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• W2016221462 abstract "RGS proteins regulate the duration of G protein signaling by increasing the rate of GTP hydrolysis on G protein α subunits. The complex of RGS9 with type 5 G protein β subunit (Gβ5) is abundant in photoreceptors, where it stimulates the GTPase activity of transducin. An important functional feature of RGS9-Gβ5 is its ability to activate transducin GTPase much more efficiently after transducin binds to its effector, cGMP phosphodiesterase. Here we show that different domains of RGS9-Gβ5 make opposite contributions toward this selectivity. Gβ5 bound to the G protein γ subunit-like domain of RGS9 acts to reduce RGS9 affinity for transducin, whereas other structures restore this affinity specifically for the transducin-phosphodiesterase complex. We suggest that this mechanism may serve as a general principle conferring specificity of RGS protein action. RGS proteins regulate the duration of G protein signaling by increasing the rate of GTP hydrolysis on G protein α subunits. The complex of RGS9 with type 5 G protein β subunit (Gβ5) is abundant in photoreceptors, where it stimulates the GTPase activity of transducin. An important functional feature of RGS9-Gβ5 is its ability to activate transducin GTPase much more efficiently after transducin binds to its effector, cGMP phosphodiesterase. Here we show that different domains of RGS9-Gβ5 make opposite contributions toward this selectivity. Gβ5 bound to the G protein γ subunit-like domain of RGS9 acts to reduce RGS9 affinity for transducin, whereas other structures restore this affinity specifically for the transducin-phosphodiesterase complex. We suggest that this mechanism may serve as a general principle conferring specificity of RGS protein action. regulators of G protein signaling rod outer segments type 6 cyclic nucleotide phosphodiesterase from ROS the inhibitory γ subunit of PDE the ninth member of the RGS protein family catalytic domain of RGS9 the long splice variant of type 5 G protein β subunit the short splice variant of type 5 G protein β subunit GTPase-activating protein high pressure liquid chromatography glutathioneS-transferase nitrilotriacetic acid Proteins of the RGS1 family regulate the duration of G protein signaling by increasing the intrinsic GTP hydrolysis rate on G protein α subunits (see Refs. 1Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (929) Google Scholar, 2De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (508) Google Scholar, 3Cowan C.W. He W. Wensel T.G. Prog. Nucleic Acids Res. Mol. Biol. 2000; 65: 341-359Crossref Google Scholar for recent reviews). At present, over 20 mammalian RGS proteins have been identified that regulate the GTPase activity of most known G protein α subunits. The abundance of both protein types, along with findings that catalytic domains of RGS proteins have a very poor ability to discriminate among different G protein α subunits, makes the problem of specificity in the RGS-G protein interaction one of the most interesting questions in G protein signaling. A striking example of the specificity in RGS protein action is described for the photoreceptor cells of the retina, where RGS9 is able to discriminate between the free activated α subunit of photoreceptor-specific G protein, transducin, and transducin bound to its effector, PDE (reviewed in Refs. 3Cowan C.W. He W. Wensel T.G. Prog. Nucleic Acids Res. Mol. Biol. 2000; 65: 341-359Crossref Google Scholar and 4Arshavsky V.Y. Pugh Jr., E.N. Neuron. 1998; 20: 11-14Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The physiological importance of this selectivity is evident from the necessity for RGS9 to promptly terminate the photoresponse after PDE activation by transducin but not to inactivate transducin before it has a chance to interact with PDE. In photoreceptors, RGS9 is represented by its short splice isoform, which exists as a constitutive complex with the photoreceptor-specific long splice isoform of Gβ5 (5Makino E.R. Handy J.W. Li T.S. Arshavsky V.