Interactions of Phosducin with the Subunits of G-Proteins
BINDING TO THE alpha  AS WELL AS THE beta gamma SUBUNITS*

Petra H. Bauer, Klaus Blüml, Stefan Schröder, Jutta Hegler, Christian Dees, and Martin J. LohseDagger

From the Institut für Pharmakologie und Toxikologie der Universität Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The high affinity interactions of phosducin with G-proteins involve binding of phosducin to the G-protein beta gamma subunits. Here we have investigated whether phosducin interacts also with G-protein alpha  subunits. Interactions of phosducin with the individual subunits of Go were measured by retaining phosducin-G-protein subunit complexes on columns containing immobilized anti-phosducin antibodies. Both the alpha  and the beta  subunits of trimeric Go were specifically retained by the antibodies in the presence of phosducin. This binding was almost completely abolished for both subunits following protein kinase A-mediated phosphorylation of phosducin and was reduced, more for alpha  than for beta  subunits, by the stable GTP analog guanosine 5'-(3-O-thio)triphosphate. Isolated alpha o was also retained on the columns in the presence of phosducin but not in the presence of protein kinase A-phosphorylated phosducin. Likewise, purified G-protein beta gamma subunit complexes as well as purified alpha  subunits of Go and Gt were precipitated together with His6-tagged phosducin with nickel-agarose; this co-precipitation occurred concentration-dependently, with apparent affinities for phosducin of 55 nM (Gbeta gamma ), 110 nM (alpha o), and 200 nM (alpha t). In functional experiments, the steady state GTPase activity of isolated alpha o was inhibited by phosducin by approx 60% with an IC50 value of approx 300 nM, whereas the GTPase activity of trimeric Go was inhibited by approx 90% with an IC50 value of approx 10 nM. Phosducin did not inhibit the GTP-hydrolytic activity of isolated alpha o as measured by single-turnover assays, but it inhibited the release of GDP from alpha o; the rate constant of GDP release was decreased approx 40% by 500 nM phosducin, and the inhibition occurred with an IC50 value for phosducin of approx 100 nM. These data suggest that phosducin binds with high affinity to G-protein beta gamma subunits and with lower affinity to G-protein alpha  subunits. We propose that the alpha  subunit-mediated effects of phosducin might increase both the extent and the rapidity of its inhibitory effects compared with an action via the beta gamma subunit complex alone.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Heterotrimeric GTP-binding proteins (G-proteins) comprise a family of regulatory proteins that couple transmembrane receptors for a variety of neurotransmitters, hormones, and other stimuli to their intracellular effectors (for reviews see Refs. 1-4). G-proteins are composed of alpha , beta , and gamma  subunits. Upon activation by agonist, receptors couple to their G-proteins and promote the exchange of bound GDP for GTP. The resultant conformational change induces dissociation of the GTP-bound alpha  subunit (Galpha )1 from the beta gamma subunit complex (Gbeta gamma ). In this dissociated state Galpha activates effectors such as adenylyl cyclases, other enzymes, or ion channels. Additionally, the free beta gamma subunits also interact with and regulate effectors (reviewed in Refs. 2 and 5). The intrinsic GTPase activity of Galpha then leads to hydrolysis of the bound GTP, and this enables reassociation of GDP-bound Galpha with Gbeta gamma . The resulting reformation of the trimeric G-protein terminates the signal.

A similar GTPase switch function occurs in the small molecular weight monomeric GTP-binding proteins. For these proteins a large array of regulatory proteins have been identified that regulate various steps in the GTPase cycle (6). More recently, regulatory proteins have also been discovered for the heterotrimeric G-proteins. Among these are the growth cone protein GAP-43 and the RGS (regulators of G-protein signaling) family members that have been shown to activate the GTPase activity of several G-protein alpha  subunits (7-10) and a number of proteins that contain the structural motif of the pleckstrin homology domain and that bind to G-protein beta gamma subunits (11), most notably the beta -adrenergic receptor kinases (12-15).

Phosducin is another type of regulator of G-protein signaling that is thought to act via the beta gamma subunits of G-proteins (16, 17). It was initially discovered as a major retinal phosphoprotein that could be copurified with the beta  and gamma  subunits of transducin, Gt (18). Its expression had been thought to be restricted to the retina and the developmentally related pineal gland (18-20), but more recently phosducin has been shown to be ubiquitously expressed (16, 21).

The molecular mechanisms of the interaction of phosducin with G-proteins are not clear. The co-purification of phosducin from the retina with the beta gamma subunit complex of Gt (18) indicated high affinity interactions between these proteins, and both the N and the C terminus of phosducin appear to contain high affinity binding sites for the beta gamma subunit complex (22-25). From these functional as well as structural observations it has been concluded that phosducin interacts exclusively with the beta gamma subunits of G-proteins. Indeed, studies on the effects of phosducin and phosducin-like protein on Gt suggest that phosducin acts by "trapping" free beta gamma subunits; the GDP-bound alpha  subunits would then be unable to find free beta gamma subunits for reassociation and would therefore stay inactive (17, 26, 27).

