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• W2010201306 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 α-subunit with GTP. α-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 γ-subunits (PDEγ) 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 PDEγ affinity for transducin, but did not affect PDEγ interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDEγ to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDEγ in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules. 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 α-subunit with GTP. α-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 γ-subunits (PDEγ) 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 PDEγ affinity for transducin, but did not affect PDEγ interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDEγ to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDEγ in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules. 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),1 1The abbreviations used are: PDErod cGMP phosphodiesterasePDEαβthe complex of PDE α- and β-subunitstrPDEαβthe complex of PDE α-and β-subunits obtained by PDE trypsinizationPDEγthe γ-subunit of PDEPDEγLYPDEγ labeled by lucifer yellow vinyl sulfoneROSrod outer segmentsGtαtransducin α-subunitGTPγSguanosine 5′-(γ-thio)triphosphateGAPGTPase activating proteinSPRsurface plasmon resonance. until bound GTP is hydrolyzed (reviewed in Stryer, 1986Stryer L. Annu. Rev. Neurosci. 1986; 9: 87-119Crossref PubMed Scopus (774) Google Scholar, Chabre and Deterre(1989), and Hurley, 1992Hurley J.B. J. Bioenerg. Biomembr. 1992; 24: 219-226Crossref PubMed Scopus (39) Google Scholar). 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, 1989Chabre M. Deterre P. Eur. J. Biochem. 1989; 179: 255-266Crossref PubMed Scopus (219) Google Scholar and Arshavsky et al., 1991Arshavsky V.Y. Gray-Keller M.P. Bownds M.D. J. Biol. Chem. 1991; 266: 18530-18537Abstract Full Text PDF PubMed Google Scholar). 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., 1987Dratz E.A. Lewis J.W. Schaechter L.E. Parker K.R. Kliger D.S. Biochem. Biophys. Res. Commun. 1987; 146: 379-386Crossref PubMed Scopus (40) Google Scholar; Wagner et al., 1988Wagner R. Ryba N. Uhl R. FEBS Lett. 1988; 234: 44-48Crossref PubMed Scopus (23) Google Scholar; Arshavsky et al., 1989Arshavsky V.Y. Antoch M.P. Lukjanov K.A. Philippov P.P. FEBS Lett. 1989; 250: 353-356Crossref PubMed Scopus (23) Google Scholar; Angleson and Wensel, 1993Angleson J.K. Wensel T.G. Neuron. 1993; 11: 939-949Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Data from several laboratories now show that transducin's interaction with PDE (Arshavsky and Bownds, 1992Arshavsky V.Y. Bownds M.D. Nature. 1992; 357: 416-417Crossref PubMed Scopus (218) Google Scholar; Pagès et al., 1992Pagès F. Deterre P. Pfister C. J. Biol. Chem. 1992; 267: 22018-22021Abstract Full Text PDF PubMed Google Scholar, Pagès et al., 1993Pagès F. Deterre P. Pfister C. J. Biol. Chem. 1993; 268: 26358-26364Abstract Full Text PDF PubMed Google Scholar; Otto-Bruc et al., 1994Otto-Bruc A. Antonny B. Vuong T.M. Biochemistry. 1994; 33: 15215-15222Crossref PubMed Scopus (24) Google Scholar), and more specifically with PDEγ (Arshavsky and Bownds, 1992Arshavsky V.Y. Bownds M.D. Nature. 1992; 357: 416-417Crossref PubMed Scopus (218) Google Scholar; Angleson and Wensel, 1994Angleson J.K. Wensel T.G. J. Biol. Chem. 1994; 269: 16290-16296Abstract Full Text PDF PubMed Google Scholar; Arshavsky et al., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar), 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, 1994Angleson J.K. Wensel T.G. J. Biol. Chem. 1994; 269: 16290-16296Abstract Full Text PDF PubMed Google Scholar; Arshavsky et al., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar; Otto-Bruc et al., 1994Otto-Bruc A. Antonny B. Vuong T.M. Biochemistry. 