The alpha -Helical Domain of Galpha t Determines Specific Interaction with Regulator of G Protein Signaling 9*

Nikolai P. Skiba, Chii-Shen YangDagger , Tao Huang, Hyunsu Bae, and Heidi E. Hamm§

From the Northwestern University Institute for Neuroscience, Department of Molecular Pharmacology and Biological Chemistry, and Department of Ophthalmology, Northwestern University, Chicago, Illinois 60611 and the Dagger  Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha  subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha  subunit of transducin, Galpha t. Here we report another unique property of RGS9: high specificity for Galpha t. The core (RGS) domain of RGS9 (RGS9) stimulates Galpha t GTPase activity by 10-fold and Galpha i1 GTPase activity by only 2-fold at a concentration of 10 µM. Using chimeric Galpha t/Galpha i1 subunits we demonstrated that the alpha -helical domain of Galpha t imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galpha t (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galpha t and Galpha i1 calculated from the direct binding and competition experiments were 185 nM and 2 µM, respectively. The gamma  subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galpha t. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galpha t suggests a unique mechanism of effector-mediated GAP function of the RGS9.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The alpha  subunits of heterotrimeric G proteins function as molecular switches that determine active and inactive states of signaling pathways. The crystal structures of Galpha t1 and Galpha i1 in their activated, inactive, and transition state forms have revealed the nature of the molecular switches (switches I, II, and III), which are local conformational changes in the regions around the nucleotide binding pocket depending on whether GTP or GDP is bound (1-4). G protein alpha  subunits are activated by seven helical membrane receptors that catalyze the exchange of GDP for GTP by decreasing the affinity of GDP for the alpha  subunit. The lifetime of the active state of an alpha  subunit is defined by the rate of intrinsic GTPase activity that converts GTP to GDP. Therefore, termination of the signal response is dependent on Galpha GTPase activity. Purified Galpha subunits typically display slow (~ 4/min) GTP hydrolysis that often cannot account for the deactivation rates of G protein-controlled processes, for example phototransduction (5) and ion channel regulation (6).

A large family of regulatory proteins that modulate the inactivation rate of Galpha subunits by accelerating their intrinsic GTPase activity has recently been identified (7, 8). These proteins, known as regulators of G protein signaling (RGS), are encoded by at least 19 genes and have been identified in mammalian tissues based on homology to the diagnostic RGS core domain of ~ 120 amino acid residues. It is not yet certain that all RGS proteins are GAPs (GTPase-accelerating proteins) because several very recently identified RGS domains of D-AKAP (9), axin (10), and Lsc (11) are less conserved, especially at the positions that correspond to the contact sites of RGS4 with Galpha i1 (12). However, the RGS domain of p115, which belongs in this less conserved RGS family, has been shown to have GAP activity for Galpha 12 and Galpha 13 but not for Gs, Gi, and Gq subfamilies of Galpha proteins (13), suggesting that these new RGS proteins may have GAP activity toward other G proteins.

The crystal structure of the RGS4·Galpha i1 complex identified the three conformational switch regions of Galpha subunits as the major structural determinants of RGS4 binding to Galpha i1 (12). Unlike Ras·GAP, which contributes a catalytic Arg (14) to the active site of Ras, the core domain of RGS4 does not contribute catalytic residues and is thought to accomplish its GAP function primarily by stabilizing the switch regions of Galpha in the transition state. The crystal structure also suggests that RGS proteins may down-regulate the activity of Galpha subunits not only by acting as GAPs but also by competing for effector binding to Galpha switch regions because these switches are involved in effector binding. Indeed, RGS4 and GAIP can block activation of phospholipase Cbeta 1 by constitutively active Galpha q·GTPgamma S (15). Similar observations have been made for RGS4, GAIP, and RGSr, which compete with the Galpha t effector, the gamma  subunit of cGMP phosphodiesterase (Pgamma ), for interaction with Galpha t (16, 17).

Most RGS proteins studied to date show relatively little specificity for members of the Gi subfamily of Galpha proteins and discriminate minimally among them. Some RGS proteins, for example RGS4 and GAIP, are not selective among several subfamilies of G proteins, being able to act as GAPs toward members of the Gq subfamily as well. Surprisingly, no RGS proteins accelerating Gs GTPase activity have been identified so far, but Galpha s can be converted into a substrate for RGS16 and RGS4 by a single mutation Asp229right-arrowSer (18). Lack of RGS specificity was studied mainly by in vitro assays using expressed RGS domains; thus, the data do not necessarily exclude higher specificity between particular RGS proteins and G proteins in the cellular environment. Lipid modification and membrane association domains can target RGS proteins to certain cellular compartments (19). For example, RGS12 contains a PDZ domain, which could specifically target it to certain G protein-coupled receptors (20). Additionally, the regions flanking the core domain of RGS proteins may possess other structural determinants for specific interaction with Galpha subunits. Finally, an effector-mediated modulation of RGS function may provide another selectivity filter.

In rod photoreceptor cells transducin GTPase activity is too slow (1-2/min) to account for the physiologically measured light response. The clear functional requirement for transducin inactivation promoted biochemical studies that demonstrated that the intrinsic rate of GTP hydrolysis of transducin is enhanced significantly by concentrated suspensions of rod outer segment membranes (21, 22). Further studies have identified that the inhibitory subunit of Pgamma , together with a membrane factor, cooperatively stimulate the GTPase rate of transducin (23-26). The COOH-terminal 25 amino acids of Pgamma possess the GAP determinants (24), and Trp70 located within this region is critical for GAP activity of Pgamma (27). A Pgamma Trp70 right-arrow Ala mutant expressed in transgenic mouse rods caused a decrease in the recovery rate of the flash response (28), suggesting that the normal deactivation of transducin in vivo, similar to its deactivation in reconstituted membranes, requires its interaction with Pgamma . Very recently the membrane factor was identified as RGS9 (29). The predicted amino acid sequence of bovine RGS9 revealed a conserved RGS domain located at the COOH terminus of the molecule. The extended NH2 terminus of RGS9 contains approximately a 190-amino acid region that is homologous to the NH2-terminal domain of RGS7 and Egl-10. Within this region there is an approximately 80-residue subdomain homologous to the consensus sequence of the DEP domain of unknown functional significance, found in a number of signaling proteins (30).