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1947-1952Crossref PubMed Scopus (194) Google Scholar). RGS9 belongs to a subfamily of RGS proteins including mammalian RGS6, RGS7, and RGS11, all abundant in neural tissues, and two Caenorhabditis elegans RGS proteins, EGL-10 and EAT-16. All members of this RGS9 subfamily can bind Gβ5 through their G protein γ subunit-like domains or GGL (6Snow B.E. Krumins A.M. Brothers G.M. Lee S.F. Wall M.A. Chung S. Mangion J. Arya S. Gilman A.G. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13307-13312Crossref PubMed Scopus (229) Google Scholar, 7Levay K. Cabrera J.L. Satpaev D.K. Slepak V.Z. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2503-2507Crossref PubMed Scopus (84) Google Scholar, 8Sondek J. Siderovski D.P. Biochem. Pharmacol. 2001; 61: 1329-1337Crossref PubMed Scopus (110) Google Scholar) and are likely to exist as constitutive complexes with Gβ5 in vivo(8Sondek J. Siderovski D.P. Biochem. Pharmacol. 2001; 61: 1329-1337Crossref PubMed Scopus (110) Google Scholar, 9Cabrera J.L. De Freitas F. Satpaev D.K. Slepak V.Z. Biochem. Biophys. Res. Commun. 1998; 249: 898-902Crossref PubMed Scopus (114) Google Scholar, 10Zhang J.H. Simonds W.F. J. Neurosci. 2000; 20 (–NIL13): RC59Crossref PubMed Google Scholar, 11Liang J.J. Chen H.H.D. Jones P.G. Khawaja X.Z. J. Neurosci. Res. 2000; 60: 58-64Crossref PubMed Scopus (26) Google Scholar, 12Witherow D.S. Wang Q. Levay K. Cabrera J.L. Chen J. Willars G.B. Slepak V.Z. J. Biol. Chem. 2000; 275: 24872-24880Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The ability of RGS9-Gβ5L to select between free transducin and transducin-PDE complex is achieved because its affinity for free transducin is lower than its affinity for transducin bound to PDE (13Skiba N.P. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2000; 275: 32716-32720Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The effect of PDE is conferred through its inhibitory γ subunit, PDEγ (14Arshavsky V.Y. Bownds M.D. Nature. 1992; 357: 416-417Crossref PubMed Scopus (218) Google Scholar, 15Angleson J.K. Wensel T.G. J. Biol. Chem. 1994; 269: 16290-16296Abstract Full Text PDF PubMed Google Scholar, 16Arshavsky 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), which directly binds to transducin. The catalytic role in activating transducin GTPase belongs to the RGS homology domain of RGS9, RGS9d, which itself can discriminate between free transducin and transducin-PDE complex (17He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 18McEntaffer R.L. Natochin M. Artemyev N.O. Biochemistry. 1999; 38: 4931-4937Crossref PubMed Scopus (25) Google Scholar, 19Skiba N.P. Yang C.S. Huang T. Bae H. Hamm H.E. J. Biol. Chem. 1999; 274: 8770-8778Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 20Sowa M.E. He W. Slep K.C. Kercher M.A. Lichtarge O. Wensel T.G. Nat. Struct. Biol. 2001; 8: 234-237Crossref PubMed Scopus (113) Google Scholar). However, PDEγ causes not more than a 2–3-fold potentiation of the activity of RGS9d, whereas the effect observed with the entire RGS9-Gβ5L complex is ∼20-fold (13Skiba N.P. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2000; 275: 32716-32720Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This implies that the major role in conferring the stimulating effect of PDEγ is played by other, noncatalytic domains of RGS9-Gβ5L. This idea has been recently supported by He et al. (21He W. Lu L.S. Zhang X. El Hodiri H.M. Chen C.K. Slep K.C. Simon M.I. Jamrich M. Wensel T.G. J. Biol. Chem. 2000; 275: 37093-37100Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), who reported that practically all domains within the RGS9-Gβ5L complex contribute to its ability to discriminate between free activated transducin and transducin bound to PDE. In this study, we addressed the kinetic mechanism by which individual domains within RGS9-Gβ5L confer its ability to specifically recognize transducin complexed with PDEγ. This was achieved by analyzing catalytic properties of a series of recombinant RGS9-Gβ5L fragments ranging from RGS9d to the full-length RGS9-Gβ5L. Our approach was similar to that of He and colleagues (21He W. Lu L.S. Zhang X. El Hodiri H.M. Chen C.K. Slep K.C. Simon M.I. Jamrich M. Wensel T.G. J. Biol. Chem. 2000; 275: 37093-37100Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), but