In contrast to the many studies on phosducin-Gbeta gamma interactions, potential interactions with G-protein alpha  subunits have not been investigated. Such direct effects on alpha  subunits may be proposed from the observation that the extent of inhibition of Go GTPase is often larger than would be expected from antagonism of the function of beta gamma subunits alone. A possible solution to this problem would be a direct interaction of phosducin with alpha  subunits. Therefore, the present study was undertaken to define the nature of interactions of phosducin with the subunits of G-proteins, using mostly Go as a model system.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Protein Purification-- Recombinant phosducin was expressed in Escherichia coli as described earlier using the plasmid pET-phosducin (16). Following lysis of the bacteria by freeze thawing and precipitation of DNA with 2% streptomycin and centrifugation at 50,000 × g for 10 min, the supernatant was applied to a MonoQ column (Amersham Pharmacia Biotech) and eluted with a 0-500 mM NaCl gradient in 10 mM Tris-HCl, pH 7.4. Peak fractions of phosducin eluting at approx 250 mM NaCl were concentrated to approx 1 ml and were then further purified by gel filtration on Superdex 200 (Amersham Pharmacia Biotech). The purity of the preparations was >95% as determined by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue. Mock preparations from E. coli transformed with "empty" vector were used as controls as described earlier (16).

Alternatively, phosducin was expressed in E. coli with a C-terminal His6-tag and purified on Ni-NTA-columns (Qiagen) as recently described (25). This resulted in apparently pure preparations as judged by Coomassie-stained SDS-polyacrylamide gels.

Go and its resolved alpha o and beta gamma subunits were purified from bovine brain according to the methods described by Sternweis and Robishaw (28). The amounts of Go or alpha o were determined by [35S]GTPgamma S binding as described by Freissmuth and Gilman (29). Contaminating beta gamma subunits were removed from the alpha o preparation by a second round of heptylamine-Sepharose chromatography. The absence of contaminating beta  subunits from these alpha o preparations was verified in Western blots using a beta common antibody kindly provided by Dr. G. Schultz (Department of Pharmacology, Free University, Berlin, Germany). The alpha  and beta gamma subunits of Gt were purified to apparent homogeneity from bovine retina according to a protocol adapted from Gierschik et al. (30).

Phosphorylation of Phosducin-- The catalytic subunit of protein kinase A was purified to apparent homogeneity from bovine heart by the method of Sugden et al. (31). 1 µg of purified phosducin was incubated with 2 units of the PKA catalytic subunit in a buffer containing 10 mM Tris-HCl, pH 6.5, 5 mM MgCl2, 0.1 mM EDTA, 70 mM KCl, 0.5 mM dithiothreitol, 0.1 mM ATP, 5 mg/ml bovine serum albumin, 0.1% Tween 80, 0.2 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin for 30 min at 0 °C and 30 min at 30 °C. At the end of the phosphorylation procedure, PKA inhibitor peptide was added to a final concentration of 10 µM. In preliminary experiments we verified that this was sufficient to completely suppress further PKA activity. Control samples were treated identically, but the PKA catalytic subunit was absent during the incubations. The extent of phosphorylation was determined by addition of a small amount of [gamma -32P]ATP and measurement of the incorporation of 32P into phosducin; phosphorylated phosducin used in the present experiments contained 0.8-1.0 mol phosphate/mol phosducin.

Binding of G-proteins and Phosducin to Anti-phosducin Antibody Columns-- An antiserum against phosducin was generated by immunization of rabbits with purified recombinant phosducin, and phosducin-specific antibodies were isolated by affinity chromatography (21, 32). 2 ml of antiserum were incubated with 1 ml of wet protein A-Sepharose (Amersham Pharmacia Biotech) for 1 h at room temperature under gentle rocking. The resin was washed twice with 10 volumes of 0.2 M sodium borate, pH 9.0, and was suspended in 10 volumes of 0.2 M sodium borate, pH 9.0. The cross-linker bis(sulfosuccinimidyl)suberate (Pierce) was then added to a concentration of 1 mg/ml. After shaking for 30 min at room temperature, the reaction was stopped by washing the resin with several volumes of 0.2 M ethanolamine, pH 8.0, followed by incubation with 0.2 M ethanolamine, pH 8.0, for 2 h at room temperature. The resin was then washed with 5 volumes of 100 mM glycine-HCl, pH 2.5, and 40 volumes of phosphate buffered saline.

