Regulation of Transducin GTPase Activity by Human Retinal RGS*

(Received for publication, March 28, 1997, and in revised form, May 7, 1997)

Michael Natochin , Alexey E. Granovsky and Nikolai O. Artemyev Dagger

From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory gamma -subunit of PDE (Pgamma ), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin alpha -subunit, Gtalpha GDPAlF4-, while its binding to Gtalpha GTPgamma S or Gtalpha GDP was weak. hRGSr and Pgamma did not compete for the interaction with Gtalpha GDPAlF4-. Affinity of the Pgamma -Gtalpha GDPAlF4- interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of Gtalpha GDPAlF4- binding to Pgamma labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (Pgamma BC). Binding of hRGSr to Gtalpha GDPAlF4- complexed with Pgamma BC resulted in a maximal ~40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for Gtalpha GDPAlF4- (Kd 35 nM). In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s-1. Addition of Pgamma to the reconstituted system reduced the GTPase level accelerated by hRGSr (kcat 0.085 s-1). The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 µM rhodopsin these rates exceeded 0.7 s-1. Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by Pgamma but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.


INTRODUCTION

In vertebrate photoreceptor cells, the signal is transduced from light-activated rhodopsin to the effector enzyme, cGMP-phosphodiesterase (PDE),1 via the heterotrimeric G-protein, transducin (Gtalpha beta gamma ). The GTP-bound alpha -subunit of transducin (Gtalpha GTP) relieves the inhibition imposed by two inhibitory PDE gamma -subunits (Pgamma ) on the enzyme catalytic alpha beta subunits (Palpha beta ). Activation of PDE leads to a closure of cGMP-gated channels in the photoreceptor plasma membranes (1-3). The inactivation of PDE is a critical component of the turnoff mechanism in the visual transduction cascade. This inactivation is controlled by the intrinsic GTPase activity of transducin which hydrolyzes GTP to GDP. The GDP-bound Gtalpha (Gtalpha GDP) has a substantially reduced affinity for Pgamma and releases Pgamma to re-inhibit Palpha beta (1, 4-6). The rate of GTP hydrolysis by transducin measured in vitro (7, 8) is too slow to account for the fast photoresponse turnoff in vivo (9, 10). The Pgamma subunit (11, 12) and a distinct membrane-associated protein factor (13, 14) have been shown to enhance transducin GTPase activity in the activated membrane-bound transducin-PDE complex to a level comparable with the rate of transducin inactivation in vivo. A recent study has shown that a retinal specific member of the RGS family, RGSr, serves as a GTPase-activating protein (GAP) for transducin, providing an additional dimension to an already complex picture of the regulation of transducin GTPase activity (15). Functional relationships between RGSr, the gamma -subunit of PDE, and a putative membrane GAP factor are currently not understood.

Here, we study the interaction between transducin and a human retinal specific RGS (hRGSr), and regulation of transducin GTPase activity by hRGSr. We examine the effects of Pgamma and photoreceptor membrane concentration on modulation of the GTPase activity by hRGSr.


EXPERIMENTAL PROCEDURES

Materials

GTP and GTPgamma S were products of Boehringer Mannheim. Blue-Sepharose CL-6B was obtained from Pharmacia. 3-(Bromoacetyl)-7-diethylaminocoumarin (BC) was purchased from Molecular Probes, Inc. [gamma -32P]GTP (>5000 Ci/mmol) was obtained from Amersham. [35S]GTPgamma S (1250 Ci/mmol) was purchased from NEN Life Sciences Products. All other chemicals were from Sigma.

Preparation of ROS Membranes, Gtalpha beta gamma , Gtalpha GTPgamma S, Gtalpha GDP, Gtbeta gamma , and Pgamma BC

Bovine ROS membranes were prepared as described previously (16). Urea-washed ROS membranes (uROS) were prepared according to protocol in Ref. 17. Hypotonically washed ROS membranes were prepared as described in Ref. 14. Transducin, Gtalpha beta gamma , was extracted from ROS membranes using GTP as described in Ref. 18. The Gtalpha GTPgamma S was extracted from ROS membranes using GTPgamma S and purified by chromatography on Blue-Sepharose CL-6B by the procedure described in Ref. 19. Gtalpha GDP was prepared and purified according to protocols in Ref. 20. Pgamma BC was obtained and purified as described in Ref. 6. The purified proteins were stored in 40% glycerol at -20 °C or without glycerol at -80 °C.