the use of an alternative kinetic method enabled us to specifically address the major catalytic parameters of each fragment: their maximal catalytic activities and their affinities for transducin with and without PDEγ. Our surprising finding is that different structures within the RGS9-Gβ5L complex make opposite contributions in establishing this selectivity. The structure, including the putative seven-bladed β-propeller core of Gβ5 connected to GGL, imposes a dramatic reduction in the affinity of RGS9d for both transducin and transducin-PDE complex. All other parts of RGS9 as well as the N terminus of Gβ5L restore this affinity for the transducin-PDEγ complex to a much greater degree than for free transducin. We suggest that this mechanism may serve as a general principle by which the specificity of RGS action is achieved in various signaling pathways. GGL-Gβ5 modules may serve as nonspecific inhibitors of RGS function, whereas other structures in the RGS-Gβ5 complex may counter the action of GGL-Gβ5 specifically toward either appropriate G protein-α subtype or appropriate G protein-α-effector complex. ROS were isolated from frozen bovine retinas (T. A. & W. L. Lawson Co., Lincoln, NE) under infrared illumination as described (22McDowell J.H. Methods Neurosci. 1993; 15: 123-130Crossref Scopus (57) Google Scholar). Urea-treated ROS membranes lacking the GTPase-activating protein (GAP) activity of RGS9 were prepared using a two-step protocol (23Nekrasova E.R. Berman D.M. Rustandi R.R. Hamm H.E. Gilman A.G. Arshavsky V.Y. Biochemistry. 1997; 36: 7638-7643Crossref PubMed Scopus (52) Google Scholar). First, photoreceptor discs were purified from the ROS in the dark (24Smith H.G. Stubbs G.W. Litman B.J. Exp. Eye Res. 1975; 20: 211-217Crossref PubMed Scopus (203) Google Scholar). Second, the residual activity of RGS9 was inactivated by treating discs with 6 murea for 30 min on ice (15Angleson J.K. Wensel T.G. J. Biol. Chem. 1994; 269: 16290-16296Abstract Full Text PDF PubMed Google Scholar). Urea was then removed from the membrane preparation by five consecutive washes of the discs with an isotonic buffer. Rhodopsin concentration in all membranes was determined spectrophotometrically from the difference in absorbance at 500 nm before and after rhodopsin bleaching using the molar extinction coefficient of 40,000 (25Bownds D. Gordon-Walker A. Gaide-Huguenin A.-C. Robinson W. J. Gen. Physiol. 1971; 58: 225-237Crossref PubMed Scopus (137) Google Scholar). Transducin was purified from bovine ROS as described (26Ting T.D. Goldin S.B. Ho Y.-K. Methods Neurosci. 1993; 15: 180-195Crossref Scopus (27) Google Scholar). Transducin concentration used in all calculations was determined based on the maximum amount of rhodopsin-catalyzed GTPγS binding performed as described in Ref. 27Fung B.B.K. Hurley J.B. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 152-156Crossref PubMed Scopus (512) Google Scholar. The PDEγ-(63–87) peptide was synthesized by the solid-phase Merrifield method on an automated peptide synthesizer and purified to homogeneity by reversed-phase HPLC. Purity and chemical formula of the peptide were confirmed by mass spectrometry and analytical reversed-phase HPLC. A schematic illustration of RGS9 constructs used in this study is presented in Fig. 1. The mammalian expression vector pcDNA3 containing mouse RGS9 cDNA (5Makino E.R. Handy J.W. Li T.S. Arshavsky V.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1947-1952Crossref PubMed Scopus (194) Google Scholar) was used as a template for PCR amplification of RGS9 cDNA and its fragments. DNA fragments encoding RGS9 residues 193–431 (GR), 193–484 (GRC), 112–431 (IGR), 112–484 (IGRC), 1–431 (DIGR), and 1–484 (DIGRC) were amplified using specific upstream and downstream primers containing BamHI and EcoRI sites, respectively. The resulting PCR products were cut with BamHI andEcoRI and ligated with the large fragment of a modified version of the baculovirus transfer vector pVL1392 (see below), digested with the same restriction enzymes. DNA sequence of all RGS9 constructs was confirmed over the PCR-amplified regions. The resulting constructs encoded recombinant proteins