (4)Department of Zoology and Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53706, and (5)Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Heterotrimeric G-proteins mediate between receptors and effectors, acting as molecular clocks. G-protein interactions with activated receptors catalyze the replacement of GDP bound to the -subunit with GTP. -Subunits then modulate the activity of downstream effectors until the bound GTP is hydrolyzed. In several signal transduction pathways, including the cGMP cascade of photoreceptor cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with corresponding effectors. In the phototransduction cascade the GTPase activity of photoreceptor G-protein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. Here we characterize the stimulation of transducin GTPase by a set of 23 mutant phosphodiesterase -subunits (PDE) containing single alanine substitutions within a stretch of the 25 C-terminal amino acid residues known to be primarily responsible for the GTPase regulation. The substitution of tryptophan at position 70 completely abolished the acceleration of GTP hydrolysis by transducin in a complex with this mutant. This mutation also resulted in a reduction of PDE affinity for transducin, but did not affect PDE interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDE to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDE in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules.


INTRODUCTION

The activation-inactivation cycle of the photoreceptor G-protein, transducin, begins when photoexcited rhodopsin catalyzes the exchange of transducin-bound GDP for GTP. Transducin then stimulates the activity of its target, rod cGMP phosphodiesterase (PDE),()until bound GTP is hydrolyzed (reviewed in Stryer(1986), Chabre and Deterre(1989), and Hurley (1992)). It has remained as a paradox for a number of years that the rate of intrinsic transducin GTPase activity measured in vitro was substantially slower that the duration of the photoresponse (reviewed in Chabre and Deterre(1989) and Arshavsky et al.(1991)). Recent studies have shown that, under more physiological conditions, for example in concentrated suspensions of disrupted ROS, the rates of transducin GTPase are high enough to cause the termination of PDE activation on the time scale of the photoresponse (Dratz et al., 1987; Wagner et al., 1988; Arshavsky et al., 1989; Angleson and Wensel, 1993). Data from several laboratories now show that transducin's interaction with PDE (Arshavsky and Bownds, 1992; Pagès et al., 1992, 1993; Otto-Bruc et al., 1994), and more specifically with PDE (Arshavsky and Bownds, 1992; Angleson and Wensel, 1994; Arshavsky et al., 1994), results in an acceleration of transducin GTPase that can exceed 20-fold. The effect of PDE requires the presence of a membrane-bound factor whose nature has not yet been identified (Angleson and Wensel, 1994; Arshavsky et al., 1994; Otto-Bruc et al., 1994).

Our previous study with synthetic peptides corresponding to different segments of PDE has shown that the epitope responsible for transducin GTPase activation is located within a stretch of 25 C-terminal amino acid residues of PDE (Arshavsky et al., 1994). Here we report the identification of amino acid residues in this region that are directly involved in the GTPase regulation. We have found that the alanine substitution of Trp results in a complete abolishment of the GTPase activation and seven other substitutions of hydrophobic residues result in the reduction of the GTPase stimulation by 50-70%. These data indicate that the transducin GTPase activation by an effector may be a result of hydrophobic interaction between relatively long stretches of these proteins.


EXPERIMENTAL PROCEDURES

Preparation of ROS, Test Membranes, and Proteins

ROS were purified from frozen retinas (TA & WL Lowson Co., Lincoln, NE) under infrared illumination by double step sucrose flotation (Smith et al., 1975). Rhodopsin concentration was determined spectrophotometrically according to Bownds et al.(1971). Test membranes used for the measurements of transducin GTPase activity were obtained as described by Arshavsky et al.(1994). Briefly, ROS were bleached on ice to achieve tight binding of transducin with rhodopsin and homogenized in a glass-to-glass homogenizer. The membranes were washed once by an isotonic buffer containing 100 mM KCl, 2 mM MgCl, 1 mM dithiothreitol, and 10 mM Tris-HCl (pH 7.5) and three times by a hypotonic buffer containing 5 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, and 1 mM dithiothreitol. Analysis of these membranes by SDS-gel electrophoresis shows that they retain >80% of their transducin and are depleted of >98% of their endogenous PDE. Before being used, test membranes were incubated for 5 h at room temperature to achieve practically irreversible binding of GTP upon transducin activation (see Arshavsky et al.(1994) for a detailed explanation).