1994; 33: 15215-15222Crossref PubMed Scopus (24) Google Scholar). rod cGMP phosphodiesterase the complex of PDE α- and β-subunits the complex of PDE α-and β-subunits obtained by PDE trypsinization the γ-subunit of PDE PDEγ labeled by lucifer yellow vinyl sulfone rod outer segments transducin α-subunit guanosine 5′-(γ-thio)triphosphate GTPase activating protein surface plasmon resonance. Our previous study with synthetic peptides corresponding to different segments of PDEγ has shown that the epitope responsible for transducin GTPase activation is located within a stretch of 25 C-terminal amino acid residues of PDEγ (Arshavsky et al., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar). 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 Trp70 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. ROS were purified from frozen retinas (TA & WL Lowson Co., Lincoln, NE) under infrared illumination by double step sucrose flotation (Smith et al., 1975Smith H.G. Stubbs G.W. Litman B.J. Exp. Eye Res. 1975; 20: 211-217Crossref PubMed Scopus (203) Google Scholar). Rhodopsin concentration was determined spectrophotometrically according to Bownds et al., 1971Bownds D. Gordon-Walker A. Gaide-Huguenin A.-C. Robinson W. J. Gen. Physiol. 1971; 58: 225-237Crossref PubMed Scopus (137) Google Scholar. Test membranes used for the measurements of transducin GTPase activity were obtained as described by Arshavsky et al., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar. 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 MgCl2, 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., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar for a detailed explanation). GtαGTPγS was eluted from the test membranes by 20 μM GTPγS, 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., 1979Baehr W. Devlin M.J. Applebury M.L. J. Biol. Chem. 1979; 254: 11669-11677Abstract Full Text PDF PubMed Google Scholar. Soluble trPDEαβ dimer lacking the isoprenylated and carboxymethylated C termini was prepared by tryptic proteolysis (Catty and Deterre, 1991Catty P. Deterre P. Eur. J. Biochem. 1991; 199: 263-269Crossref PubMed Scopus (50) Google Scholar). 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. trPDEαβ was then purified to >95% purity by gel filtration of a Superose-6 column (Pharmacia Biotech Inc.). Protein concentration was determined by the Bradford, 1976Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar assay using bovine serum albumin as the standard. To obtain PDEγ and its mutants the coding sequence of the PDEγ from the synthetic gene for the fusion protein (Brown and Stryer, 1989Brown R.L. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4922-4926Crossref PubMed Scopus (55) Google Scholar) was subcloned into an expression vector pET-11a (Novagen) under control of the isopropyl β-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 β-D-thiogalactoside. PDEγ or its mutants were then purified by a combination of cation-exchange and reverse-phase chromatography (Brown and Stryer, 1989Brown R.L. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4922-4926Crossref PubMed Scopus (55) Google Scholar). The purity of PDEγ was estimated to be >95%; the PDEγ 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, 1976Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar method using the wild type PDEγ as the standard. Both methods yielded identical results. The preparation of PDEγLY and the W70A mutant labeled by lucifer yellow vinyl sulfone was performed as described by Artemyev et al., 1992Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar. Transducin GTPase activity was determined by a single-turnover technique described in detail by Arshavsky et al., 1991Arshavsky V.Y. Gray-Keller M.P. Bownds M.D. J. Biol. Chem. 1991; 266: 18530-18537Abstract Full Text PDF PubMed Google Scholar. 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 MgCl2. The reaction was started by mixing 20 μl of the test membranes (20 μM final rhodopsin concentration) with 20 μl of [γ-32P]GTP (~4 × 104 dpm/pmol, 0.2 μM final concentration) supplemented by various concentrations of PDEγ or its mutants. The reaction was stopped by the addition of 100 μl of 6% perchloric acid. 32Pi formation was measured according to a modified method of Godchaux and Zimmerman, 1979Godchaux III, W. Zimmerman W.F J. Biol. Chem. 1979; 254: 7874-7884Abstract Full Text PDF PubMed Google Scholar described by Arshavsky et al., 1991Arshavsky V.Y. Gray-Keller M.P. Bownds M.D. J. Biol. Chem. 1991; 266: 18530-18537Abstract