To study binding of G-proteins or their subunits to phosducin, equal molar amounts of phosducin and Go (or alpha o as indicated) were preincubated for 10 min at room temperature and then added to 0.5 ml of the anti-phosducin resin together with 200 µl of 20 mM Hepes, pH 7.4, 5 mM MgSO4, 0.1% Lubrol. The suspension was incubated under gentle rocking for 12 h at 4 °C and the resin was subsequently washed with 5 ml of phosphate buffered saline/0.5% Tween 20, followed by a buffer change to 10 mM Tris-HCl, pH 6.8 (5 ml). The entire washing procedure was completed in less than 5 min. Phosducin and proteins bound to phosducin were then eluted with 1 ml of 100 mM glycine-HCl, pH 2.5. After neutralization and drying, these proteins were resolved by SDS-polyacrylamide gel electrophoresis and identified by Western blotting with specific antibodies against the alpha  or beta  subunit (Signal Transduction Laboratories). Peroxidase-coupled secondary antibodies and enhanced chemoluminescence reagents (Amersham) were used to develop the blots.

Co-precipitation of Phosducin-His6 and G-protein Subunits with Ni-NTA-Agarose-- As an alternative to antibody immobilization of phosducin in the phosducin/G-protein binding assays we used immobilization of hexahistidine-tagged phosducin on Ni-NTA-agarose. Purified phosducin-His6 (20 nM) was incubated with various concentrations of Gbeta gamma or alpha o purified from bovine brain, or alpha t purified from bovine retina, in phosphate-buffered saline containing 0.05% cholate (140 mM NaCl, 30 mM KCl, 6.5 mM Na2HPO4, pH 7.3) for 30 min at 4 °C. The mixture was centrifuged at 14,000 rpm for 10 min, and 40 µl of Ni-NTA resin (Qiagen) were added to the supernatant to bind phosducin-His6 (plus associated proteins). After 10 min at 4 °C, the resin was pelleted by centrifugation and washed twice in the same buffer with intervening short centrifugations. G-protein subunits retained together with phosducin-His6 were detected by taking up the beads in SDS-sample buffer followed by SDS-polyacrylamide gel electrophoresis and Western blotting as above.

Determination of GTPase Activity-- The steady state GTPase activity of Go or alpha o was measured as described earlier by determining the formation of 32Pi from [gamma -32P]GTP (33). 0.1 pmol (2 nM) of Go or alpha o was assayed in buffer containing 0.1% Lubrol and 25 mM MgSO4. Phosducin was present at various concentations. The incubations lasted for 30 min at 30 °C and were terminated by addition of 500 µl of 1% charcoal in 2 mM NaH2PO4, pH 2.

The catalytic activity of alpha o was measured in single-turnover assays adapted from Freissmuth and Gilman (29). alpha o (1 pmol/time point) was incubated with 1 µM [gamma -32P]GTP (5 × 105 cpm/time point) in a buffer containing 50 mM Hepes, pH 7.6, 1 mM EDTA, 0.2 mM AppNHp, 1 mg/ml bovine serum albumin, 0.2 mM dithiothreitol, and 0.1% Lubrol in a volume of 50 µl/time point for 15 min at 20 °C. Phosducin was present in a final concentration of 2 µM. Aliquots were withdrawn at the begin of the incubation and after 15 min, and then the hydrolysis reaction was started by addition of MgSO4 and GppNHp to final concentrations of 10 mM and 100 µM, respectively. 50-µl aliquots were withdrawn at the indicated time intervals, and the reactions were stopped by pipetting the aliquots into 500 µl of 1% charcoal (w/v) in 2 mM NaH2PO4, pH 2. The amount of 32Pi formed was determined as above.

Determination of [alpha -32P]GDP Release-- alpha o (0.5 pmol/time point) was incubated with 500 nM (or the indicated concentrations) phosducin and 1 µM [alpha -32P]GTP (105 cpm/time point) in a buffer containing 50 mM Hepes, pH 7.6, 0.1% Lubrol, 1 mg/ml bovine serum albumin, and either 100 µM or 25 mM free Mg2+, added as MgSO4, for 30 min at 20 °C, to allow binding of GTP and hydrolysis to GDP. The [alpha -32P]GDP release reaction was started by addition of GppNHp to a concentration of 100 µM. Aliquots of 50 µl were withdrawn at the indicated intervals, and the reaction was stopped by addition to ice-cold stop solution (10 mM Hepes, pH 7.6, 50 mM NaCl, 5 mM MgSO4, 5 mM NaF, 10 mM AlCl3). Samples were applied to BA-85 nitrocellulose filters (Schleicher & Schuell, 0.45 µm), the tubes were rinsed twice with 2 ml of stop solution, and each rinse was applied to the filter. Filters were then washed two times with 20 ml of stop solution and counted by liquid scintillation counting. Phosducin was present in these assays either in varying concentrations as indicated or, for kinetic studies, in 50-fold excess over alpha o.

Data Analysis-- Kinetic data were fitted assuming exponential functions as described earlier (34). Concentration response curves were fitted to the Hill equation, and binding data were analyzed with a nonlinear binding analysis program as described (34, 35).