GGTPS was eluted from the test membranes by 20 µM GTPS, and then purified to at least 95% homogeneity by gel filtration on a Superose-12 column (Pharmacia Biotech Inc.). PDE was extracted from ROS as described by Baehr et al.(1979). Soluble PDE dimer lacking the isoprenylated and carboxymethylated C termini was prepared by tryptic proteolysis (Catty and Deterre, 1991). PDE extract containing 1 mg/ml PDE was incubated with 40 µg/ml trypsin for 90 min at 20 °C which resulted in a complete enzyme activation. The proteolysis was terminated by an addition of soybean trypsin inhibitor at a final concentration of 400 µg/ml. PDE was then purified to >95% purity by gel filtration of a Superose-6 column (Pharmacia Biotech Inc.). Protein concentration was determined by the Bradford(1976) assay using bovine serum albumin as the standard.

Preparation of PDE and Its Mutants

To obtain PDE and its mutants the coding sequence of the PDE from the synthetic gene for the fusion protein (Brown and Stryer, 1989) was subcloned into an expression vector pET-11a (Novagen) under control of the isopropyl -D-thiogalactoside-sensitive promoter T7. The alanine substitutions were introduced by a ``cassette mutagenesis'' strategy. For each mutation two complementary oligonucleotides containing desired mutations and protruding ends matching appropriate restriction sites were annealed and ligated with the vector replacing the wild type sequence. The vectors were transfected into Escherichia coli BL21-DE3 strain. Protein expression was induced by isopropyl -D-thiogalactoside. PDE or its mutants were then purified by a combination of cation-exchange and reverse-phase chromatography (Brown and Stryer, 1989). The purity of PDE was estimated to be >95%; the PDE concentration was determined spectrophotometrically at 280 nm using a molar extinction coefficient of 7,100. The concentration of the W70A mutant whose absorbance at 280 nm is small was determined either based on the results of a complete amino acid analysis of this protein or by the Bradford(1976) method using the wild type PDE as the standard. Both methods yielded identical results. The preparation of PDELY and the W70A mutant labeled by lucifer yellow vinyl sulfone was performed as described by Artemyev et al.(1992).

GTPase Measurements

Transducin GTPase activity was determined by a single-turnover technique described in detail by Arshavsky et al.(1991). All the measurements were conducted at room temperature (22-25 °C) in a buffer containing 10 mM Tris-HCl (pH 7.8), 100 mM NaCl, and 8 mM MgCl. The reaction was started by mixing 20 µl of the test membranes (20 µM final rhodopsin concentration) with 20 µl of [-P]GTP (4 10 dpm/pmol, 0.2 µM final concentration) supplemented by various concentrations of PDE or its mutants. The reaction was stopped by the addition of 100 µl of 6% perchloric acid. P formation was measured according to a modified method of Godchaux and Zimmerman(1979) described by Arshavsky et al.(1991). The GTPase rate constant was determined by the exponential fit of the time course of P formation.

Determination of GGTPS Affinity to PDE and Its Mutants by Fluorescent Assay

Fluorescent measurements were performed as described earlier (Artemyev et al., 1992) on a Perkin Elmer LS5B spectrofluorometer in a buffer containing 10 mM HEPES, 100 mM NaCl, and 1 mM MgCl. The excitation wavelength was 430 nm, and the emission was measured at 520 nm. Fluorescence of 25 nM PDELY in the presence of 50 nM GGTPS was measured before and after additions of increasing concentrations of PDE or its mutants. PDE or mutants cause a decrease in the fluorescence due to their competition with PDELY for binding to GGTPS. The K values in all cases were calculated from the competition curves considering 36 nM as the K value for the PDELYGGTPS complex (Artemyev et al., 1992).