where RGS9 sequence was preceded in frame by the sequence MAHHHHHHGLVPRGS containing His6 tag and thrombin cleavage site. Baculovirus expression constructs encoding Gβ5S or Gβ5L (modified by a Met-43 to Leu point mutation to block an internal translation initiation site) (28Watson A.J. Aragay A.M. Slepak V.Z. Simon M.I. J. Biol. Chem. 1996; 271: 28154-28160Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) employed the mouse cDNAs ligated between the EcoRI andXbaI cloning sites in pVL1392. Finished constructs in baculovirus transfer vectors were used to generate recombinant baculoviruses using a custom service provided by Pharmingen (San Diego, CA). Positive viral clones were isolated by the plaque assays followed by immunoblotting detection of the expressed RGS9 constructs using anti-RGS9 antibodies (5Makino E.R. Handy J.W. Li T.S. Arshavsky V.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1947-1952Crossref PubMed Scopus (194) Google Scholar). Plaque isolated recombinant viruses were amplified to obtain high titer stocks (>108 plaque-forming units/ml). In our choice of the protein expression systems we followed the strategies reported by He et al. (21He W. Lu L.S. Zhang X. El Hodiri H.M. Chen C.K. Slep K.C. Simon M.I. Jamrich M. Wensel T.G. J. Biol. Chem. 2000; 275: 37093-37100Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).Escherichia coli vector pGEX2T encoding GST-His6-RGS9d fusion protein was a generous gift from T. Wensel (Baylor College of Medicine). GST-His6-RGS9d from the soluble fraction of E. coli lysate was purified using affinity chromatography on glutathione-agarose (Sigma) as described (21He W. Lu L.S. Zhang X. El Hodiri H.M. Chen C.K. Slep K.C. Simon M.I. Jamrich M. Wensel T.G. J. Biol. Chem. 2000; 275: 37093-37100Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The fusion protein was cleaved with thrombin followed by the separation of His6-RGS9d from the GST domain and thrombin on Ni2+-NTA-agarose. All other RGS9 constructs in their complexes with either Gβ5L or Gβ5S were produced in the Sf9/baculovirus expression system. We found, as did others (21He W. Lu L.S. Zhang X. El Hodiri H.M. Chen C.K. Slep K.C. Simon M.I. Jamrich M. Wensel T.G. J. Biol. Chem. 2000; 275: 37093-37100Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), that the expression of all RGS9 constructs containing the GGL domain in soluble form was possible only in the presence of Gβ5. We therefore co-expressed all GGL domain-containing RGS9 constructs with either Gβ5L or Gβ5S. For a standard purification, Sf9 cells (4–5 liters) were cultured in suspension in SF900 II SFM (Life Technologies Inc.) containing 0.1% Pluronic F68, 1% fetal calf serum, and 50 μg/ml gentamicin at 27 °C with constant shaking (125 rpm). Cells (1.5–2 × 106 cells/ml) were co-infected with amplified recombinant baculoviruses encoding desired RGS9 construct and the recombinant virus encoding Gβ5 at a multiplicity of infection of 3. Cells were harvested 72 h after infection and resuspended in 100 ml of ice-cold 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, two tablets of EDTA-free protein inhibitors mixture (Roche Molecular Biochemicals) (buffer A). Remaining procedures were carried out at 4 °C unless otherwise specified. Cells were disrupted by nitrogen cavitation using a Parr bomb using two 30-min compression/decompression cycles at 600 p.s.i. Cell lysates were centrifuged at 100,000 × g for 1 h, and supernatants were collected and loaded onto a 2-ml Ni2+-NTA-agarose column, which had been equilibrated in buffer A. The column was sequentially washed with 20 ml of buffer A and then 20 ml of buffer A containing 20 mmimidazole. Recombinant proteins were eluted off the column with 5 ml of buffer A containing 250 mm imidazole. Imidazole was removed from the protein preparation, and buffer composition was changed to 20 mm Tris-HCl, pH 7.4, 0.1 m NaCl, 5 mm dithiothreitol by a sequential dilution and concentration of the eluate using an Amicon concentrator. The resulting samples were further purified on a Mono S column (Amersham Pharmacia Biotech) using a gradient of NaCl concentration (from 0.1 to 0.4m). The eluted RGS9-Gβ5 complexes were dialyzed against buffer A containing 50% glycerol and stored at −20 °C without significant loss of their functional activity. The purity of the recombinant RGS9 constructs was no less than 80%. The concentration of recombinant proteins was measured spectrophotometrically based on the theoretically calculated values of extinction coefficients at 280 nm. Transducin GTPase activity was determined by using either a multiple turnover ([GTP] > [transducin]) or single turnover ([GTP] < [transducin]) technique as described previously (13Skiba N.P. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2000; 275: 32716-32720Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). GTPase assays were conducted at room temperature (22–24 °C) in a buffer containing 10 mm Tris-HCl (pH 7.8), 100 mm NaCl, 8 mm MgCl2, and 1 mm dithiothreitol. Urea-treated ROS membranes, lacking endogenous GAP activity of RGS9, were used as a source for rhodopsin for assaying GAP activity of the recombinant RGS9 constructs. Rhodopsin in these membranes was activated by illuminating them on ice immediately before the experiments. The reaction was started by the addition of 10 μl of [γ-32P]GTP at desired concentration (∼105dpm/sample) to 20 μl of membranes (20 μm rhodopsin final concentration) reconstituted with proteins of choice. The reaction was stopped by the addition of 100 μl of 6% perchloric acid. 32Pi formation was measured with activated charcoal as described (29Cowan C.W. Wensel T.G. Arshavsky V.Y. Methods Enzymol. 2000; 315: 524-538Crossref PubMed Google Scholar). All data fitting and statistical analyses were performed with the Sigmaplot software, version 6. 10 μl of Ni2+-NTA-agarose beads were equilibrated with the binding buffer: 20 mmTris-HCl, pH 8.0, 300 mm NaCl, 0.25% lauryl sucrose, and 50 μg/ml bovine serum albumin. The beads were then incubated on ice for 20 min with 50 μl of 5 μm His-tagged RGS9 constructs and then washed twice with 200 μl of binding buffer. Washed beads were mixed with 50 μl of binding buffer containing 1 μg of transducin. 10 mm NaF and 30 μmAlCl3 (AlF4−) and/or PDEγ at the final concentration of 1 μm were added when necessary. We used full-length PDEγ in this assay, since its affinity for transducin is higher than that of the PDEγ-(63–87) peptide, allowing better transducin-PDEγ complex retention upon agarose washes. Samples were incubated on ice for 20 min with occasional shaking. The agarose beads were spun down and washed twice with 1 ml of the binding buffer containing 20 mm imidazole and also 10 mm NaF and 30 μm AlCl3 when required. Bound transducin was eluted from the beads with 40 μl of SDS-polyacrylamide gel electrophoresis sample buffer. 10-μl aliquots of eluates were then subjected to SDS-polyacrylamide gel electrophoresis. The α subunit of transducin was detected using monoclonal antibodies 4A (a generous gift from Heidi Hamm, Vanderbilt University). Schematic illustrations of RGS9 domain composition and protein constructs used in this study are presented in Fig. 1. The N-terminal domain of RGS9 containing ∼110 amino acids is called DEP because it is also present in Disheveled, EGL-10, andpleckstrin (30Ponting C.P. Bork P. Trends Biochem. Sci. 1996; 21: 245-246Abstract Full Text PDF PubMed Scopus (144) Google Scholar). The function of this domain in each of these proteins remains unknown, although a recent report on the three-dimensional structure of DEP from Disheveled (Dvl) confirms that this domain exists as a unique, independently folded structure (31Wong H.C. Mao J. Nguyen J.T. Srinivas S. Zhang W. Liu B. Li L. Wu D. Zheng J. Nat. Struct. Biol. 2000; 7: 1178-1184Crossref PubMed Scopus (126) Google Scholar). In the RGS9 sequence, DEP is followed by an ∼80-amino acid stretch, which we will call interdomain. It has the lowest degree of homology among the members of the RGS9 subfamily. The next domain, composed of ∼80 residues, is the GGL domain, providing the binding site for Gβ5 in all representatives of the RGS9 subfamily. In RGS9, GGL binds to Gβ5L, whereas other subfamily members are usually found in a complex with the short splice isoform of Gβ5, Gβ5S (7Levay K. Cabrera J.L. Satpaev D.K. Slepak V.Z. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2503-2507Crossref PubMed Scopus (84) Google Scholar, 8Sondek J. Siderovski D.P. Biochem. Pharmacol. 2001; 61: 1329-1337Crossref PubMed Scopus (110) Google Scholar, 9Cabrera J.L. De Freitas F. Satpaev D.K. Slepak V.Z. Biochem. Biophys. Res. Commun. 1998; 249: 898-902Crossref PubMed Scopus (114) Google Scholar, 10Zhang J.H. Simonds W.F. J. Neurosci. 2000; 20 (–NIL13): RC59Crossref PubMed Google Scholar, 11Liang J.J. Chen H.H.D. Jones P.G. Khawaja X.Z. J. Neurosci. Res. 2000; 60: 58-64Crossref PubMed Scopus (26) Google Scholar, 12Witherow D.S. Wang Q. Levay K. Cabrera J.L. Chen J. Willars G.B. Slepak V.Z. J. Biol. Chem. 2000; 275: 24872-24880Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). GGL is followed by the RGS homology domain of ∼120 residues. RGS9, both the retinal isoform employed here and the brain isoform (32Rahman Z. Gold S.J. Potenza M.N. Cowan C.W. Ni Y.G. He W. Wensel T.G. Nestler E.J. J. Neurosci. 1999; 19: 2016-2026Crossref PubMed Google Scholar), also contains a prominent C-terminal extension, unique among other members of this subfamily. This extension is often called the C-terminal domain. The domain composition of Gβ5L appears more simple. It is thought to have a core seven-bladed β-propeller structural domain characteristic of all G protein β subunits and mostly α-helical N-terminal region, including a photoreceptor-specific 42-amino acid extension, which is absent in Gβ5S and in other known G protein β subunits (28Watson A.J. Aragay A.M. Slepak V.Z. Simon M.I. J. Biol. Chem. 1996; 271: 28154-28160Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The unique opportunity in studying G protein signaling with the components of the phototransduction cascade is that transducin could be supplied with an essentially unlimited amount of its activated receptor, photoexcited rhodopsin. This enabled us to study the catalytic properties of RGS9-Gβ5L and its fragments under multiple turnover steady-state conditions, where the GTP hydrolysis by transducin was immediately followed by transducin's return to its GTP-bound form (13Skiba N.P. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2000; 275: 32716-32720Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 23Nekrasova E.R. Berman D.M. Rustandi R.R. Hamm H.E. Gilman A.G. Arshavsky V.Y. Biochemistry. 1997; 36: 7638-7643Crossref PubMed Scopus (52) Google Scholar). As illustrated in the Reaction 1 below, our approach could be considered a modification of the Michaelis-Menten method. We considered RGS9-Gβ5L constructs as the enzymes, activated α subunit of transducin (Gαt-GTP) as their substrate, and Gαt-GDP and inorganic phosphate as the reaction products. RGS+Gαt­GTP↔k−1k+1RGS·Gαt­GTP→kcatRGS+Gαt­GDP+PiREACTION 1 The power of this approach is that it allowed us to assess both maximal catalytic activities of the RGS9-Gβ5L constructs and their affinities for transducin with and without PDEγ, after calculating corresponding values of kcat and apparentKm. In the context of this study, this approach is preferential over the single turnover approach utilized in most previous studies of transducin GTPase (reviewed in Ref. 29Cowan C.W. Wensel T.G. Arshavsky V.Y. Methods Enzymol. 2000; 315: 524-538Crossref PubMed Google Scholar). Although the single turnover method was extremely productive in establishing the basic observations that GAP for transducin exists in rods (33Arshavsky V.Y. Antoch M.P. Lukjanov K.A. Philippov P.P. FEBS Lett. 1989; 250: 353-356Crossref PubMed Scopus (23) Google Scholar, 34Angleson J.K. Wensel T.G. Neuron. 1993; 11: 939-949Abstract Full Text PDF PubMed Scopus (83) Google Scholar) and that PDEγ is an activator of transducin GTPase (14Arshavsky V.Y. Bownds M.D. Nature. 