Cloning and Expression hRGSr

A BLAST search at NCBI (Bethesda, MD) to compare the mouse mRGSr cDNA sequence against DNA sequence data bases revealed a human homologue, A28-RGS14p (the GenBank accession number U70426), which is 85% identical to mRGSr. DNA prepared from the amplified human retinal cDNA lambda gt10 library (kindly provided by J. Nathans, Johns Hopkins University) was used as a template for the polymerase chain reaction amplification with primers that were synthesized based on the A28-RGS14p sequence. The polymerase chain reaction was performed in 30 µl of reaction mixture containing 100 ng of the template DNA and 0.5 µM of the following primers: ATACTCTAGACATGTGCCGCACCCTGGC (5') and ATGCCTCGAGACTCAGGTGTGTGAGG (3'). The polymerase chain reaction product (620 bp) was digested with XbaI and XhoI (the restriction sites are underlined) and subcloned into the pGEX-KG vector (21) for GST-hRGSr fusion protein expression. The DNA sequence was verified by automated DNA sequencing at the University of Iowa DNA Core Facility using the 3'-pGEX sequencing primer (Pharmacia) and the 5'-primer shown above. The subcloned sequence was different from A28-RGS14p at two nucleotide positions of the open reading frame (C125 right-arrow T and A160 right-arrow G), leading to substitutions of amino acid residues Ser42 right-arrow Phe and Asn54 right-arrow Asp. Typically, expression host E. coli DH5alpha cells were grown on 2 × TY medium and induced at OD600 = 0.5 by addition of isopropyl-1-thio-beta -D-galactopyranoside (0.4 mM final concentration). After a 4-h induction at 37 °C, cells were harvested and sonicated in 20 mM Tris-HCl (pH 8.0) buffer containing 100 mM NaCl, 2 mM MgCl2, 6 mM beta -mercaptoethanol, and 5% glycerol (buffer A). The supernatant (100,000 × g, 1 h) was loaded on a glutathione-agarose column. GST-hRGSr was eluted from the column using 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM glutathione. GST-hRGSr was then passed through a PD-10 column (Pharmacia) to separate glutathione and digested with thrombin (0.25 NIH units/mg) for 90 min at room temperature. hRGSr was reapplied on a glutathione-agarose column to remove GST.

Binding of Transducin to GST-hRGSr-agarose

Gtalpha GDP or Gtalpha GTPgamma S (6 µM final concentration) were mixed with glutathione-agarose retaining ~10 µg of hRGSr in 40 µl of 20 mM HEPES buffer (pH 7.6), 100 mM NaCl, and 2 mM MgCl2 (buffer B). Where indicated, the buffer contained 30 µM AlCl3, 10 mM sodium fluoride, and 10-30 µM Pgamma . After incubation for 20 min at room temperature, the agarose beads were spun down, washed with 1 ml of buffer B, and the bound proteins were eluted with a sample buffer for SDS-polyacrylamide gel electrophoresis.

Fluorescence Assays

Fluorescence assays were performed on a F-2000 Fluorescence Spectrophotometer (Hitachi) in 1 ml of buffer B essentially as described in Ref. 6. Where indicated, the buffer contained 30 µM AlCl3 and 10 mM sodium fluoride. Typically, hRGSr was added to Pgamma BC prior to addition of transducin. The assays were carried out at equilibrium which was reached in less than 3 s after mixing of the components except when Gtalpha GDP and AlF4- were used. In the latter experiments the equilibrium due to Gtalpha GDP activation by AlF4- was reached in less than 15 s. Fluorescence of Pgamma BC was monitored with excitation at 445 nm and emission at 495 nm. Concentration of Pgamma BC was determined using epsilon 445 = 53,000.