Determination of GGTPS and PDE Binding with PDE and Its Mutants on the BIAcore Sensor Chip

PDE was covalently attached to the surface of the BIAcore sensor chip (Pharmacia Biosensor) via primary amines following the activation of the carboxymethyl groups of dextran on the chip. Briefly, the CM5 chip was activated at the flow rate of 5 µl/min with 30 µl of 0.2 MN-(3-dimethylaminopropyl)-N-ethylcarbodiimide and 0.4 M of N-hydroxysuccinimide, and then 15-45 µl of 0.5 µM PDE in 100 mM NaCl with 10 mM sodium formate (pH 4.3) were flown through the activated surface. Unbound groups were blocked by 30 µl of 1 M ethanolamine (pH 8.5). The noncovalently bound PDE was then removed by a 5-µl pulse of 6 M guanidine, 100 mM NaCl, 1 mM dithiothreitol, and 10 mM Tris-HCl (pH 8.0). For kinetics studies 35 µl of varying concentrations of GGTPS or PDE were injected at a flow rate of 5 µl/min in a buffer containing 120 mM NaCl, 8 mM MgCl, 1 mM dithiothreitol, 0.05 mg/ml bovine serum albumin, and 10 mM HEPES-KOH (pH 7.5). Each injection was followed by a buffer flow for 7 min to monitor the dissociation of the complex. For regeneration the cycle was concluded by a 5-µl pulse of 6 M guanidine, 100 mM NaCl, 1 mM dithiothreitol, and 10 mM Tris-HCl (pH 8.0).

The data were analyzed after subtraction of background signal (blank injections) with the BIAevaluation software (Pharmacia Biosensor). The kinetic parameters of the PDEGGTPS interaction were determined by fitting the data to the general rate equation:

On-line formulae not verified for accuracy

where R is the PDEGGTPS complex concentration (it is proportional to the amplitude of the SPR signal), t is time, T is GGTPS concentration (which remains constant during each injection due to the constant flow of fresh solution through the reaction cell), P is the total amount of immobilized PDE, k and k are the association and dissociation rate constants. As seen from the rearranged equation, the change in the response (dR/dt) is linearly related to the response amplitude (R) with the slope proportional to transducin concentration. The values of the slopes for the lines dR/dt versus R obtained at different transducin concentrations were then replotted as a function of transducin concentration. The k for PDE and each of the mutants could now be determined as slopes of these lines, while k is the ordinate intercept. Alternatively the values of k were determined from the exponential analysis of the SPR signal decay after replacement of the GGTPS solution by the buffer. The k values determined by these two methods did not differ more than 3-fold. The second method, however, provided more reliable data, so it was used for the calculations of the K values presented in this study. The same analysis was performed in the case of PDE.


RESULTS

Alanine scanning mutagenesis (Gibbs and Zoller, 1991) was used to determine the residues on PDE which are responsible for the stimulation of GTPase activity of the rod G-protein, transducin. A previous study (Arshavsky et al., 1994) had shown that the segment comprised of the 25 C-terminal amino acid residues of PDE, DITVICPWEAFNHLELHELAQYGII, is able to stimulate transducin GTPase practically to the same extent as full-length PDE, and thus the mutagenesis was limited to this area. Since two alanine residues are present in this segment, the total number of mutants was 23. Their ability to stimulate transducin GTPase was compared with that of the wild type PDE in the test system containing photoreceptor membranes with most of their transducin, but depleted of endogenous PDE. A single turnover approach described in detail in our previous publications (Arshavsky et al., 1989, 1991, 1994) was used to monitor the rate of transducin GTPase. Briefly, the GTPase reaction was initiated by the addition of [-P]GTP in the amount less than transducin. GTP was quickly bound to transducin due to a relatively high concentration of transducin in this assay, so the subsequent formation of the P reflected a single synchronized turnover of transducin GTPase. This approach is illustrated in Fig. 1showing a family of the GTP hydrolysis curves obtained with increasing concentrations of PDE.