RGS9 is expressed predominantly in the retina at levels significantly higher in cones than in rods (31). RGS9 is tightly associated with membranes and can be solubilized at high concentrations of detergent. There is no evidence for lipid modification or membrane-spanning regions for RGS9, based on analysis of its primary structure. However, Cowan et al. (31) have proposed that a strong electrostatic interaction with the membranes might be the dominant force in its membrane localization. Several other RGSs found in the retina have been shown to stimulate the GTPase activity of transducin: RGSr/16 (16, 32), RGS4, GAIP (17), and RET-RGS1 (33). However, unlike other retinal RGS proteins, the GAP activity of RGS9 is substantially (3-fold) accelerated by Pgamma (29). Immunodepletion of RGS9 from detergent extracts of rod outer segments (ROS) demonstrated that RGS9 is the predominant source of GAP activity in ROS. All of the immunological and biochemical data (29, 31) indicate that RGS9 is the membrane-associated Galpha t GAP that acts cooperatively with Pgamma in stimulation of Galpha t GTPase (24, 25, 34).

Very recently another important function has been defined for RGS9 in photoreceptor cells. Retinal RGS9 is able to inhibit the activity of guanylyl cyclase, thus controlling the levels of cGMP (35). This finding suggests an additional modulatory role of RGS9 downstream of the effector, cGMP phosphodiesterase, as the linker between phosphodiesterase and guanylyl cyclase.

In this study we focus on further biochemical characterization of RGS9. First, we have examined the specificity of RGS9 GAP activity using homologous Galpha t and Galpha i1 as the substrates and found that RGS9 is a more potent GAP for Galpha t. An analysis of various Galpha t/Galpha i1 chimeras for their ability to be substrates for RGS9 has revealed that the GAP-responsive determinants reside within the alpha -helical domain of Galpha t. Kinetic analysis of RGS9 binding to the Galpha t·Pgamma complex shows a distinct, nonoverlapping pattern of a cooperative interaction of RGS9 and Pgamma with Galpha t, providing the structural basis for the acceleration of RGS9 GAP activity by Pgamma .

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- GTP, GTPgamma S, GDP, deoxyribonucleotides, and imidazole were purchased from Boehringer Mannheim. Restriction and DNA modification enzymes were obtained from Boehringer Mannheim or Life Technologies, Inc. Ni-NTA agarose was a product of Qiagen Inc. [gamma -32P]GTP (30 Ci/mmol) was obtained from NEN Life Science Products. All other reagents were from Sigma or other sources described previously (36).

Preparation of ROS Membranes, Gt, Galpha tGDP, Galpha tGTPgamma S, Gbeta 1gamma 1, Galpha i1, and Pgamma -- Gt, Galpha tGTPgamma s, Galpha tGDP, Gbeta 1gamma 1 and rhodopsin containing ROS membranes treated with urea were prepared as described (37). Galpha i1, NH2-terminally modified with His6-tag, was expressed in Escherichia coli and purified as described by Skiba et al. (36). Wild type gamma  subunit of phosphodiesterase was expressed in E. coli and purified as described (38).

Preparation of Galpha t/Galpha i1 Chimeras-- Chi6b is a derivative of Chi6 described by Skiba et al. (36) in which amino acid residues 216-295 of Galpha t are replaced with the corresponding region from Galpha i1 (residues 220-299). Chi6b was generated by changing Cys347 in Chi6 to Ser. Mutagenesis, E. coli expression, and labeling of Chi6b with a thiol-specific fluorescent reagent Lucifer Yellow vinyl sulfone (LY) were carried out as described by Yang et al. (39). The stoichiometry of Chi6b labeling with LY, calculated as a ratio of the concentration of LY and the concentration of chimera in the labeled sample, was 1:1.

Chimera Gi/GtH is a derivative of His6-Galpha i1 in which residues 60-177 of Galpha i1 encompassing the alpha -helical domain are replaced with the corresponding region of Galpha t, residues 56-173. The chimeric gene was constructed by introduction of unique restriction enzyme sites flanking the DNA fragments of Galpha i1 cDNA and Galpha t cDNA which encode the alpha -helical domain. A MluI restriction enzyme site (3'-end of the fragment) was inserted in both Galpha t and Galpha i1 cDNA, and a BstXI site, which is present in Galpha i1 gene, was inserted only in Galpha t cDNA (5'-end of the fragment) using PCR-based mutagenesis with corresponding oligonucleotide primers-mutagenes. The BstXI-MluI DNA fragment of Galpha t was inserted into the Galpha i1 cDNA after cutting off the corresponding fragment of Galpha i1 with BstXI and MluI restriction enzymes.

Chimera Chi6/GiH contains the alpha -helical domain of Galpha i1 in the context of Chi6. The Galpha t alpha -helical domain of this chimera (residues 56-174) was replaced with the corresponding region of Galpha i1 using the same approach as described for the construction of chimera Gi/GtH. The schematic structures of Galpha t/Galpha i1 chimeras are shown in Table I.