The concentration response curve of the GTPase inhibition of Go by phosducin (see Fig. 6) was also analyzed assuming an interaction of phosducin with both the alpha  subunit and the beta gamma subunit complex.
A=100−[I<SUB>&bgr;&ggr;</SUB>∗P/(K<SUB>&bgr;&ggr;</SUB>+P)+I<SUB>&agr;</SUB>∗P/(K<SUB>&agr;</SUB>+P)] (Eq. 1)
where A denotes the GTPase activity (in the percentage of the control activity), Ialpha and Ibeta gamma denote the maximal inhibition caused via the alpha  or beta gamma subunits, respectively, Kalpha and Kbeta gamma denote the affinities of phosducin for the alpha  or beta gamma subunits, respectively, and P is the concentration of phosducin. All data are derived from at least three independent experiments and represent means ± S.E. unless stated otherwise.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

To demonstrate directly the binding of phosducin to the subunits of Go, we incubated phosducin with Go or alpha o in solution and then immobilized phosducin to detect which subunits were bound to phosducin. This was done by covalently coupling phosducin-specific antibodies raised in rabbits (32) to protein A-Sepharose, which allowed specific retention of phosducin (and proteins bound to phosducin) on small columns. In preliminary experiments we verified that the antibodies showed no cross-reactions with any of the subunits of Go, so that the Go subunits should only be retained on the columns if they were bound to phosducin. Phosducin (plus associated proteins) was then eluted from the columns with 100 mM glycine-HCl, pH 2.5, and the eluates were analyzed for the presence of alpha  and beta  subunits of Go in Western blots. (We did not analyze the presence of gamma  subunits due to a lack of high affinity antibodies and also because the beta gamma subunit complex does not appear to dissociate under physiological conditions.) Fig. 1A shows that using trimeric Go the alpha  as well as beta  subunits were retained on the column in the presence of phosducin and that there was virtually no retention of either alpha  or beta  subunits when mock preparations were used instead of phosducin. The retention of the alpha  as well as the beta  subunits was sensitive to phosphorylation of phosducin by PKA (Fig. 1B). For both subunits, PKA-mediated phosphorylation decreased the amount of retention by more than 80% as determined by densitometric analysis. This corresponds roughly to the 80-100% stoichiometry of phosducin phosphorylation that was achieved with PKA in these assays (see "Experimental Procedures") and suggests that phosphorylated phosducin binds the subunits of Go weakly if at all.


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Fig. 1.   Binding of phosducin to the subunits of Go. Phosducin and Go or alpha o were preincubated and then loaded onto an anti-phosducin antibody column. After washing, specifically bound proteins were eluted with 100 mM glycine-HCl, pH 2.5, and the eluates were analyzed for the presence of alpha  and beta  subunits of Go by Western blotting. The data shown are representative of three to five independent experiments. A, specificity of the binding reaction. Go (200 pmol) was incubated with (+) or without (-) an equal amount of phosducin and then loaded onto the anti-phosducin antibody column. The left lanes containing a mock preparation from E. coli with empty vector (-) indicate the nonspecific retention of alpha  and beta  subunits to the column, and the right lanes indicate the phosducin-dependent retention. B, inhibition of Go-phosducin binding by PKA-mediated phosphorylation of phosducin. Phosducin was phosphorylated by PKA (+) or incubated under identical conditions without PKA (-) prior to addition of Go. Incubation and retention were done as in A. C, effects of the dissociation of Go induced by GTPgamma S on the binding to phosducin. Go was preincubated with 100 µM GTPgamma S for 5 min before addition of phosducin.

Preincubation of Go with GTPgamma S to dissociate alpha o from the beta gamma subunits markedly reduced the amount of retained alpha o but caused only a minor reduction of retained beta  subunits (Fig. 1C). This result is compatible with the notion that phosducin binds to the beta gamma subunit complex with high affinity, whereas binding to alpha o is of low affinity. However, this experiment does not indicate whether the residual binding of alpha o is due to incomplete dissociation of Go by GTPgamma S or whether it does indeed represent direct low affinity binding of alpha o to phosducin.

Direct low affinity binding of alpha o to phosducin was then demonstrated with isolated alpha o. The preparations of alpha o used in these experiments were devoid of residual beta gamma subunits by the criterion of Western blots (data not shown). Incubation of such alpha o with phosducin led to the retention of alpha o on the column, but, as expected for a low affinity interaction, the amounts of alpha o retained were small (Fig. 2). Again, this binding was essentially abolished when PKA-phosphorylated phosducin was used (Fig. 2).


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Fig. 2.   Binding of phosducin to alpha o and inhibition of this binding by PKA-mediated phosphorylation of phosducin. The experiment was carried out as that shown in Fig. 1B using 100 pmol of purified alpha o (instead of trimeric Go) and an equal amount of control or PKA-phosphorylated phosducin. No beta  subunits could be detected in the alpha o preparation by Western blotting (not shown). The data are representative of three independent experiments.