Full Text PDF PubMed Google Scholar. The GTPase rate constant was determined by the exponential fit of the time course of Pi formation. Fluorescent measurements were performed as described earlier (Artemyev et al., 1992Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar) on a Perkin Elmer LS5B spectrofluorometer in a buffer containing 10 mM HEPES, 100 mM NaCl, and 1 mM MgCl2. The excitation wavelength was 430 nm, and the emission was measured at 520 nm. Fluorescence of 25 nM PDEγLY in the presence of 50 nM GtαGTPγS was measured before and after additions of increasing concentrations of PDEγ or its mutants. PDEγ or mutants cause a decrease in the fluorescence due to their competition with PDEγLY for binding to GtαGTPγS. The KD values in all cases were calculated from the competition curves considering 36 nM as the KD value for the PDEγLY·GtαGTPγS complex (Artemyev et al., 1992Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar). PDEγ 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 PDEγ 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 PDEγ 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 GtαGTPγS or trPDEαβ were injected at a flow rate of 5 μl/min in a buffer containing 120 mM NaCl, 8 mM MgCl2, 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 PDEγ·GtαGTPγS interaction were determined by fitting the data to the general rate equation: dRdt=kass⋅T⋅(P−R)−kdiss⋅R or dRdt=kass⋅T⋅P−R⋅(kass⋅T+kdiss) where R is the PDEγ·GtαGTPγS complex concentration (it is proportional to the amplitude of the SPR signal), t is time, T is GtαGTPγS 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 PDEγ, kass and kdiss 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 kass for PDEγ and each of the mutants could now be determined as slopes of these lines, while kdiss is the ordinate intercept. Alternatively the values of kdiss were determined from the exponential analysis of the SPR signal decay after replacement of the GtαGTPγS solution by the buffer. The kdiss 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 KD values presented in this study. The same analysis was performed in the case of trPDEαβ. Alanine scanning mutagenesis (Gibbs and Zoller, 1991Gibbs C.S. Zoller M.J. Methods: A Companion to Methods Enzymol. 1991; 3: 165-173Crossref Scopus (9) Google Scholar) was used to determine the residues on PDEγ which are responsible for the stimulation of GTPase activity of the rod G-protein, transducin. A previous study (Arshavsky et al., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar) had shown that the segment comprised of the 25 C-terminal amino acid residues of PDEγ, 63DITVICPWEAFNHLELHELAQYGII87, is able to stimulate transducin GTPase practically to the same extent as full-length PDEγ, 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 PDEγ 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., 1989Arshavsky V.Y. Antoch M.P. Lukjanov K.A. Philippov P.P. FEBS Lett. 1989; 250: 353-356Crossref PubMed Scopus (23) Google Scholar, Arshavsky et al., 1991Arshavsky V.Y. Gray-Keller M.P. Bownds M.D. J. Biol. Chem. 1991; 266: 18530-18537Abstract Full Text PDF PubMed Google Scholar, Arshavsky et al., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar) was used to monitor the rate of transducin GTPase. Briefly, the GTPase reaction was initiated by the addition of [γ-32P]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 32Pi reflected a single synchronized turnover of transducin GTPase. This approach is illustrated in Fig. 1 showing a family of the GTP hydrolysis curves obtained with increasing concentrations of PDEγ. 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 PDEγ. 