                              
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Table I
Structure and functional properties of Galpha t, Galpha i1 and chimeras

Cloning and Expression of RGS9-- Total RNA as a template for cDNA synthesis was purified from fresh frozen bovine retinas using RNeasy Total RNA kit (Qiagen Inc.). Random cDNA for PCR was synthesized using Advantage RT-for-PCR kit (CLONTECH) with an oligo(dT) primer. A DNA fragment encoding residues 284-461 of the bovine retinal RGS9 core domain was amplified by PCR using the specific primers 5'-AAAGGATCCCTGGTGGACATCCCAACCAAG (upstream) and 5'-TTTAAGCTTACGTGGTGGCCGCCTCCCGC (downstream) containing BamHI and HindIII restriction sites respectively (underlined). The resulting PCR product (550 base pairs) was cut with BamHI and HindIII and ligated with the large fragment of the expression vector pQE30 (Qiagen) digested with the same restriction enzymes. The DNA sequence of this construct was confirmed by DNA sequencing over the PCR-amplified region using type III/IV and reverse sequencing primers (Qiagen). The subcloned sequence contained one nucleotide substitution (T right-arrow A) which resulted in the conservative Ser400 right-arrow Thr mutation. The resulting construct (RGS9) encodes a protein where the RGS sequence is preceded by the sequence MRGSHHHHHHGS containing a His6-tag and RGS-His antibody (Qiagen) epitope. Recombinant protein was expressed in E. coli JM109 and purified as described by He et al. (29). The final yield of RGS9 ranged from 5 to 10 mg of more than 85% pure protein/liter of bacterial culture.

GTPase Assay-- Single turnover GTPase reactions were performed under conditions described by He et al. (29) with some minor modifications. Freshly illuminated urea-washed ROS membranes (final concentration 15 µM) were reconstituted with 1 µM Gt or 1 µM Galpha t and 1 µM Gbeta 1gamma 1 in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 0.02 mM AMP-PNP and incubated for 10 min in the dark at room temperature. The reaction was started by the addition of RGS9 and 200 nM [gamma -32P]GTP (~ 105 cpm/pmol) to the reconstituted membranes and quenched by the addition of 100 µl of 7% perchloric acid. Nucleotides were removed by activated charcoal, and free 32Pi was measured by scintillation counting.

Fluorescent Assay-- Binding of RGS9 and/or Pgamma to Chi6b-LY as well as competition among Galpha i1, Galpha t, chimeras, and Chi6b-LY for binding to RGS9 was monitored by the fluorescent change of a single reporter group attached to Cys210 located in the switch II region. Fluorescent measurements were performed on an Aminco-Bowman Series 2 Luminescence Spectrometer (SLM Aminco) at room temperature in 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM MgCl2 (buffer A) using excitation at 430 nm and emission at 520 nm. In the direct binding experiment Chi6b-LY (50 nM) was initially activated by 10 mM NaF and 30 µM AlCl3. The fluorescence of activated Chi6b-LY in the absence of RGS9 or Pgamma is defined as the base line. The fluorescence increase upon the addition of an interacting protein is expressed as a percent of the initial fluorescence of activated Chi6b-LY after the addition of the reactant. Data were best fit to Equation 1,
Y=Y<SUB>0</SUB>+(Y<SUB><UP>max</UP></SUB>−Y<SUB>0</SUB>)/1+10<SUP>(<UP>logEC</UP><SUB>50</SUB><UP>−</UP>X)*H</SUP> (Eq. 1)
where Y0 and Ymax represent, respectively, the initial and maximum values of the binding function which describes the normalized fluorescence at a given concentration of interacting protein; X is a logarithm of the protein concentration; and H is the Hill coefficient. For competition measurements Chi6b-LY (50 nM) activated with AlF4- was mixed with 100 nM RGS9 in buffer A. A typical increase in the fluorescence was 50-60% of the initial. The fluorescence of the Chi6b-LY-AlF4-·RGS9 complex was set to 100%. The fluorescent change (decrease) was monitored after the addition of increasing concentrations Galpha t, Galpha i1, or chimera and normalized as the percent of maximal fluorescence. The initial fluorescence of Chi6b-LY activated with AlF4- was set to 0%. Data were best fit to Equation 1. Kd values for the binding of Galpha t, Galpha i1, and chimeras to RGS9 were determined from their binding or competition curves based on calculated EC50 values, the concentration of Chi6b-LY in the assay (50 nM), and the Kd value for Chi6-LY-AlF4-·RGS9 complex (190 nM, see Fig. 5B).

General Methods-- Protein concentration of Galpha subunits, Pgamma , RGS9, and Gbeta gamma were determined spectrophotometrically using calculated extinction coefficients based on the number of Trp and Tyr residues. The measured concentrations of alpha  subunits were corrected for the amount of functional protein based on a fluorescent assay detecting an AlF4--dependent increase in Trp fluorescence, as described in Ref. 36. To monitor an intrinsic AlF4--dependent conformational change of Galpha t, Galpha i1, and chimeras, tryptophan fluorescence was determined with excitation at 280 nm and emission at 340 nm. The fluorescence of Galpha (200 nM) in buffer A was measured before and after the addition of 10 mM NaF and 30 µM AlCl3.

SDS-polyacrylamide gel electrophoresis of proteins was performed according to the method of Laemmli (40).

Curve fitting of the experimental data and kinetic analysis were performed using Prism 2.01 for Windows 95 from GraphPad.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of the RGS9 RGS Domain-- The sequence corresponding to the RGS domain of the bovine retinal RGS9 (RGS9) (residues 284-461) was amplified from total retinal RNA using the gene-specific primers based on its primary structure (29), NH2-terminally modified with His6-tag, and expressed in E. coli. It was purified on Ni-NTA resin under denaturing conditions (8 M urea) followed by a renaturation step using a slow stepwise dialysis to remove denaturant. Despite significant losses of the RGS9 during renaturation (~60-70% of total protein before dialysis) caused by reaggregation, the yield of the remaining soluble protein/liter of the bacterial culture ranged from 5 to 10 mg. The resulting protein migrated as a 25-kDa band corresponding to its calculated molecular mass and was more that 85% pure (Fig. 1).


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Fig. 1.   Expression and purification of RGS9. SDS-polyacrylamide gel (10-20%) stained with Coomassie is shown. Lane 1, molecular weight markers. Lane 2, purified on the Ni-NTA agarose column and renaturated His6-RGS9. of RGS9 (1 µM) and Pgamma at 5 s. The reaction was stopped at 10 s by the addition of perchloric acid. Data points represent mean percent GTP hydrolysis ± S.E. (n = 3).