Analytical Methods

Single turnover GTPase activity measurements were carried out essentially as described in Ref. 22. The reaction was initiated by mixing bleached ROS membranes with 200 nM [gamma -32P]GTP (~5 × 104 dpm/pmol) in a total volume of 20 µl. The reaction was quenched by addition of 100 µl of 7% perchloric acid. Nucleotides were then precipitated using charcoal, and 32Pi formation was measured by liquid scintillation counting. Concentrations of transducin in different preparations of ROS membranes were determined using the [35S]GTPgamma S binding assay. ROS membranes were incubated with 2 µM GTPgamma S (105 dpm/pmol) for 10 min at room temperature in 20 µl of buffer B, and the mixture was applied onto GF/B filters (Millipore). The filters were washed with 3 ml of 40 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl and 4 mM MgCl2 and counted in a liquid scintillation counter. To determine if hRGSr or Pgamma may affect the nucleotide binding to Gtalpha beta gamma , 200 nM [35S]GTPgamma S was added to uROS membranes (5 µM rhodopsin) reconstituted with 0.4 µM Gtalpha beta gamma and 1 µM hRGSr (and/or 1 µM Pgamma ) in 20 µl of buffer B. The mixture was immediately (<2 s) diluted into 3 ml of cold 40 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl, 4 mM MgCl2, and 2 µM GTPgamma S. The GTPgamma S binding was then measured as described above. The PDE activity was measured using the proton-evolution assay as described in Ref. 23. Protein concentrations were determined by the method of Bradford (24) using IgG as a standard or using calculated extinction coefficients at 280 nm. SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (25) in 12% acrylamide gels. Rhodopsin concentrations were measured using the difference in absorbance at 500 nm between "dark" and bleached ROS preparations. The Kd and IC50 values were calculated as described in Ref. 26. The results are expressed as the mean ± S.E. of triplicate measurements.


RESULTS

Expression and Purification of hRGSr

The sequence encoding for the human homologue of mouse retinal RGSr protein was polymerase chain reaction amplified from the retinal cDNA library, subcloned into the pGEX-KG vector, and expressed as a GST fusion protein as described under "Experimental Procedures." Eluate of GST-hRGSr from a column with glutathione-agarose is shown in Fig. 1 (lane 2). GST-hRGSr migrated on SDS gels with the expected mobility of a 52-kDa protein. Appearance of an additional doublet at ~28-30 kDa, which most likely contains GST polypeptide, is typical for the GST fusion expression system (27). hRGSr, purified after cleavage of the fusion protein GST-hRGSr with thrombin, migrated as a 25-kDa protein (Fig. 1, lane 3). The yield of hRGSr was ~6 mg/liter of culture.


Fig. 1. Expression and purification of hRGSr. SDS-polyacrylamide gel (12%) stained with Coomassie Blue. Lane 1, soluble fraction after sonication of the E. coli cells induced with isopropyl-1-thio-beta -D-galactopyranoside as described under "Experimental Procedures." Lane 2, GST-hRGSr fusion protein. Lane 3, hRGSr purified after cleavage of GST-hRGSr with thrombin.
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Binding of GST-hRGSr to Gtalpha GDPAlF4-, Gtalpha GTPgamma S, and Gtalpha GDP

A number of RGS proteins (RGS1, RGS4, and mouse RGSr) have been shown to interact preferentially with a transitional Galpha GDPAlF4- conformation of Galpha subunits (15, 28, 29). We examined the ability of GST-hRGSr bound to glutathione-agarose to co-precipitate Gtalpha in different conformations. Fig. 2A shows that GST-hRGSr precipitated stoichiometric amounts of Gtalpha GDPAlF4-, while amounts of Gtalpha GTPgamma S and Gtalpha GDP that co-precipitated with GST-hRGSr were significantly lower. In control experiments, Gtalpha GDPAlF4-, Gtalpha GTPgamma S, and Gtalpha GDP did not co-precipitate with glutathione-agarose that contained no bound GST-hRGSr (not shown). The results suggest that the affinity of hRGSr for the Gtalpha conformations decreases in the following order: Gtalpha GDPAlF4- >>  Gtalpha GTPgamma S > Gtalpha GDP.