Figure 1: Activation of transducin GTPase by increasing concentrations of PDE. The GTPase reaction was started by mixing equal volumes (20 µl) of the test membranes (40 µM rhodopsin) with 0.4 µM [-P]GTP, and the time course of the P formation was determined after quenching samples by perchloric acid. In all experiments, besides the control, GTP solution contained PDE concentrations indicated in the figure. The rate constants of transducin GTPase were determined by single exponential fit of the data. The data are taken from one of three similar experiments.



Based on their ability to stimulate transducin GTPase the mutants can be separated into three groups (Fig. 2A). The first group of 15 mutants has GTPase activating ability similar to that of wild type PDE. The seven members of the second group, V66A, F73A, L76A, L78A, L81A, I86A, and I87A, retain only 35-50% of their GAP activity. The third group includes only one mutant, W70A, in which the ability to activate transducin GTPase is abolished. Interestingly, all the mutations leading to a change of phenotype have substitutions of hydrophobic rather than charged or polar amino acid residues.


Figure 2: PDE mutants: acceleration of transducin GTPase (A) and binding affinities for transducin (B). The GTPase measurements in the test membranes and the determinations of binding affinities with the fluorescent assay were performed as described under ``Experimental Procedures.'' A, saturating, 2 µM concentrations of PDE and its mutants were used routinely. No GTPase acceleration was observed with up to 30 µM W70A mutant. The bars represent mean ± S.D. for at least two independent determinations. The extent of GTPase regulation by the wild type PDE was taken as 100%. B, the bars represent mean ± S.D. for three independent determinations. For the wild type PDE the K was 10 ± 2 nM.



In principle, the reduction of the PDE mutants' ability to stimulate transducin GTPase might be caused by two mechanisms. It might be the result of lower efficiency of formation of the complex between transducin and PDE or inability of the mutant PDE to accelerate GTP hydrolysis after forming a complex with transducin. To decide which of these is correct, we studied transducin association with PDE mutants by two complementary techniques. The first is based on the ability of transducin's -subunit complex with GTPS (GGTPS) to enhance the fluorescence of PDE labeled with lucifer yellow vinyl sulfone (Artemyev et al., 1992). The K for PDELY binding with GGTPS was determined from the measurements of fluorescence changes, and then the K values for the wild type PDE and all the mutants were calculated from the analysis of their competition with PDELY for binding to GGTPS. Fig. 2B shows that the only mutation causing a substantial (4-5-fold) reduction of the PDE affinity for transducin was W70A. Direct measurements of GGTPS binding to the W70A mutant labeled with lucifer yellow vinyl sulfone revealed a similar, 10-fold loss in affinity (data not shown). This is in general agreement with an earlier observation that a W70F substitution results in 100-fold reduction of PDE affinity for transducin (Otto-Bruc et al., 1993).

The second approach to analysis of transducin-PDE interaction was direct monitoring of complex formation by surface plasmon resonance on Pharmacia Biosensor's BIAcore instrument (Jonsson et al., 1991; Schuster et al., 1993). PDE (wild type and one representative of each group of mutants, P69A, F78A, and W70A) was covalently attached on the dextran layer of the sensor chip, and different concentrations of GGTPS were applied. The binding of GGTPS with immobilized PDE or PDE mutants was monitored as an increase of the SPR signal (Fig. 3, upper panels). After 7 min the flow of transducin solution was exchanged for a flow of buffer, initiating GGTPS dissociation from the chip. The kinetic parameters of the PDEGGTPS interaction were determined as described under ``Experimental Procedures.'' The data obtained with the SPR measurements are in a good agreement with the determinations of PDELY fluorescence changes. The only mutant showing a substantial, 25-fold, reduction of the affinity to transducin was W70A. This reduction is due to the decrease of the association rate; the dissociation rate for this mutant is not affected. The K values for PDE and its mutants obtained with BIAcore were higher than those with the lucifer yellow method most likely reflecting differences in the properties of immobilized PDE and free PDE in solution.