RGS9 Is a More Potent Stimulator of GTPase Activity of Galpha t than Galpha i1-- The effect of RGS9 on the Galpha t GTPase activity was measured using a single turnover GTPase assay. 4 M urea is known to inactivate endogenous GAP activity in the ROS membranes without its physical removal (25, 31). The rate of GTP hydrolysis by purified Galpha t reconstituted with purified Gbeta 1gamma 1 and urea-washed ROS was 0.028 ± 0.001 s-1 (Fig. 2A), which is in agreement with previously published data (0.022 s-1) (29, 32). The addition of the RGS9 resulted in a dose-dependent stimulation of transducin GTPase activity with an approximate 10-fold increase at the maximal dose (10 µM RGS9, k = 0.30 ± 0.007, Fig. 2A). A similar stimulatory effect of RGS9 on GTPase activity of Galpha t was observed when holo Gt was reconstituted with urea-washed ROS membranes (data not shown). To evaluate the specificity of RGS9 for different Galpha s we have determined its effect on the GTPase activity of Galpha i1, a close structural homolog of Galpha t. It is known that rhodopsin can catalyze GDP/GTP exchange on Galpha i1 in the presence of Gbeta 1gamma 1 with a rate similar to that of Galpha t (36, 41). Indeed, Galpha i1 GTPase activity in the presence of urea-washed ROS membranes and beta 1gamma 1 (k = 0.031 ± 0.05 s-1, Fig. 2B and Table I) was in good agreement with the kinetic parameters determined in the single turnover GTPase assay using a nucleotide autoexchange assay (42, 43). Surprisingly, RGS9 produced only a 2-fold enhancement (k = 0.058 ± 0.004 s-1) of Galpha i1 GTPase activity at 10 µM, the concentration that maximally stimulated the GTPase activity of Galpha t (Fig. 2B). However, at higher concentrations, stimulatory effects of RGS9 on Galpha i1 increased, reaching approximately 5-fold at 40 µM (Fig. 2B and Table I). This finding demonstrates a high GTPase-activating specificity of RGS protein for Galpha from the Gi subfamily of heterotrimeric G proteins.


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Fig. 2.   Stimulation of GTPase activity of Galpha t (panel A) and Galpha i1 (panel B) by RGS9 and effect of Pgamma (panel C). Time courses of GTP hydrolysis were determined in a single turnover GTPase assay as described under "Experimental Procedures." Urea-washed ROS membranes (15 µM rhodopsin) were reconstituted with 1 µM beta gamma t and 1 µM Galpha t or 1 µM Galpha i1 in the presence of different concentrations of RGS9. The reaction was started at time 0 with 200 nM [gamma -32P]GTP. At the indicated times the reaction mixtures were quenched with perchloric acid. The GTPase reactions were analyzed using an exponential function: % GTP hydrolyzed = 100(1 - e-kt), where k is a rate constant of GTP hydrolysis. Panel C shows the effect of Pgamma on activation of Galpha t GTPase by RGS9. Urea-washed ROS membranes (15 µM) were reconstituted with 1 µM Galpha t and 1 µM beta gamma t. The GTPase reaction was initiated at time 0 by the addition of 200 nM [gamma -32P]GTP followed by the addition

The Switch III Region of Galpha t Is Not Involved in the Selective Interaction with RGS9-- The crystal structure of the RGS4·Galpha i1 complex combined with the data on mutational analysis of Galpha s indicate that the three conformational switch regions of Galpha are the major structural determinants of the RGS-Galpha interface. Although there is a high degree of conservation between Galpha i1 and Galpha t in the switch regions, switch III is the most divergent. To analyze the role of the switch III region in the Galpha t GTPase-activating specificity for RGS9, we measured the ability of RGS9 to stimulate the GTPase activity of the functional analog of Galpha t (Chi6) in which the switch III region of Galpha t was replaced with the corresponding region of Galpha i1 (residues 216-295). Functional analysis of Chi6 has revealed its similarity to Galpha t in interaction with rhodopsin and Gbeta gamma t (36). RGS9 stimulated the GTPase activity of Chi6 to an extent similar to that of Galpha t (approximately 8-fold) (Table I), suggesting that the switch III region of Galpha t plays little if any role in defining the specificity of Galpha t GTPase acceleration by RGS9. Chi6 expressed in E. coli lacks an NH2-terminal myristoyl group. The ability of RGS9 to stimulate the GTPase activity of Chi6 to an extent similar to that of myristoylated Galpha t indicates that the lipid group does not play a critical role in this process.

The Molecular Determinants of Specific GTPase Stimulation by RGS9 Reside within the Helical Domain of Galpha t-- Besides the switch III region of Galpha t, which does not contribute to its sensitive response to RGS9, switches I and II can be considered as the main GAP-responsive regions of Galpha subunit because they have a number of contacts with RGS protein according to the crystal structure of the Galpha i1·RGS4 complex (44). The nearly identical conformation of the switch regions for activated and transition state forms of Galpha t and Galpha i1 (1, 4, 45) suggests that only a difference in the primary structure of the switches could account for the different GTPase stimulation effects of RGS9 on Galpha t and Galpha i1. However, residues in the switch II region are identical between Galpha i1 and Galpha t, whereas in the switch I region only Val185 of Galpha i1, which contacts RGS4, is replaced with Ile in Galpha t. To probe the role of other regions of Galpha t in specifying the interaction with RGS9 we have replaced the alpha -helical domain of Galpha i1 with the corresponding domain of Galpha t and vice versa and evaluated the ability of RGS9 to stimulate GTPase activity of the resulting proteins. Replacement of the alpha -helical domain of Galpha i1 with the alpha -helical domain of Galpha t (chimera Gi/GtH) resulted in an increased stimulation of its GTPase activity by RGS9. The intrinsic rate of GTP hydrolysis for chimera Gi/GtH was 0.032 s-1. A maximal stimulation effect by RGS9 for this chimera was approximately 8-fold at 10 µM RGS9 (Table I), similar to the RGS9 effect on Galpha t and Chi6 (Fig. 2A and Table I). Thus, the presence of the alpha -helical domain of Galpha t in the Galpha i1 context is sufficient to provide the Galpha t-like specificity for RGS9. The GTPase activity of the complementary chimera (Chi6/GiH), where the alpha -helical domain of Galpha t in Chi6 was replaced with the corresponding region of Galpha i1, was Galpha i1-like in its interaction with RGS9 (Table I). These data indicate that the alpha -helical domain of Galpha t possesses the determinants of RGS9 specificity.