Fig. 2. Binding of GST-hRGSr to Gtalpha . SDS-polyacrylamide gel (12%) stained with Coomassie Blue. A, binding of Gtalpha GDP, Gtalpha GDPAlF4-, and Gtalpha GTPgamma S to GST-hRGSr immobilized on glutathione agarose was carried out as described under "Experimental Procedures." Lanes: 1, Gtalpha GDP; 2 and 3, Gtalpha GDP and Gtalpha GDPAlF4- bound to GST-hRGSr, respectively; 4, Gtalpha GTPgamma S; 5, Gtalpha GTPgamma S bound to GST-hRGSr. B, effects of Pgamma on binding of Gtalpha GDPAlF4- to GST-hRGSr. Lanes: 1, Gtalpha GDP; 2, Gtalpha GDPAlF4- bound to GST-hRGSr; and 4, Gtalpha GDPAlF4- bound to GST-hRGSr in the presence of 10 and 30 µM Pgamma , respectively.
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Effects of Pgamma on the Interaction between hRGSr and Gtalpha GDPAlF4-

To determine if Pgamma can compete with hRGSr for the interaction with Gtalpha GDPAlF4-, we initially tested effects of Pgamma on Gtalpha GDPAlF4- binding to GST-hRGSr. Even at high concentrations (up to 30 µM) Pgamma did not affect binding of Gtalpha GDPAlF4- to GST-hRGSr immobilized on glutathione-agarose (Fig. 2B). We next investigated effects of hRGSr on the interaction between Pgamma and Gtalpha GDPAlF4- or Gtalpha GTPgamma S using a fluorescence assay. Addition of Gtalpha GDPAlF4- to a fluorescently labeled Pgamma , Pgamma BC, produced an approximately 7.5-fold maximal increase in the BC fluorescence (Fig. 3A), while Gtalpha GTPgamma S enhanced the fluorescence of Pgamma BC by more than 6-fold (not shown). The Kd values for the Gtalpha GDPAlF4- and Gtalpha GTPgamma S binding to Pgamma BC were 2.8 ± 0.1 and 2.1 ± 0.1 nM, respectively. The affinity of Gtalpha GDPAlF4- binding to Pgamma BC was somewhat higher in the presence of 100 nM hRGSr (Kd 1.2 ± 0.1 nM), suggesting that hRGSr and Pgamma bind to Gtalpha GDPAlF4- noncompetitively (Fig. 3A). Addition of hRGSr had no effect on the fluorescence of Pgamma BC alone (not shown), but resulted in a dose-dependent decrease in the fluorescence enhancement of Pgamma BC caused by the latter binding to Gtalpha GDPAlF4- (Fig. 3B). The fluorescence was decreased maximally by ~40% with an IC50 of ~35 nM. Since hRGSr and Pgamma interact with Gtalpha GDPAlF4- noncompetitively, this IC50 value may serve as an estimate for the affinity of hRGSr interaction with Gtalpha GDPAlF4-. In control experiments, hRGSr did not affect the fluorescence of the Gtalpha GTPgamma S·Pgamma BC complex (not shown).