To confirm direct binding of alpha  subunits to phosducin, a second strategy involving another method to purify and to immobilize phosducin was used. To this end, a hexahistidine tag was added to the C terminus of phosducin, and phosducin-His6 was purified to apparent homogeneity via binding of this hexahistidine tag to Ni-NTA-agarose. When 50 nM phosducin-His6 were co-incubated with various concentrations of purified Gbeta gamma and then pelleted with Ni-NTA-agarose, Gbeta gamma were co-precipitated as detected by Western blots (Fig. 3A). Much less Gbeta gamma was precipitated in the absence of phosducin, indicating a relatively low nonspecific binding of Gbeta gamma to the resin. A semiquantitative analysis of this experiment by densitometry (Fig. 3B) revealed that the binding of Gbeta gamma to phosducin-His6 was saturable with an apparent affinity of 25 nM. Analysis of four similar experiments gave an average apparent affinity of 55 ± 22 nM.


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Fig. 3.   Binding of Gbeta gamma to phosducin-His6. Phosducin-His6 (20 nM) was preincubated with the indicated concentrations of bovine brain Gbeta gamma and then precipitated with Ni-NTA-agarose. After washing, co-precipitated Gbeta was detected by Western blotting. The data shown are representative of four independent experiments. A, Western blot of Gbeta precipitated in the presence (phosducin) or the absence (control) of phosducin-His6. B, analysis of the total (presence of phosducin, open circles), nonspecific (absence of phosducin, open squares), and specific (total - nonspecific, filled circles) binding of Gbeta gamma to phosducin. The data were obtained by densitometric scanning of Western blot bands as shown in A. Analysis of the binding curve by nonlinear curve fitting gave an apparent affinity of 25 nM. Four similar experiments gave a mean value of 55 ± 22 nM.

Analogous experiments were then done with purified alpha o (Fig. 4). Again, there was a phosducin-His6-dependent precipitation of alpha o with Ni-NTA-agarose. A semiquantitative analysis showed saturable binding of alpha o to phosducin-His6 with an apparent affinity of 85 nM (Fig. 4), and seven similar experiments gave an average value of 110 ± 12 nM.


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Fig. 4.   Binding of alpha o to phosducin-His6. Binding of bovine brain alpha o to phosducin-His6 was analyzed as described in Fig. 3 for Gbeta gamma . The data shown are representative of seven independent experiments. A, Western blot of alpha o precipitated in the presence (phosducin) or the absence (control) of phosducin-His6. B, analysis of the total (open circles), nonspecific (open squares), and specific (filled circles) binding of alpha o to phosducin. Analysis of the binding curve by nonlinear curve fitting gave an apparent affinity of 84 nM. Seven similar experiments gave a mean value of 110 ± 12 nM.

Because Gt and its subunits can be purified better than Go (and also because phosducin was initially discovered in the visual system) such experiments were also carried out with alpha t, which had been purified to apparent homogeneity from bovine retina (Fig. 5). These experiments gave essentially similar results as those obtained with alpha o, but higher concentrations of alpha t were required. The apparent affinity was 160 nM in the experiment shown in Fig. 5 and 200 ± 53 nM in a total of four separate experiments.


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Fig. 5.   Binding of alpha t to phosducin-His6. Binding of alpha t from bovine retina to phosducin-His6 was analyzed as described in the legend to Fig. 3 for Gbeta gamma . The data shown are representative of three independent experiments. A, Western blot of alpha t precipitated in the presence (phosducin) or the absence (control) of phosducin-His6. B, analysis of the total (open circles), nonspecific (open squares), and specific (filled circles) binding of alpha t to phosducin. Analysis of the binding curve by nonlinear curve fitting gave an apparent affinity of 160 nM. Three similar experiments gave a mean value of 200 ± 53 nM.

These data provide direct evidence for binding of phosducin not only to the beta gamma subunit complex but also to G-protein alpha  subunits. Functional assays were then used to determine the effects of such binding to alpha  subunits, again using alpha o because of its substantial intrinsic GTPase activity. Fig. 6 shows the effects of phosducin on the steady state GTPase activity of alpha o and trimeric Go. Phosducin inhibited the steady state GTPase activity of alpha o in a concentration-dependent manner; maximal inhibition was approx 60%, and the IC50 value was somewhat higher (approx 300 nM) than expected from the direct binding assays. As already shown earlier for trimeric Go (16) no such inhibition was seen with PKA-phosphorylated phosducin (data not shown). These results demonstrate a direct inhibitory effect of phosducin on the GTPase activity of alpha o in agreement with its direct binding.


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Fig. 6.   Concentration-dependent inhibition alpha o and Go GTPase activity by phosducin. The steady state GTPase activity of trimeric Go or isolated alpha o (2 nM) in solution was assayed in the presence of the indicated concentrations of phosducin using an incubation time of 30 min and a reaction volume of 100 µl. Nonlinear curve fitting using the Hill equation gave the following estimates: alpha o: IC50 299 ± 26 nM; maximal inhibition, 61 ± 2%; Go: IC50 10 ± 1 nM; maximal inhibition, 90 ± 1%. The latter set of data was fitted significantly better (dotted line, p < 0.01 by F-test) assuming a beta gamma -dependent and an alpha -dependent component (see "Data Analysis") with the following estimates: beta gamma component: IC50, 7 ± 1 nM; maximal inhibition, 73 ± 3%; alpha  component: IC50 120 ± 37 nM, maximal inhibition, 19 ± 3%. Data are the means ± S.E. from fourteen (alpha o) and eight (Go) independent experiments.