Pgamma Potentiates Galpha t GTPase Stimulation by RGS9-- Pgamma is known to cooperate with another protein to stimulate the GTPase rate of Galpha t (24, 34). We have shown previously that the gamma  subunit of cGMP phosphodiesterase inhibits the ability of RGS4 and GAIP to stimulate the GTPase activity of Galpha t, suggesting overlapping binding sites on Galpha t. In contrast, RGS9 acts cooperatively with Pgamma to stimulate GTP hydrolysis (29), thus potentially implicating Pgamma in the physiological regulation of the active lifetime of Galpha t in rods. The effect of Pgamma on the Galpha t GTPase stimulation by RGS9 was examined in the single turnover GTPase assay in the presence of a concentration of RGS9 which gives intermediate GTPase acceleration (1 µM). Pgamma noticeably enhanced Galpha t GTPase acceleration by RGS9 (Fig. 2C). The maximal effect of Pgamma observed at a concentration of 500 nM was approximately a 2.5-fold enhancement (k = 0.11 s-1). In the absence of RGS9, Pgamma did not accelerate Galpha t GTPase up to a concentration of 5 µM when reconstituted with urea-washed ROS membranes.

The Affinity of RGS9 for Galpha t, Galpha i1, and Chimeras-- To compare the functional effects of RGS9 with its affinity for different substrates we have developed a sensitive fluorescent assay. As we reported recently (39), Cys210 located at the distal end of the switch II region of Galpha t can be labeled selectively with the thiol-specific fluorescent reagent Lucifer Yellow. Galpha t and Chi6 have only two cysteine residues accessible for modification with LY, located at positions 210 and 347. We have replaced Cys347 of Chi6 with Ser. The resulting mutant (Chi6b) was labeled selectively at the only accessible cysteine (Cys210) with the fluorescent group. LY at Cys210 of Chi6b was a reporter of the activating conformational change in the switch II region. The addition of AlF4- to the labeled protein resulted in a profound increase in LY fluorescence (200 ± 20%; Fig. 3). The addition of RGS9 increased the fluorescence of Chi6b-LY in a dose-dependent manner (see Fig. 5B). The maximal fluorescence increase of AlF4--activated Chi6b-LY was 108 ± 5% in the presence of 2 µM RGS9 (Fig. 3). The binding was completely reversible by adding an excess of Galpha t (Fig. 4A) or Chi6 (not shown). In the absence of AlF4-, the addition of 2 µM RGS9 to Chi6b-LY caused no detectable fluorescence change (data not shown). The Kd of the Chi6b-LY·RGS9 complex calculated from the binding curve was 190 ± 9 nM (Fig. 5B).


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Fig. 3.   Binding of RGS9 to Chi6b-LY. Sequential fluorescence increase of Chi6b-LY (50 nM) (1 above the arrow) upon the addition of 10 mM NaF and 30 µM AlCl3 (2), followed by the addition of 2 µM RGS9 (3). The AlF4--dependent increase of Chi6b-LY fluorescence was 200 ± 20%. The maximal fluorescent increase of AlF4--activated chimera in the presence of 2 µM RGS9 was 104 ± 5%. The fluorescence trace represent one of five independent similar experiments.


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Fig. 4.   Binding of Galpha t, Galpha i1, and chimeras to RGS9. Panel A, competition between Galpha tGDP-AlF4- or Galpha tGTPgamma S and Chi6b-LY for binding to RGS9. The fluorescence of the complex of RGS9 (100 nM) with Chi6b-LY (50 nM) in the presence of AlF4- was measured before and after the addition of increasing concentrations of Galpha tGDP-AlF4- (squares) or Galpha tGTPgamma S (triangles). Panel B, competition between Galpha i1 or chimeras for binding to RGS9. The fluorescence of Chi6b-LY-AlF4- (50 nM) in the presence of 100 nM RGS9 was measured before and after the addition of increasing concentrations of Galpha i1 (squares), chimera Gi/GtH (triangles), or Chi6/GiH (inverted triangles). The fluorescence change is expressed as a percent of maximal change (100% was fluorescence Chi6b-LY-AlF4--RGS9 complex before adding Galpha subunit, 0% was the initial fluorescence of Chi6b-LY before adding RGS9) and plotted against the Galpha concentration using a four-parameter logistic function (sigmoidal curve) as described under "Experimental Procedures." Data points represent the mean percent fluorescent change ± S.E. (n = 3).


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Fig. 5.   Interaction of Pgamma and RGS9 with Chi6b-LY. Panel A, the increase in fluorescence of AlF4--activated Chi6b-LY (50 nM) was measured after the addition of increasing concentrations of Pgamma in the absence (triangles) or in the presence (squares) of 500 nM RGS9. The initial fluorescence of the Chi6-LY-AlF4- or Chi6b-LY-AlF4-·RGS9 complex, before the addition of Pgamma , was set at 0. Fluorescence change, expressed as a percent of the initial fluorescence, is plotted against the Pgamma concentration. The solid lines represent the best fit to the four-parameter logistic (sigmoidal) equation as described under "Experimental Procedures." The best parameter values for the curve 1 (triangles) are: Kd = 100 ± 11 nM; Delta Fmax, 99%; Hill slope, 1.2; for curve 2 (squares): Kd = 70 ± 8 nM; Delta Fmax, 104%: Hill slope, 1.3, p = 0.31. Panel B, the increase in fluorescence of AlF4--activated Chi6b-LY (50 nM) was measured after the addition of increasing concentrations of RGS9 in the absence (triangles) or in the presence (squares) of 500 nM Pgamma . Data were plotted as described in panel A. The calculated kinetic parameters are: curve 1 (triangles) Kd = 190 ± 9 nM; Delta Fmax, 114%; Hill slope, 1.12; curve 2 (squares) Kd = 67 ± 5 nM; Delta Fmax, 103%; Hill slope, 1.15; p = 0.035. Data points represent the mean percent fluorescent increase ± S.E. (n = 3).