Fig. 3. Effects of hRGSr on the interaction between Gtalpha GDPAlF4- and Pgamma BC. A, the relative increase in fluorescence (F/Fo) of Pgamma BC (5 nM) alone (squares) or in the presence of 50 (triangles) and 100 nM (circles) hGGSr was determined after addition of increasing concentrations of Gtalpha GDP and is plotted as a function of the free Gtalpha GDPAlF4- concentration. The assay buffer contained 30 µM AlCl3 and 10 mM sodium fluoride. The binding curve characteristics are: squares, Kd = 2.8 ± 0.1 nM, maximum F/Fo = 7.5 ± 0.2, r = 0.99; triangles, Kd = 1.6 ± 0.1 nM, maximum F/Fo = 5.2 ± 0.2, r = 0.98; circles, Kd = 1.2 ± 0.1 nM, maximum F/Fo = 3.9 ± 0.1, r = 0.98. B, the relative increase in fluorescence (F/Fo) of Pgamma BC (5 nM) in the presence of increasing concentrations of hRGSr was determined after addition of 10 nM Gtalpha GDP. The assay buffer contained 30 µM AlCl3 and 10 mM sodium fluoride. The fluorescent change (F/Fo) is plotted as a function of hRGSr concentration. The IC50 value of 35 nM is calculated from the curve.
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Effects of hRGSr on Transducin's GTPase Activity

Single turnover measurements of GTPase activity were carried out as described under "Experimental Procedures." Under these conditions ([GTP] < [Gt]) the GTPase reaction can be analyzed using an exponential function: % GTP hydrolyzed = 100(1-e-kt), where k is a rate constant for GTP hydrolysis. First, transducin GTPase activity was measured in the reconstituted system with uROS membranes. According to previous reports, uROS membranes lack the GAP activity of a membrane factor, and the Pgamma subunit did not affect the GTPase activity of transducin when uROS membranes were used (14). The calculated rate of GTP hydrolysis by transducin (0.4 µM) reconstituted with uROS containing 5 µM rhodopsin was 0.022 ± 0.001 s-1 (Fig. 4A). Addition of 1 µM hRGSr resulted in acceleration of the GTPase activity by more than 10-fold (k = 0.23 ± 0.01 s-1). The basal GTPase activity of transducin was not significantly altered in the presence of 1 µM Pgamma (k = 0.019 ± 0.003 s-1). However, Pgamma substantially reduced the accelerated GTPase activity of the Gtalpha ·hRGSr complex (k = 0.085 ± 0.005 s-1). In control experiments, hRGSr, Pgamma , or the two proteins combined had no notable effect on the binding of [35S]GTPgamma S to transducin under similar conditions (not shown). The reaction was complete in less than 2 s.


Fig. 4. The time course of GTP hydrolysis in suspensions of ROS membranes was determined as described under "Experimental Procedures." The reaction mixtures contained: A, uROS (5 µM rhodopsin) reconstituted with: squares, 0.4 µM Gtalpha beta gamma alone; circles, 0.4 µM Gtalpha beta gamma and 1 µM Pgamma ; triangles, 0.4 µM Gtalpha beta gamma and 1 µM hRGSr; and diamonds, 0.4 µM Gtalpha beta gamma , 1 µM Pgamma , and 1 µM hRGSr; B, untreated ROS (5 µM rhodopsin) containing 0.41 µM Gtalpha beta gamma alone (squares) or in the presence of 1 µM hRGSr (triangles). C, dROS membranes (5 µM rhodopsin) containing 0.38 µM Gtalpha beta gamma alone (squares) or in the presence of: circles, 1 µM Pgamma ; triangles, 1 µM hRGSr; diamonds, 1 µM Pgamma and 1 µM hRGSr. D, untreated ROS (30 µM rhodopsin) containing 2.46 µM Gtalpha beta gamma alone (squares) or in the presence of 1 µM hRGSr (triangles).
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The basal GTPase activity of transducin in the suspension of untreated native ROS membranes containing 5 µM rhodopsin and 0.41 µM transducin was higher (k = 0.052 ± 0.002 s-1) than in the reconstituted system with uROS (Fig. 4B). However, the elevation of transducin GTPase activity by hRGSr was only ~3.3-fold (k = 0.17 ± 0.01 s-1). Perhaps, the presence of PDE containing the Pgamma subunit in native ROS preparations prevented a larger enhancement of the GTPase activity by hRGSr as seen using uROS. To test this idea we have prepared hypotonically washed dROS membranes depleted of PDE. The GTPase activity in suspensions of dROS containing 5 µM rhodopsin and 0.38 µM transducin (k = 0.031 ± 0.001 s-1) was accelerated by ~7.5-fold (k = 0.23 ± 0.02 s-1) with addition of hRGSr (Fig. 4C). The Pgamma subunit increased transducin's GTPase activity by more than 2-fold to a rate of 0.075 ± 0.003 s-1. Effects of hRGSr and Pgamma on transducin were not additive. Moreover, the GTPase activity of transducin was lower in the presence of both hRGSr and Pgamma (k = 0.18 ± 0.01 s-1) than in the presence of hRGSr alone. Interestingly, hRGSr enhanced the GTPase rates in suspensions of untreated ROS to levels similar to those in suspensions of dROS membranes reconstituted with Pgamma .