The GTPase activity of trimeric Go under the same conditions (i.e. basal activity in 0.1% Lubrol), was inhibited by phosducin up to approx 90% with an IC50 value of approx 10 nM (Fig. 6). Assuming that this inhibition is caused both by a direct effect of phosducin on alpha o and the well known effect via Gbeta gamma , we also analyzed this inhibition curve, assuming that it has a beta gamma -dependent and an alpha -dependent component as described under "Data Analysis." This analysis gives a fit indicating that the beta gamma -dependent component causes 73% inhibition with an IC50 value of approx 7 nM, whereas the alpha -dependent component causes an additional 19% inhibition with an IC50 value of approx 120 nM. The latter affinity is in the range of the IC50 values determined from the direct effects of phosducin on alpha o; likewise, the extent of inhibition (approx 19/(100-73), i.e. approx 70%) is similar to the approx 60% inhibition seen in the GTPase assays with alpha o alone. This two-component fit is statistically significantly better (p < 0.01 by F-test) than the simple one-component fit, but the differences between the two curves are very small.

The GTPase cycle contains two main steps: hydrolysis of GTP to GDP and release of GDP (followed by very rapid binding of GTP). The effects of phosducin on the first step in alpha o were assayed in single-turnover experiments. In these assays, [gamma -32P]GTP is bound to Go in the absence of Mg2+, conditions that suppress the enzymatic activity of G-proteins. The addition of Mg2+ plus a large excess of a stable GTP analog then allows the monitoring of the hydrolysis of the bound [gamma -32P]GTP. Fig. 7 shows that even a high concentration (2 µM) of phosducin had no appreciable effect on this catalytic step. GTP hydrolysis occurred with a rate constant of 4.8 ± 0.4 min-1 in the absence and 3.9 ± 0.4 min-1 in the presence of phosducin. The amount of [gamma -32P]GTP hydrolyzed was almost identical under the two conditions (0.51 ± 0.01 pmol/pmol Go under control conditions versus 0.56 ± 0.02 in the presence of phosducin). These data indicate that the GTP-hydrolytic step of Go is not significantly affected by phosducin.


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Fig. 7.   Lack of effect of phosducin on the hydrolytic step of the GTPase cycle of alpha o. alpha o (1 pmol/50-µl aliquot) was preloaded with [gamma -32P]GTP in a magnesium-free buffer in the absence (open circles) or the presence (filled circles) of 2 µM recombinant phosducin. GTP hydrolysis was started after 15 min by the addition of 10 mM Mg2+ and 100 µM GppNHp (final concentrations). 50-µl aliquots were withdrawn at the indicated times, and the amount of 32Pi formed was determined. Data are means of three independent experiments. Nonlinear curve fitting assuming an exponential time course gave kcat values of 4.8 ± 0.4 min-1 (control) and 3.9 ± 0.4 min-1 (phosducin).

However, phosducin did impair the GDP release step of isolated alpha o. The presence of 0.5 µM phosducin reduced the rate constant of GDP release from isolated alpha o from 0.23 ± 0.01 to 0.14 ± 0.01 min-1 (Fig. 8). Concentration response curves for this inhibitory effect were done by monitoring the amount of GDP remaining bound to alpha o 5 min after initiation of the release reaction (Fig. 9). Phosducin caused a concentration-dependent increase in the amount of bound GDP, and this effect was half-maximal at approx 100 nM. This concentration is in agreement with the apparent affinity of phosducin for alpha o determined in the direct binding assays (Fig. 4B).


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Fig. 8.   Inhibition of [alpha -32P]GDP release from isolated alpha o by phosducin. GDP release from alpha o (0.5 pmol in a reaction volume of 50 µl) was determined in the absence and the presence of 25 pmol (0.5 µM) phosducin. Shown is the amount of [alpha -32P]GDP that remained bound to alpha o at the indicated times after initiation of the release reaction. koff values calculated by nonlinear curve fitting were 0.23 ± 0.01 min-1 under control conditions and 0.14 ± 0.01 min-1 in the presence of phosducin. Data are the means ± S.E. of four independent experiments.