To determine the affinity of RGS9 for different Galpha s we used a competition approach. Fig. 4A shows that unlabeled Galpha t completely displaces the Chi6-LY from its complex with RGS9 in a dose-dependent manner. The Kd of the Galpha t-AlF4-·RGS9 complex calculated from the competition curve was 185 ± 8 nM. Chi6 activated with AlF4- had a similar affinity for RGS9 (Kd 174 ± 11 nM, Table I), closely corresponding to the affinity of this complex calculated in the direct binding experiment (Fig. 5B). However, Galpha tGTPgamma S was less potent in its ability to compete with Chi6b-LY for binding with RGS9 compared with Galpha tGDP-AlF4-. The Kd of the Galpha tGTPgamma S·RGS9 complex calculated from the competition curve was 0.9 µM (Fig. 4A). These data provide an accurate measurement of the difference in affinity of RGS protein for Galpha s in GTP-bound and transition state analog forms, which was reported earlier based on immunoprecipitation (46, 47) and bead precipitation (32) of the Galpha ·RGS complexes.

Galpha i1 was also able to compete with Chi6b-LY for binding to RGS9 and displaced the labeled protein from the complex. The affinity of Galpha i1 for RGS9 calculated from the competition curve was more than 10-fold lower (Kd 2 ± 0.25 µM, Fig. 4B) than that of Galpha t (185 nM). The decreased affinity of Galpha i1 for RGS9 is consistent with its decreased ability to stimulate Galpha i1 GTPase activity.

To determine the region of Galpha t which is responsible for the increased affinity to RGS9 we used Galpha t/Galpha i1 chimeras with exchanged helical domains (Table I) in the fluorescent competition assay (Fig. 4B). Replacement of the alpha -helical domain of Galpha i1 with the corresponding region of Galpha t (chimera Gi/GtH) resulted in a more than 10-fold increase in its affinity for RGS9 (Kd 170 ± 7 nM) compared with Galpha i1 (Kd 2 ± 0.25 µM). On the other hand, the reciprocal chimera where the alpha -helical domain of Galpha t was replaced with the corresponding domain of Galpha i1 (chimera Chi6/GiH) exhibited decreased affinity for RGS9 (Kd 1.6 ± 0.1 µM) compared with Galpha t or Chi6, but similar to Galpha i1 (Table I). Comparison of the functional and binding data indicates that the GTPase stimulation activity of RGS9 correlates well with its affinity for the substrate.

Effect of RGS9 on the Interaction of Pgamma with Chi6b-- Pgamma potentiates the RGS9-mediated stimulation of Galpha t GTPase. However, the mechanism responsible for this effect is not yet known. Different structural events may cause this effect. First, Pgamma could participate in the trimeric complex by binding directly to RGS9. Alternatively, Pgamma could induce a conformational change on Galpha t resulting in a higher affinity for RGS9. Third, Pgamma could participate directly in stabilizing the transition state of the Galpha t·GTP complex. To understand how Pgamma potentiates the GAP effect of RGS9, we have studied the interaction of Pgamma with the Chi6b-LY·RGS9 complex in the fluorescent assay.

We first studied Pgamma interaction with Chi6b-LY. Pgamma increased the fluorescence of Chi6b-LY in the presence of AlF4- in a dose-dependent manner. The binding was specific and completely reversible by the addition of the unlabeled chimera or trypsin-activated phosphodiesterase (data not shown). The maximal fluorescence increase at saturation was 104 ± 5%. The Kd of the Pgamma ·Chi6b-LY·AlF4- complex calculated from the binding curve was 100 ± 11 nM (Fig. 5A). Thus, LY at Cys210 in the switch II region of Galpha t is a sensitive reporter of Pgamma binding. The binding of Pgamma to Chi6b-LY was activation-dependent, since no appreciable change in fluorescence was observed in the absence of AlF4- (data not shown).

To determine the effect of RGS9 on Pgamma binding to Chi6b-LY in the fluorescent assay, we first formed the RGS9·Chi6b-LY complex by mixing RGS9 (500 nM) with the labeled chimera (50 nM). The fluorescence increase was an indicator of complex formation. Under these conditions more than 90% of the chimera was in complex with RGS9 as determined from the binding curve in Fig. 5B (triangles) and, therefore, the effect of Pgamma binding to free Chi6b-LY (less than 10%) is negligible. In the presence of RGS9, Pgamma further increased the fluorescence of LY-labeled Chi6b in a dose-dependent manner reaching a maximal effect (Delta Fmax) of 99% from the initial fluorescence of Chi6b-LY-AlF4- similar to the Delta Fmax of Pgamma binding to free Chi6b-LY (Fig. 5A, 104%). RGS9, prebound to Chi6b-LY, did not change the affinity of Pgamma for the chimera significantly (Kd 70 ± 8 nM, p = 0.31, Fig. 5A). The similar affinity of Pgamma for Chi6 with or without RGS9 present indicates as well that Pgamma and RGS9 binding sites on Galpha t do not overlap.