The GTPase activity of transducin has been shown to elevate with increasing concentrations of ROS membranes presumably due to the action of a putative membrane GAP factor (11-14). At low concentrations of ROS membranes the Gtalpha GTP·Pgamma complex dissociates from Palpha beta and is mainly soluble (30, 31). Increasing the membrane concentration shifts the equilibrium toward the membrane bound complex (Gtalpha GTP)2Palpha beta gamma 2 (32-34) allowing the interaction of Gtalpha GTP with a putative GAP factor. The calculated rate for the GTP hydrolysis in the suspension of native ROS membranes containing 30 µM rhodopsin was ~3-fold higher (k = 0.16 ± 0.02 s-1) than that seen using 5 µM rhodopsin (Fig. 4D). hRGSr was at least as effective at higher concentrations of ROS membranes as it was in diluted ROS suspensions. We estimate that the rate of GTP hydrolysis in ROS suspensions containing 30 µM rhodopsin in the presence of 1 µM hRGSr is >0.7 s-1.

Acceleration of PDE Inactivation by hRGSr

We measured PDE inactivation in suspensions of bleached native ROS membranes using the proton evolution assay (23). The activation and inactivation of PDE was monitored after addition of GTP under single turnover conditions using a pH microelectrode. The PDE activity was maximal in less than 1 s after addition of GTP. Therefore, we were unable to resolve the activation phase. As shown earlier, the rate of PDE inactivation can be well approximated by fitting the inactivation phase with a single exponential decay function (14). The PDE activity in the assay is proportional to the concentration of active Gtalpha GTP. The change of pH due to hydrolysis of cGMP under single GTP turnover conditions can be described using an exponential function: pH = Delta pHmax(1-e-kt) or [cGMP]hydrolyzed = Delta [cGMP]max(1-e-kt). The PDE activity represents a derivative of this function, and it decays with the inactivation constant k from the equation above. In diluted suspensions of native ROS (5 µM rhodopsin), PDE was inactivated with a rate of 0.096 s-1. In the presence of hRGSr, PDE inactivation was enhanced to a rate of 0.31 s-1 (Fig. 5A). The increase in kinact (>3-fold) correlated well with the decrease in maximal amounts of cGMP hydrolyzed in the presence of hRGSr (Fig. 5A). Furthermore, the acceleration of PDE inactivation caused by hRGSr was proportional to the elevation of transducin GTPase activity. We next tested effects of ROS membrane concentration on the modulation of PDE inactivation by hRGSr. The PDE inactivation rate (0.26 s-1) was significantly higher in suspensions of ROS membranes containing 30 µM rhodopsin than in diluted ROS suspensions (Fig. 5B). However, this enhanced rate was not accompanied by an equivalent decrease in the maximal amount of hydrolyzed cGMP because the initial PDE activity after addition of GTP was higher. It has been shown previously that PDE activation by transducin is more efficient at higher concentrations of photoreceptor membranes (32-34). Addition of 1 µM hRGSr to ROS membranes containing 30 µM rhodopsin resulted in ~3-fold increase in the PDE inactivation rate. The PDE inactivation rate was as high as 0.75 s-1 and correlated well with the GTPase rate (>0.7 s-1) measured under the same conditions. To determine if hGRSr can serve as an antagonist for PDE and block the enzyme activation, we have measured effects of hRGSr on GTPgamma S-induced PDE activity in suspensions of ROS membranes containing 5 µM rhodopsin. The rates of the GTPgamma S-induced cGMP hydrolysis were not significantly affected by hRGSr. In the presence of relatively high concentrations of hRGSr (5 µM) PDE activity was suppressed by only ~15% (not shown).