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Fig. 9.   Concentration-dependent inhibition of GDP release from isolated alpha o by phosducin. Shown is the amount of [alpha -32P]GDP remaining bound to alpha o (8 nM) 5 min after initiation of the release reaction as described in the legend to Fig. 8. Phosducin was present at the indicated concentrations. Nonlinear curve fitting gave an EC50 value of 100 ± 17 nM. Data are the means ± S.E. from four independent experiments.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Phosducin is a widely distributed inhibitor of the GTPase activity of trimeric G-proteins (16, 17, 21, 22, 26). In addition to inhibition of the enzymatic activity of G-proteins themselves, it has also been shown to inhibit G-protein-mediated activation of adenylyl cyclase in the beta -adrenergic receptor system (16) and of cGMP phosphodiesterase in the rhodopsin system (17). In intact cells its overexpression impairs cAMP production induced by stimulation of beta -adrenergic receptors (32). In the present study we have addressed the role of the G-protein subunits in this process and in particular whether in addition to the well demonstrated interactions with G-protein beta gamma subunits phosducin might also have effects on the alpha  subunits.

Our data indicate that phosducin can indeed interact directly with G-protein alpha  subunits. This was shown in direct binding experiments as well as in functional assays. In the direct binding experiments two different factors were used to immobilize phosducin: specific affinity-purified antibodies and a C-terminal hexahistidine tag. These two strategies first permitted the use of two different initial purification procedures for phosducin and second should result in very different kinds of nonspecific binding of the G-protein subunits. With the antibody method no detectable amounts of G-protein subunits were retained with the use of mock preparations from E. coli instead of phosducin (Fig. 1A), indicating first that the antibodies showed no significant reactivity toward any of the G-protein subunits and second that the retention of G-protein subunits was not due to a contaminating protein. We conclude that these assays did indeed measure the interaction of phosducin with G-protein subunits. With the hexahistidine tag method there was detectable nonspecific precipitation of both Galpha and Gbeta gamma (Figs. 4-6), presumably due to the fact that the washing conditions could not be chosen as harsh as in the antibody method. However, this nonspecific binding was relatively low and allowed the clear detection of saturable specific binding of Gbeta gamma , alpha o, and alpha t to phosducin. The demonstration of direct binding to alpha t as well as alpha o is important for two reasons. First, alpha t can be purified to apparent homogeneity, indicating that no other proteins are required to effect phosducin-Galpha interactions. Second, it indicates that phosducin might affect G-protein alpha  subunits also in the visual system.

The analysis of these binding assays by densitometry can be only semiquantitative due to the nonlinearity of the detection method. However, the apparent affinities correlate fairly well with those determined in functional assays; the affinities of phosducin for Gbeta gamma reported by various groups using various methods is in the range of 8-80 nM (16, 22, 23, 36-38). The value found in our direct binding assay was 55 nM, which is in the upper range of the reported functional values. It is about 10-fold better than the previously obtained values utilizing Gbeta gamma directly immobilized on microtiterplates, which presumably resulted in distortion of Gbeta gamma (37).

The apparent affinity of phosducin for alpha o determined in the direct binding assay was 110 nM, whereas in the functional assays IC50 values of 100 nM (GDP release) and 300 nM (GTPase) were obtained; the analysis of the inhibition of Go-GTPase with a beta gamma - and an alpha -dependent component gave an IC50 value of the alpha -component of 120 nM. These affinities agree within the accuracy of the methods used.

The affinity of phosducin for alpha o is lower than that for the beta gamma subunit complex. However, an affinity in the range of 100-300 nM is still below the concentration of phosducin in most tissues, which is about 1 µM (21). Thus, this affinity is sufficient to allow phosducin-Galpha interactions in vivo. Likewise, the high concentrations of phosducin in the retina (18-21) are above the levels required for the low affinity interaction with alpha t. Furthermore it appears that these interactions do indeed contribute to the inhibition of G-protein function by phosducin; the Go GTPase activity was inhibited by phosducin by up to 90% (Fig. 6). Under the conditions of these assays (in particular 0.1% Lubrol and high Mg2+), Gbeta gamma causes a 2-4-fold activation of the GTPase activity of alpha o (39). Thus, if phosducin acted only to "trap" Gbeta gamma , it should inhibit the GTPase activity of Go by at most 50-75%. This is clearly less than the observed inhibition of 90% and suggests an additional mode of inhibition. The analysis of this inhibition curve with two components (alpha  and beta gamma ) did indeed result in a significantly better fit. Even though the improvement is only small, this two-component fit is entirely compatible in qualitative and in quantitative terms with the other data obtained here; it gives a Gbeta gamma -mediated inhibition by 73% with an IC50 value of 7 nM, compatible with the ranges given above, and a Galpha -mediated inhibition of the remaining activity by 70% with an IC50 value of 120 nM, compatible with the affinities and the extent of inhibition seen in assays with isolated alpha o.

The functional effects of phosducin on alpha o consist in an inhibition of GDP release but no effect on the catalytic step of the GTPase cycle. Thus, the effects of phosducin on Go appear to be composed of a direct inhibition of GDP release from alpha o, and an antagonism of the effects of the beta gamma subunit complex on the function of alpha o. Because the beta gamma subunits have stimulatory effects on alpha o under activating conditions, such as in the presence of high Mg2+ concentrations, mastoparan, or active receptors (39), the direct and the Gbeta gamma -mediated effects of phosducin on alpha o are additive under the conditions of stimulated Go activity.