Effect of Pgamma on the Interaction Between RGS9 and Chi6b-LY-- To evaluate whether Pgamma can modulate the binding of RGS9 to Galpha , we determined the affinity of RGS9 to free and Pgamma -complexed Chi6b-LY in the fluorescent assay. The fluorescence experiment was set up similarly to that described in the previous section. This time, we preformed the complex of Chi6b-LY (50 nM) with Pgamma (500 nM). The increase in the fluorescence of Chi6b-LY after the addition of Pgamma indicated that more than 90% of the chimera was in complex with Pgamma . The addition of increasing concentrations of RGS9 enhanced the fluorescence of the chimera·Pgamma complex (Fig. 5B). The affinity of RGS9 for the chimera·Pgamma complex, as calculated from the binding curve, was 67 ± 5 nM, nearly 3-fold higher than the affinity of RGS9 for the chimera alone (Kd 190 ± 8 nM, p = 0.035, Fig. 5B). The maximum increase in fluorescence of Chi6b-LY upon binding of RGS9 in the presence of Pgamma (104 ± 2%) was similar to its effect on the chimera alone (maximal fluorescent change 114 ± 4%). This indicates that the environmental change around the LY group at Cys210 of Chi6b as a result of RGS9 binding is the same regardless of whether Pgamma is in the complex or not.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Visual stimuli produce very rapid activation of rod photoreceptors, and the inactivation must be very rapid as well for perception of movement. Early biochemical measurements of the GTPase rate of the rod G protein transducin showed the same slow GTPase rate as other G proteins. Because of the clear functional requirement for rapid turn-off, transducin was the first heterotrimeric G protein whose GTPase activity was shown to be regulated. The intrinsic rate of GTP hydrolysis of transducin is enhanced significantly by concentrated suspensions of ROS membranes, providing the initial evidence for GAPs in the ROS (21, 22). Biochemical analysis showed that the inhibitory subunit (Pgamma ) of the visual cascade effector, cGMP phosphodiesterase, could accelerate the GTP hydrolysis rate of transducin (23). Further studies demonstrated that Pgamma alone is not the transducin GAP and requires another unknown membrane protein to activate GTP hydrolysis by transducin (24, 26, 34). Together Pgamma and the membrane factor cooperatively accelerate the GTPase rate of transducin. Very recently this unknown membrane protein was identified to be a member of a large family of RGS proteins, RGS9 (29).

Retinal RGS9 is a unique GAP in its ability to act synergistically with Pgamma . Stimulation of the Galpha t GTPase activity by RGS9 is potentiated by Pgamma (29), unlike other RGS proteins found in the retina RGSr, RET-RGS1, RGS4, GAIP (17, 32, 33). Does this imply that the effector-mediated mechanism of GTPase stimulation by RGS9 is different from that for other effector-independent RGS proteins highlighted by the crystal structure of the RGS4·Galpha ia1 complex? To answer this question we first studied the specificity of GTPase activation by retinal RGS9. The majority of mammalian RGS proteins studied to date can stimulate GTP hydrolysis of several members of the Galpha i family proteins. However, accurate comparisons of kinetic parameters of a particular RGS protein interaction with different G proteins have been difficult because of a lack of methods to compare binding and functional effects. Comparison of the affinity of RGS for structurally homologous proteins may provide important information on the molecular principles of this interaction.

Specificity Determinants of RGS9 Interaction with Galpha t-- Our data indicate that the core domain of the RGS9 is a more potent accelerator of the Galpha t GTPase than of its close structural homolog Galpha i1. The same stimulation effect of RGS9 on Galpha t was detected at 10-fold lower concentrations than on Galpha i1. The difference in the maximal effect by RGS9 on Galpha t (10-fold) and Galpha i1 (5-fold) is also evident. The less potent Galpha i1 GTPase stimulation by RGS9 compared with Galpha t is a result of its decreased affinity for Galpha i1. What is the structural basis for such a specificity for RGS9? The crystal structure of the RGS4-Galpha i1 complex combined with the data on mutational analysis of Galpha s (12, 43, 49, 50) indicates that the three conformational switch regions of Galpha are the major structural determinants of the RGS-Galpha interface. These switch regions are highly homologous in Galpha i1 and Galpha t, and we showed that they are not responsible for the specificity. We found instead that the helical domain determines the specificity of RGS-Galpha interaction. We can switch the specificity by switching the helical domains. This region is less homologous between Galpha i1 and Galpha t.

There are several structural differences in the alpha -helical domain of Galpha t and Galpha i1 which potentially could participate in contacts with RGS9. One of the local differences is evident in the conformation of residues 108-120 (Galpha i1), which correspond to the distal end of helix B and the following loop (4, 45) (Fig. 6). It is noteworthy that Glu116, the only contact of the helical domain of Galpha i1 with RGS4, is located in this region. Glu116 is conserved in Galpha i1 and Galpha t and interacts with Glu161 and Arg166 of RGS4 which correspond to Lys387 and Ala392 of RGS9. Therefore, the different conformation of Glu116 in the alpha -helical domain of Galpha i1 and Galpha t (Fig. 6) may result in abolishing this contact with RGS9 for Galpha i1. Alternatively, the slight difference in the packing of the helical and GTPase domains of Galpha t and Galpha i1 may result in extended orientation of the switch regions and potential RGS9 contact forming residues in the helical domain for Galpha i1 compared with Galpha t. However, conserved interdomain contacts for Galpha i1 (Asp150-Lys270 and Arg178-Glu43) and Galpha t (Asp146-Lys266 and Arg174-Glu39) most likely assure a similar domain packing in both chimeras.


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Fig. 6.   Superposition of alpha -helical domains of Galpha tGDP-AlF4- (45) and Galpha i1GDP-AlF4- (4). Blue shows Galpha i1, red indicates Galpha t. The side chains depicted as ball-and-stick models represent Glu112 of Galpha t (yellow) and Glu116 of Galpha i1 (cyan). The image was generated using WebLab ViewerLite 3.1 from Molecular Simulation Inc.