Fig. 5. Effects of hRGSr on the PDE inactivation rates. PDE activity in suspensions of bleached ROS containing 5 µM (A) and 30 µM (B) rhodopsin was measured in the absence (I) or in the presence of 1 µM hRGSr (II) using the proton-evolution assay (23). The pH meter outputs were recorded after addition of 150 nM GTP and fitted with an exponential function [cGMP]hydrolyzed = Delta [cGMP]max(1-e-kt). Results of a typical experiment are shown. Each curve fitted the data with an r value better than 0.998. The calculated PDE inactivation rates are: A: I, 0.096 ± 0.03 s-1; II, 0.31 ± 0.02 s-1; B: I, 0.26 ± 0.02 s-1; II, 0.75 ± 0.05 s-1.
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DISCUSSION

Recent findings have established that members of a new family of RGS proteins serve as GAPs for heterotrimeric G-proteins and attenuate G-protein-mediated signal transduction (28, 35, 36). Evidence suggests that RGS proteins accelerate the rate of GTP hydrolysis by Galpha subunits but do not affect GDP/GTP exchange induced by activated G-protein-coupled receptors (35). Precise mechanisms of RGS GAP activity are not yet clear. It has been demonstrated that at least some RGS proteins (RGS1, RGS4, GAIP, and mRGSr) interact preferentially with the AlF4- bound conformation of Galpha subunits and thus may accelerate GTP hydrolysis through stabilization of the transitional state of G-proteins (15, 28, 29). In several signaling systems the GTPase activity of G-proteins is enhanced by their effector enzymes (11, 37). Regulation of the visual G-protein appears to be even more complicated. The gamma  subunit of PDE in concert with an unidentified membrane factor accelerate GTPase activity of transducin (11-14). The mouse retinal specific RGS (mRGSr) protein has been recently identified and preliminarily characterized. mRGSr was shown to enhance GTPase activity under steady-state conditions (15).

We have investigated the regulation of transducin GTPase activity by human RGSr and examined effects of Pgamma and photoreceptor membrane concentration on the functional activity of hRGSr. Our results support the data that retinal RGS binds tightly to the transitional conformation of transducin, Gtalpha GDPAlF4- (15), and extend this observation by showing that it also weakly interacts with Gtalpha GTPgamma S and Gtalpha GDP. Using a fluorescence assay of interaction between Pgamma BC and Gtalpha GDPAlF4-, we demonstrated that Pgamma and hRGSr interact with Gtalpha GDPAlF4- noncompetitively. Furthermore, the affinity of the Pgamma /Gtalpha GDPAlF4- interaction was modestly enhanced in the presence of hRGSr. This increase in affinity may reflect a stabilization of the Gtalpha GDPAlF4- conformation which interacts with Pgamma with high affinity. Binding of hRGSr to Gtalpha GDPAlF4- affected the Gtalpha conformation resulting in a decrease of the maximal fluorescence enhancement caused by Gtalpha GDPAlF4- binding to Pgamma BC. We used this effect to estimate the affinity of the hRGSr/Gtalpha GDPAlF4- interaction (~35 nM). Interestingly, Gtalpha GTPgamma S and Gtalpha GDPAlF4- had similar high affinities for Pgamma and significantly different affinities for hRGSr, supporting the conclusion that Gtalpha GDPAlF4- has distinct interfaces for interaction with Pgamma and hRGSr. Comparison of the crystal structures of Gtalpha GTPgamma S and Gtalpha GDPAlF4- shows limited conformational differences in the switch I and II regions of transducin (38). This suggests that hRGSr interacts with the switch I and/or switch II regions of Gtalpha GDPAlF4-. Indeed, a crystal structure of RGS4 bound to Gialpha 1AlF4-, that has been published during the review of this paper, shows that the RGS4-binding site is formed by the three switch regions of Gialpha 1 (39). The switch III, alpha 3/beta 5, and alpha 4/beta 6 regions of transducin have been earlier implicated in Gtalpha interaction with Pgamma (5, 40-43). The Pgamma and hRGSr-binding sites on Gtalpha may possibly overlap, particularly, at the switch III region. Because the interactions between the switch III region and RGS protein are not extensive (39), such potential overlap would not cause a significant effect of hRGSr on apparent affinity of Pgamma binding to Gtalpha .