Figure 3: GGTPS binding with PDE (WT) and three mutants (P69A, F78A, and W70A) on the BIAcore sensor chip. The upper panel shows groups of the binding-dissociation curves obtained with increasing concentrations of GGTPS. The x axis is time, the y axis is the SPR response in resonance units. In the lower panel the data are replotted for the determination of rate constants. The x axis is the GGTPS concentration, the y axis is the value of the slopes of the lines dR/dt versus R (see ``Experimental Procedures'') determined for corresponding GGTPS concentration. The data are taken from one of four (W70A mutant), three (wild type PDE), or two (P69A and F78A mutants) similar experiments. The K values for the PDEGGTPS complex were 37 ± 8 nM (mean ± S.D.) for the wild type PDE, 46 ± 2 nM for the P69A mutant, 94 ± 4 nM for the F78A mutant, and 980 ± 450 nM for the W70A mutant.



An important conclusion from the analysis of PDE binding to transducin is that it was completely saturated at the mutant concentrations (30 µM for W70A and 2 µm for all other mutants) used in the GTPase assays. Therefore, a reduction of GTPase stimulation by all the mutants from the second and the third groups shown in Fig. 2A does not simply reflect lower efficiency of the transducin-PDE complex formation but results from an altered ability of these mutants to activate GTP hydrolysis in a complex with transducin.

The W70A mutation, although decreasing PDE interaction with transducin, does not alter PDE interaction with PDE catalytic subunits. The ability of this mutant to inhibit the activity of PDE was identical to that of the wild type PDE (not shown). The kinetics of the W70A mutant interactions with the PDE catalytic subunits was measured with the BIAcore instrument (Fig. 4). In contrast to transducin, the rate of the W70A mutant association with PDE was identical to that for the PDE wild type. The dissociation of PDE from the sensor chip appears to be the same for both mutant and wild type PDE. It is slower than the resolution limit of the instrument, 0.0005 s, so the value of the K could be only estimated to be less than 3 nM.


Figure 4: trPDE binding with PDE (A) and W70A mutant (B) on the BIAcore sensor chip. Panels A and B show groups of the binding-dissociation curves obtained with increasing concentrations of PDE. In Panel C the data are replotted for determinations of k (see ``Experimental Procedures''). The data are taken from one of two similar experiments. Closed symbols represent PDE; open symbols, W70A mutant. The k values were 1.80 ± 0.12 10M s for PDE and 1.75 ± 0.18 10M s for the mutant.




DISCUSSION

The Functional Topography of PDE

PDE regulates the activity of two central components of the phototransduction cascade. First, it inhibits the catalytic activity of the nonactivated PDE. This inhibition is released upon PDE activation by the GTP-bound form of transducin's -subunit. The second function of PDE is to stimulate the rate of transducin-bound GTP hydrolysis, thus regulating the lifetime of PDE activation. This function is most likely to be a result of coordinated action of PDE and another membrane-associated factor whose nature remains unidentified (see below). Two domains on PDE are shown to be involved in both of these interactions. The first domain is located within the C-terminal third of the molecule. The site of PDE inhibition resides mainly within the C-terminal sequence Gly-Ile-Ile (Lipkin et al., 1988; Brown, 1992; Skiba et al., 1995), while the residues between Asp and Leu (Skiba et al., 1995) participate in binding to transducin. Our previous study (Arshavsky et al., 1994) showed that a peptide corresponding to the sequence between Asp and Ile is capable of stimulating transducin GTPase to the same extent as PDE. Another part of PDE which participates in the binding with both PDE and transducin is the lysine-rich area between residues Arg and Gly (Morrison et al., 1987, 1989; Lipkin et al., 1988; Artemyev and Hamm, 1992; Takemoto et al., 1992). The most likely role of this segment is to increase the affinity of PDE to both PDE and transducin by providing an additional binding site for these interactions.