Pgamma Accelerates Galpha t GTPase Activity Stimulated by RGS9 by Increasing Its Affinity for Galpha t-- Among the many RGS proteins found in the retina only RGS9 can stimulate the GTPase activity of Galpha t synergistically with Pgamma . Unlike RGS9, other retinal RGSs are either not responsive to (33) or are inhibited by Pgamma (17, 32). In some cases it was shown that Pgamma competes with RGS proteins for binding to Galpha t, indicating that Pgamma and RGS binding sites on Galpha t may overlap (16, 17). Our data demonstrate that RGS9 did not significantly change the binding of Pgamma to the labeled derivative of Galpha t (p value = 0.31, t test), indicating that Pgamma and RGS9 binding sites on Galpha t do not overlap. The lack of effect of RGS9 on Pgamma binding to Galpha t also indicates that there is no direct interaction of Pgamma with RGS9 in the Galpha ·Pgamma ·RGS9 complex. On the other hand, Pgamma in complex with Galpha increases the binding of RGS9 to the complex approximately 3-fold (p value = 0.035, t test). The increased affinity of RGS9 to the Chi6b·Pgamma complex closely corresponds to the stimulatory effect of Pgamma on enhancement of Galpha t GTPase by RGS9. The two distinct effects of Pgamma and RGS9 on the binding of each other to Galpha suggest an allosteric effect of Pgamma on binding of RGS9 to Galpha t.

The crystal structure of the Galpha i1·RGS4 complex complemented by the mutational analysis suggests a mechanism by which RGS proteins stimulate GTPase reaction by Galpha . According to this mechanism RGS binds to the switch regions of Galpha and stabilizes the transition state of the Galpha GTP. Unlike for Ras·GAP, no residues of RGS4 contribute catalytically to the active site of Galpha . It is appropriate to assume based on the sequence similarity (35% identity, 58% homology) that the core domain of RGS9 has a similar fold to that of RGS4 and analogous to the Galpha i1·RGS4 interface with Galpha t. What is the structural basis for the Pgamma effect on the RGS GAP activity? It is known that Pgamma contacts Galpha t at alpha -helices 3 and 4 as well as alpha 3-beta 5 and alpha 4-beta 6 loops and switch II and III regions of the alpha  subunit (36, 51, 52). Two residues from the switch II region (Trp207, Ile208) have been identified to interact directly with Pgamma (49, 53). The increased fluorescence of the reporter group attached to Cys210 indicates that RGS9 contacts the switch II region as well. Our data indicate, however, that there is no steric conflict in the trimeric Galpha t·RGS9·Pgamma complex. Earlier we demonstrated that the last 25 COOH-terminal amino acid residues of Pgamma are critical for this GTPase activation (24) and that this region binds to the switch regions of Galpha t (36). Trp70 located within this region plays a critical role in the Pgamma activation of the transducin GTPase rate (27). This suggests that the structural basis for enhancement of RGS9 GAP activity by Pgamma could be a conformational change in the vicinity of the switch II region induced by the COOH terminus of Pgamma which increases the affinity of RGS9 for Galpha t.

Natochin et al. (49) showed that Pgamma and RGS16 binding sites on Galpha t do not overlap. However, Pgamma does not synergize with RGS16. Thus, a nonoverlapping pattern of RGS and Pgamma interaction with Galpha t is not sufficient for cooperative stimulation of the transducin GTPase function. We speculate that the determinants of RGS9 specificity located in the alpha -helical domain of Galpha t distinguish the mechanism of cooperative interaction of Pgamma and RGS9 with transducin (Fig. 6).

Arshavsky et al. (24) showed that phosphodiesterase-depleted ROS membranes, even at high concentrations, cannot accelerate the GTPase activity of transducin. Thus, full-length RGS9 present in ROS is not a transducin GAP in the absence of Pgamma . On the other hand, the RGS domain of RGS9 can on its own stimulate GTPase activity of Gt reconstituted with the membranes (29, this work). These seemingly contradictory observations may suggest a role of the NH2-terminal domain of RGS9 in attenuation of GAP function. One of the possible roles of Pgamma in cooperating with RGS9 in vivo might be to relieve the inactive state of RGS9. The inactive state of RGS9 in the physiological environment thus may assure transducin interaction with Pgamma and phosphodiesterase activation leading to the photoresponse before the rapid inactivation of transducin by RGS. Further characterization of full-length RGS9 and its NH2-terminal domain in vitro and in vivo will provide valuable information for understanding the unique mechanism of effector-potentiated GAP activity of RGS protein.

Snow et al. (54) have very recently demonstrated that RGS11 specifically binds to Gbeta 5 in a region homologous to G protein gamma  subunits. This domain was defined as the GGL domain (G protein gamma  subunit-like domain) and is found in RGS11, 9, 5, 7 and Egl-10. This RGS11·Gbeta 5 complex may exist in vivo because the expression of mRNA for RGS11 and Gbeta 5 in human tissues overlaps. Finding the RGS7·Gbeta 5 complex in cytosolic fractions of photoreceptor cells supports this idea (48). The RGS11·Gbeta 5 complex functions as a GAP that selectively stimulates GTPase activity of Galpha o. It is not clear whether only Gbeta 5 can form complexes with RGS proteins or whether this is a common property for many Gbeta subunits. It is also important to understand if Gbeta 5 is associated with Ggamma in its complex with RGS. Analysis of the functional properties of different Gbeta ·RGS complexes will allow us to understand possible roles of Gbeta interaction with RGS proteins in signaling processes.

    ACKNOWLEDGEMENTS

We thank Jennifer Connor for preparation of Gt, Galpha t and Gbeta gamma t and Theresa Vera and Tarita Thomas for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants EY06062 and EY10291.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.

§ To whom correspondence should be addressed: Northwestern University, Institute for Neuroscience, Searle Bldg., Rm. 5-555, 320 E. Superior St., Chicago, IL 60611. Tel.: 312-503-1109; Fax: 312-503-7345; E-mail: h-hamm{at}nwu.edu.

    ABBREVIATIONS

The abbreviations used are: Galpha t, G protein alpha  subunit of transducin; RGS, regulator of G protein signaling; GAP, GTPase-accelerating protein; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; Pgamma , gamma subunit of cGMP phosphodiesterase; ROS, rod outer segment(s); LY, Lucifer Yellow; PCR, polymerase chain reaction; AMP-PNP, adenosine 5'-(beta ,gamma -imino)triphosphate; GAIP, Galpha -interacting protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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