Binding of RGS proteins to the switch regions of G-proteins supports the idea that RGS proteins may serve as antagonists for some effectors (39). RGS4 has been shown to block activation of phospholipase Cbeta by Gqalpha GTPgamma S (44). In our experiments, initial rates of cGMP hydrolysis in suspensions of ROS membranes after addition of GTP were unaffected in the presence of hRGSr (Fig. 5). Likewise, hRGSr has little effect on PDE activity in ROS membranes stimulated by GTPgamma S. It appears that the main mode of RGS action in the transducin/PDE signaling system is to accelerate G-protein and effector inactivation rather than to block effector activation.

Measurements of GTPase activity under single turnover conditions presented in this study demonstrate that hRGSr can dramatically increase the kcat of GTP hydrolysis by transducin. Effects of hRGSr on transducin's GTPase activity were attenuated but not eliminated by Pgamma . Even in the presence of Pgamma , hRGSr accelerated the GTPase activity by ~3-fold. Similar inhibitory effects of Pgamma on stimulation of transducin GTPase activity by mouse RGSr have just been reported (45). However, our data indicate that Pgamma attenuates effects of RGSr allosterically rather than competitively as suggested by Wieland et al. (45). In agreement with earlier observations, the rates of GTP hydrolysis were significantly higher in more concentrated suspensions of photoreceptor membranes (11-14). hRGSr was equally potent as a GAP for transducin regardless of the membrane concentration, indicating that: (a) a putative membrane GAP factor for transducin represents a distinct non-RGS-like protein, and (b) the membrane factor does not compete with hRGS for binding to transducin. A newly discovered second retina-specific RGS protein (RET-RGS1) is a membrane-bound protein and therefore is a potential candidate for a putative GAP factor (46). Our study indicates that RET-RGS1 and an unidentified GAP factor are likely to be different proteins.

The PDE inactivation rates were increased by hRGSr proportionally to the acceleration of the GTPase rates. The evidence that transducin's GTPase activity represents a major mechanism for inactivation of PDE in the turnoff of visual signals (11, 13, 47) has been disputed (48, 49). Our data support the conclusion that transducin's GTPase activity controls PDE inactivation. The rates of GTP hydrolysis and PDE inactivation (>0.7 s-1) observed at relatively high concentrations of photoreceptor membranes in the presence of RGS are adequate to explain fast turnoff kinetics in the visual transduction cascade in vivo.


FOOTNOTES

*   This work was supported by National Eye Institute Grant EY-10843, the services provided by the Diabetes and Endocrinology Research Center of the University of Iowa were supported by National Institutes of Health Grant DK-25295.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Iowa College of Medicine, 5-660 Bowen Science Bldg., Iowa City, IA 52242. Tel.: 319-335-7864; Fax: 319-335-7330; E-mail: Nikolai-Artemyev{at}UIOWA.EDU.
1   The abbreviations used are: PDE, rod outer segment cGMP phosphodiesterase; Gtalpha beta gamma , rod GTP-binding protein transducin; Palpha beta and Pgamma , alpha , beta , and gamma  subunits of PDE; ROS, rod outer segment(s); uROS, urea-stripped ROS membranes; dROS, hypotonically washed ROS membranes; Pgamma BC, Pgamma labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (BC); GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); GST, glutathione S-transferase; h, human; m, mouse.

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