1992; 357: 416-417Crossref PubMed Scopus (218) Google Scholar, 15Angleson J.K. Wensel T.G. J. Biol. Chem. 1994; 269: 16290-16296Abstract Full Text PDF PubMed Google Scholar, 16Arshavsky 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), it has at least three major limitations that make detailed kinetic analysis of the catalytic properties of RGS9-Gβ5L constructs essentially impossible. First, accurate resolution of GTPase rates above ∼1 turnover/s cannot be achieved by conventional applications of this technique. As a result, maximal rates of GTP hydrolysis stimulated by the most active RGS9-Gβ5L constructs cannot be determined by the single turnover method. Second, the single turnover approach does not provide a means to distinguish whether PDEγ affects the affinity between RGS and transducin or if it directly changes the catalytic rate of an RGS protein. Third, as GTPase rates approach saturation at high RGS concentrations, this method does not resolve whether these rates are determined by the catalytic properties of RGS or by the intrinsic limitations of transducin to form a conformation competent for RGS interaction. To the contrary, the multiple turnover GTPase approach allowed us to determine two essential catalytic parameters of this reaction, kcat and apparentKm, for each RGS construct used. Each individual experiment utilizing the multiple turnover methodology included three data sets obtained at various transducin concentrations: 1) the basal activity of transducin GTPase measured without RGS9, which was a linear function of transducin concentration; 2) GTPase activity in the presence of a small fixed concentration of a given RGS9 construct; 3) GTPase activity in the presence of the same construct and PDEγ at saturating concentration. The basal GTPase activity was then subtracted from the data obtained with RGS9 constructs, which allowed us to analyze the properties of “accelerated” GTPase alone. Both data sets were fitted by hyperbolas, yielding the kinetic parameters of activated GTPase, kcat, and apparentKm. In order to further simplify this analysis, we used saturating concentrations of GTP to ensure that the rate of GTP hydrolysis was not dependent on the GTP concentration. We also substituted PDEγ by its C-terminal PDEγ-(63–87) peptide because PDEγ-(63–87) retains all GAP properties of the full-length PDEγ (13Skiba N.P. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2000; 275: 32716-32720Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 16Arshavsky 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) but, unlike PDEγ, does not inhibit rhodopsin-catalyzed recycling of Gαt-GDP to Gαt-GTP, as discussed in Ref.13Skiba N.P. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2000; 275: 32716-32720Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar. The effects of PDEγ on the catalytic parameters of RGS9d were studied in the experiment illustrated in Fig. 2A. The kinetic parameters, kcat and apparentKm, determined from the hyperbolic fits to the data are summarized in Table I. PDEγ-(63–87) affected two parameters of GTP hydrolysis. First, it decreased the value of apparent Km by ∼3-fold. This is in a good agreement with the 2–3-fold increase in the affinity between RGS9d and transducin by PDEγ observed in direct affinity measurements (19Skiba N.P. Yang C.S. Huang T. Bae H. Hamm H.E. J. Biol. Chem. 1999; 274: 8770-8778Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Second, PDEγ caused an ∼10-fold decrease in thekcat value. This effect was completely unexpected and appeared to contradict an established concept that PDEγ is an activator, not an inhibitor, of both native RGS9-Gβ5L (13Skiba N.P. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2000; 275: 32716-32720Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and RGS9d (17He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 19Skiba N.P. Yang C.S. Huang T. Bae H. Hamm H.E. J. Biol. Chem. 1999; 274: 8770-8778Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 21He W. Lu L.S. Zhang X. El Hodiri H.M. Chen C.K. Slep K.C. Simon M.I. Jamrich M. Wensel T.G. J. Biol. Chem. 2000; 275: 37093-37100Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).Table ISummary of the catalytic parameters obtained with various RGS9-Gβ5L constructs in multiple turnover GTPase assaysConstructkcatKmKmratio+PDEγ-(63–87)−PDEγ-(63–87)+PDEγ-(63–87)−PDEγ-(63–87)μm GTP/μmRGS/sμm transducinRGS9d0.12 ± 0.021.1 ± 0.052.7 ± 1.58.9 ± 1.13.3 ± 1.4GR-Gβ5L>3>>30>>305.3 ± 0.21-aFor these constructs, the value represents the ratio of the slopes of the linear fits to th" @default.
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