Here we report the identification of amino acid residues within the Asp-Ile segment of PDE that are directly involved in the regulation of transducin GTPase. Eight hydrophobic residues are important for this function. The alanine substitutions of seven of them, Val, Phe, Leu, Leu, Leu, Ile, and Ile, result in a 2-3-fold reduction of their ability to stimulate transducin GTPase. The binding affinity of these mutants to transducin is the same as that of the wild type PDE. The alanine substitution of Trp results in a reduction of the mutant's affinity for transducin (in agreement with the report of Otto-Bruc et al., 1993) and also leads to a complete abolishment of the mutant's ability to stimulate GTP hydrolysis after forming a complex with transducin. Interestingly, this mutation is crucial only for PDE interaction with transducin. No differences in the interaction between the W70A mutant and PDE were revealed in this study.

How Could PDE Regulate Transducin GTPase Activity?

Until recently the regulation of transducin GTPase activity in ROS remained as one of the most controversial aspects of the phototransduction biochemistry (see Arshavsky et al. (1994) for a more detailed discussion). However, recent data from three laboratories (Angleson and Wensel, 1994; Arshavsky et al., 1994; Otto-Bruc et al., 1994) led the authors to a consensus conclusion that the acceleration of transducin GTPase is a result of coordinate action of PDE (or PDE) and another factor, most likely protein, tightly associated with the photoreceptor membranes. The only minor discrepancy which remains to be resolved is whether the factor itself is capable of causing some acceleration of transducin GTPase in the absence of PDE. This discrepancy may be apparent and simply reflect different amounts of residual PDE in the membrane preparations used in these studies. In any case, it does not appear to be possible to determine the exact role of PDE and the membrane factor on the GTP hydrolysis before the factor is characterized. It may be noted, however, that the mechanism of the PDE action is most likely to be distinct from the intrinsic G-protein GTPase or from the action of GTPase activating proteins (GAPs) that regulate the small GTP-binding proteins. Specifically, while a conserved arginine and glutamine are required for the intrinsic hydrolysis of GTP by the -subunits of heterotrimeric G-proteins (Markby et al., 1993; Sondek et al., 1994; Coleman et al., 1994; Kleuss et al., 1994), and a functionally similar arginine is presumably supplied by GAPs (Brownbridge et al., 1993), transducin GTPase acceleration by PDE requires the action of eight hydrophobic amino acid residues. Along with recent observations that G-protein -subunits interact with their effectors by multiple sites not directly involved in GTP-binding (summarized by Artemyev and Hamm(1994)), our data indicate that PDE action may be a result of hydrophobic interactions between long sequences of PDE and transducin. The consequences of such interactions may include an optimal positioning of the residues directly involved in GTP hydrolysis or better exclusion of bulk water leading to a decrease of the dielectric content of the catalytic center. Alternatively, PDE binding with transducin may be necessary for a further interaction of the complex with the membrane factor. These questions will be addressed in future research.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants EY-10336 (to V. Y. A.), EY-00463 (to M. D. B.), EY-06062 (to H. E. H.), and GM-34236 (to M. I. Simon). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a NARSAD Young Investigator Award.

Damon Ranyon-Walter Winchell postdoctoral fellow.

**
To whom correspondence should be addressed: Harvard Medical School/MEEI, 243 Charles St., Boston, MA 02114. Tel.: 617-573-3311.

The abbreviations used are: PDE, rod cGMP phosphodiesterase; PDE, the complex of PDE - and -subunits; PDE, the complex of PDE -and -subunits obtained by PDE trypsinization; PDE, the -subunit of PDE; PDELY, PDE labeled by lucifer yellow vinyl sulfone; ROS, rod outer segments; G, transducin -subunit; GTPS, guanosine 5`-(-thio)triphosphate; GAP, GTPase activating protein; SPR, surface plasmon resonance.


ACKNOWLEDGEMENTS

We thank Dr. M. I. Simon for many helpful discussions and Dr. R. Swanson for help with the BIAcore instrument.


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