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. In principle, the reduction of the PDEγ 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 PDEγ or inability of the mutant PDEγ to accelerate GTP hydrolysis after forming a complex with transducin. To decide which of these is correct, we studied transducin association with PDEγ mutants by two complementary techniques. The first is based on the ability of transducin's α-subunit complex with GTPγS (GtαGTPγS) to enhance the fluorescence of PDEγ labeled with lucifer yellow vinyl sulfone (Artemyev et al., 1992Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar). The KD for PDEγLY binding with GtαGTPγS was determined from the measurements of fluorescence changes, and then the KD values for the wild type PDEγ and all the mutants were calculated from the analysis of their competition with PDEγLY for binding to GtαGTPγS. Fig. 2B shows that the only mutation causing a substantial (4-5-fold) reduction of the PDEγ affinity for transducin was W70A. Direct measurements of GtαGTPγS 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 PDEγ affinity for transducin (Otto-Bruc et al., 1993Otto-Bruc A. Antonny B. Vuong T.M. Chardin P. Chabre M. Biochemistry. 1993; 32: 8636-8645Crossref PubMed Scopus (65) Google Scholar). The second approach to analysis of transducin-PDEγ interaction was direct monitoring of complex formation by surface plasmon resonance on Pharmacia Biosensor's BIAcore instrument (Jönsson et al., 1991Jönsson U. Fägerstam L. Ivarsson B. Johnsson B. Karlsson R. Lundh K. Löfs S. Persson B. Roos H. Rönnberg I. Sjölander S. Stenberg E. Sthlberg R. Urbaniczky C. stlin H. Malmqvist M. BioTechniques. 1991; 11: 620-627PubMed Google Scholar; Schuster et al., 1993Schuster S.C. Swanson R.V. Alex L.A. Bourret R.B. Simon M.I. Nature. 1993; 365: 343-347Crossref PubMed Scopus (229) Google Scholar). PDEγ (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 GtαGTPγS were applied. The binding of GtαGTPγS with immobilized PDEγ or PDEγ 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 GtαGTPγS dissociation from the chip. The kinetic parameters of the PDEγ·GtαGTPγS interaction were determined as described under “Experimental Procedures.” The data obtained with the SPR measurements are in a good agreement with the determinations of PDEγLY 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 KD values for PDEγ and its mutants obtained with BIAcore were higher than those with the lucifer yellow method most likely reflecting differences in the properties of immobilized PDEγ and free PDEγ in solution. An important conclusion from the analysis of PDEγ 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-PDEγ 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 PDEγ interaction with transducin, does not alter PDEγ interaction with PDE catalytic subunits. The ability of this mutant to inhibit the activity of trPDEαβ was identical to that of the wild type PDEγ (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 trPDEαβ was identical to that for the PDEγ wild type. The dissociation of trPDEαβ from the sensor chip appears to be the same for both mutant and wild type PDEγ. It is slower than the resolution limit of the instrument, 0.0005 s−1, so the value of the KD could be only estimated to be less than 3 nM. PDEγ 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 α-subunit. The second function of PDEγ 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 PDEγ and another membrane-associated factor whose nature remains unidentified (see below). Two domains on PDEγ 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 PDEαβ inhibition resides mainly within the C-terminal sequence Gly85-Ile86-Ile87 (Lipkin et al., 1988Lipkin V.M. Dumler I.L. Muradov K.G. Artemyev N.O. Etingof R.N. FEBS Lett. 1988; 234: 287-290Crossref PubMed Scopus (57) Google Scholar; Brown, 1992Brown R.L. Biochemistry. 1992; 31: 5918-5925Crossref PubMed Scopus (61) Google Scholar; Skiba et al., 1995Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), while the residues between Asp63 and Leu86 (Skiba et al., 1995Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) participate in binding to transducin. Our previous study (Arshavsky et al., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar) showed that a peptide corresponding to the sequence between Asp63 and Ile87 is capable of stimulating transducin GTPase to the same extent as PDEγ. Another part of PDEγ which participates in the binding with both PDEαβ and transducin is the lysine-rich area between residues Arg24 and Gly46 (Morrison et al., 1987Morrison D.F. Rider M.A. Takemoto D.J. FEBS Lett. 1987; 222: 266-270Crossref PubMed Scopus (16) Google Scholar, Morrison et al., 1989Morrison D.F. Cunnick