The effects of phosducin on Galpha should not only cause an increased inhibition of G-protein function compared with Gbeta gamma -mediated effects alone, but they should also increase the rapidity of this inhibition. This is because trapping of Gbeta gamma subsequent to G-protein activation would leave GTP-bound Galpha free to interact with effectors and disrupt the G-protein cycle only after the first round of GTP hydrolysis. In contrast, a direct effect on Galpha might already affect signaling in this first cycle of G-protein activation. Taken together these data suggest that the interactions of phosducin with alpha o are of functional relevance.

Direct interactions of phosducin with G-protein alpha  subunits were not observed by Lee et al. (17) and Yoshida et al. (26) in their investigations on transducin (Gt). These authors found no comigration of phosducin and alpha t on gel filtration columns and no effect of phosducin on the binding of alpha t to rod outer segment membranes. There are two possible explanations for this discrepancy. First, we found that the affinity of phosducin for alpha t is lower than that for alpha o. Second, the assays used by these authors, which involve physical separation of proteins by chromatography or by centrifugation and washing, may be less sensitive to interactions of low affinity than the assays used here. In fact, gel filtration chromatography of phosducin has been shown to involve interactions with the matrix (26) that might well result in disruption of low affinity phosducin-alpha subunit interactions, and the affinity of phosducin for Gt reported in binding assays with rod outer segment membranes (26) is approx 10-fold lower than that found in our assays.

In our hands, phosphorylation of phosducin by PKA impaired binding to Gbeta gamma as well as to alpha o. This was seen both in direct binding and in GTPase assays. The data about the effect of phosphorylation on the phosducin/transducin-beta gamma interaction are conflicting, depending on the assay used. Phosphorylated phosducin no longer coeluted with transducin-beta gamma from gel filtration columns but still inhibited transducin-beta gamma binding to rod outer segment membranes (26). It was concluded that phosphorylation might not alter the affinity of phosducin for transducin-beta gamma but rather affect the character of the interaction (26). However, Hawes et al. (22) observed a loss of Gbeta gamma binding upon phosphorylation of phosducin similar to our data. Because phosphorylation of phosducin would be required to impair Gbeta gamma binding by the C-terminal as well as the N-terminal binding sites, it is plausible to assume that it involves a major alteration of the structure of phosducin (24). Under these circumstances it is not surprising that phosphorylation of phosducin would also impair its interactions with Galpha .

The molecular mechanisms of the interaction between phosducin and Galpha remain to be elucidated. The crystal structure of phosducin complexed with the beta gamma subunits of Gt (24) suggests that phosducin binds with its C-terminal domain, particularly with an essential stretch of a few essential amino acids close to the C terminus (25), to the side of the beta -propeller, whereas its less well defined N terminus is stretched out on the face of the propeller covering sites where Gbeta gamma interacts with Galpha . Detailed studies will be required to elucidate Galpha -binding sites in phosducin. Furthermore, we do not know whether the interactions of phosducin with Go involve two separate binding events (one with Gbeta gamma and another one with alpha o), two-step binding (first to Gbeta gamma and then to the alpha beta gamma trimer), or a single composite reaction to form a tetrameric phosducin-alpha beta gamma complex.

In summary, we believe that our data support interactions of phosducin with Gbeta gamma as well as Galpha . At low concentrations, phosducin appears to act preferentially by binding to Gbeta gamma and by neutralizing Gbeta gamma effects on Go activation. At higher concentrations, a direct inhibition of alpha o causes additional inhibition of G-protein function. Both types of interactions occur with affinities in the lower range of physiological phosducin concentrations in many tissues. Direct effects of phosducin on Galpha are predicted to have two major effects compared with the previously presumed exclusive action via Gbeta gamma ; they would increase the extent as well as the rapidity of its inhibitory effects. Our observations suggest that complex interactions of phosducin with G-protein subunits play a role under physiological circumstances. These interactions provide an additional level to the many mechanisms (40) that regulate transmembrane signaling via G-protein-coupled receptors.

    ACKNOWLEDGEMENTS

We thank Stefan Müller for purified alpha o, Werner Schnepp for purified alpha t, and Cornelius Krasel for purified PKA. Some Galpha and Gbeta gamma antibodies were kindly provided by G. Schultz and R. Schulz. We thank M. Heck and K. P. Hofmann for helpful discussions regarding the curve fitting of the GTPase experiments.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.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. Tel.: 49-931-2015400; Fax: 49-931-2013539.

1 The abbreviations used are: Galpha , G-protein alpha  subunit; Gbeta gamma , G-protein beta gamma subunit complex; GTPgamma S, guanosine 5'-(3-O-thio)triphosphate; PKA, protein kinase A; Ni-NTA, nickel-nitrilotriacetic acid.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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