J.M. Oppert B. Takemoto D.J. J. Biol. Chem. 1989; 264: 11671-11681Abstract Full Text PDF PubMed Google Scholar; Lipkin et al., 1988Lipkin V.M. Dumler I.L. Muradov K.G. Artemyev N.O. Etingof R.N. FEBS Lett. 1988; 234: 287-290Crossref PubMed Scopus (57) Google Scholar; Artemyev and Hamm, 1992Artemyev N.O. Hamm H.E. Biochem. J. 1992; 283: 273-279Crossref PubMed Scopus (72) Google Scholar; Takemoto et al., 1992Takemoto D.J. Hurt D. Oppert B. Cunnick J. Biochem. J. 1992; 281: 637-643Crossref PubMed Scopus (42) Google Scholar). The most likely role of this segment is to increase the affinity of PDEγ to both PDEαβ and transducin by providing an additional binding site for these interactions. Here we report the identification of amino acid residues within the Asp63-Ile87 segment of PDEγ 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, Val66, Phe73, Leu76, Leu78, Leu81, Ile86, and Ile87, 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 PDEγ. The alanine substitution of Trp70 results in a reduction of the mutant's affinity for transducin (in agreement with the report of Otto-Bruc et al., 1993Otto-Bruc A. Antonny B. Vuong T.M. Chardin P. Chabre M. Biochemistry. 1993; 32: 8636-8645Crossref PubMed Scopus (65) Google Scholar) 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 PDEγ interaction with transducin. No differences in the interaction between the W70A mutant and PDEαβ were revealed in this study. 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., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar for a more detailed discussion). However, recent data from three laboratories (Angleson and Wensel, 1994Angleson J.K. Wensel T.G. J. Biol. Chem. 1994; 269: 16290-16296Abstract Full Text PDF PubMed Google Scholar; Arshavsky et al., 1994Arshavsky V.Y. Dumke C.L. Zhu Y. Artemyev N.O. Skiba N.P. Hamm H.E. Bownds M.D. J. Biol. Chem. 1994; 269: 19882-19887Abstract Full Text PDF PubMed Google Scholar; Otto-Bruc et al., 1994Otto-Bruc A. Antonny B. Vuong T.M. Biochemistry. 1994; 33: 15215-15222Crossref PubMed Scopus (24) Google Scholar) led the authors to a consensus conclusion that the acceleration of transducin GTPase is a result of coordinate action of PDE (or PDEγ) 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 PDEγ. 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 PDEγ and the membrane factor on the GTP hydrolysis before the factor is characterized. It may be noted, however, that the mechanism of the PDEγ 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 α-subunits of heterotrimeric G-proteins (Markby et al., 1993Markby D.W. Onrust R. Bourne H.R. Science. 1993; 262: 1895-1901Crossref PubMed Scopus (142) Google Scholar; Sondek et al., 1994Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (535) Google Scholar; Coleman et al., 1994Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (753) Google Scholar; Kleuss et al., 1994Kleuss C. Raw A.S. Lee E. Sprang S.R. Gilman A.G. Proc. Natl. Acad Sci. U. S. A. 1994; 91: 9828-9831Crossref PubMed Scopus (94) Google Scholar), and a functionally similar arginine is presumably supplied by GAPs (Brownbridge et al., 1993Brownbridge G.G. Lowe P.N. Moore K.J.M. Skinner R.H. Webb M.R. J. Biol. Chem. 1993; 268: 10914-10919Abstract Full Text PDF PubMed Google Scholar), transducin GTPase acceleration by PDEγ requires the action of eight hydrophobic amino acid residues. Along with recent observations that G-protein α-subunits interact with their effectors by multiple sites not directly involved in GTP-binding (summarized by Artemyev and Hamm, 1994Artemyev N.O. Hamm H.E. Nat. Struct. Biol. 1994; 1: 752-754Crossref PubMed Scopus (3) Google Scholar), our data indicate that PDEγ action may be a result of hydrophobic interactions between long sequences of PDEγ 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, PDEγ 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. We thank Dr. M. I. Simon for many helpful discussions and Dr. R. Swanson for help with the BIAcore instrument." @default.
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• W2010201306 title "An Effector Site That Stimulates G-protein GTPase in Photoreceptors" @default.
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