©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interactions of Phosducin with Defined G Protein -Subunits (*)

(Received for publication, December 18, 1995; and in revised form, February 21, 1996)

Stefan Müller (§) Annette Straub Stefan Schröder Petra H. Bauer Martin J. Lohse (¶)

From the Laboratory of Molecular Biology, University of Munich, 82152 Martinsried, Germany and Institute of Pharmacology, University of Würzburg, 97078 Würzburg, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phosducin has recently been identified as a cytosolic protein that interacts with the beta-subunits of G proteins and thereby may regulate transmembrane signaling. It is expressed predominantly in the retina but also in many other tissues, which raises the question of its potential specificity for retinal versus nonretinal beta-subunits. We have therefore expressed and purified different combinations of beta- and -subunits from Sf9 cells and have also purified transducin-beta from bovine retina and a mixture of beta complexes from bovine brain. Their interactions with phosducin were determined in a variety of assays for beta function: support of ADP-ribosylation of alpha(o) by pertussis toxin, enhancement of the GTPase activity of alpha(o), and enhancement of rhodopsin phosphorylation by the beta-adrenergic receptor kinase 1 (betaARK1). There were only moderate differences in the effects of the various beta complexes alone on alpha(o), but there were marked differences in their ability to support betaARK1 catalyzed rhodopsin phosphorylation. Phosducin inhibited all beta-mediated effects and showed little specificity toward specific defined beta complexes with the exception of transducin-beta (beta(1)(1)), which was inhibited more efficiently than the other beta combinations. In a direct binding assay, there was no apparent selectivity of phosducin for any beta combination tested. Thus, in contrast to betaARK1, phosducin does not appear to discriminate strongly between different G protein beta- and -subunits.


INTRODUCTION

Guanine nucleotide-binding proteins (G proteins) are transducers between heptahelical receptors and various effectors. Traditionally, G proteins have been classified according to their alpha-subunits, but in the last years a plethora of effects have been assigned to the beta-subunits. Thus, they have been shown to regulate adenylyl cyclases, phospholipases C-beta and A(2), PI3-kinase, the ADP-ribosylation factor and several ion channels (for reviews, see (1) and (2) ). In addition, beta-subunits provide a membrane attachment site for the beta-adrenergic receptor kinase (betaARK) (^1)which allows translocation of the kinase from the cytosol to the cell membrane and thus in close proximity to its substrate receptors(3, 4) . Phosphorylation of G protein-coupled receptors by betaARK is thought to represent the first step of homologous desensitization; it is followed by binding of proteins of the arrestin family which results in uncoupling of receptors and their G proteins(5, 6) .

Another cytosolic protein which interacts with G protein beta-dimers is phosducin, a phosphoprotein originally purified from bovine retina as a complex with the beta-subunits of G(t)(7) . By binding to G protein beta-subunits phosducin can inhibit G protein-mediated signaling(8, 9) . Furthermore, phosducin can compete with betaARK for the beta-subunits and can thereby impair betaARK-mediated phosphorylation of receptors(10) . The interaction of phosducin with G proteins is markedly reduced following its phosphorylation by protein kinase A(8, 11) .

The possibility of a common motif for the interaction of all these proteins with beta-subunits has attracted much recent interest. Binding to distinct types of adenylyl cyclases, phospholipase C-beta3, atrial K channels, and betaARK has been found to include a defined stretch of amino acids(12) . However, this sequence cannot be found in phosducin. Binding to betaARK involves the kinase's C terminus, which partially overlaps with the pleckstrin-homology domain (PH domain) of betaARK(13, 14) . The binding site of phosducin responsible for interaction with beta-subunits has been mapped to its N terminus (15, 16) which contains neither a PH domain nor the sequence mentioned above. Thus, the interaction of phosducin with G protein beta-subunits must be different from those with other beta-binding proteins.

Molecular cloning techniques have revealed multiple isoforms of beta- and -subunits. The most recent data indicate the existence of five distinct beta- and 10 -subunits(17, 18) . Compared to the highly conserved beta-subunits, the -subunits are more divergent, suggesting them to be the specificity-determining factor. The functional differences of defined beta combinations are limited; regulation of particular types of adenylyl cyclases or stimulation of phospholipase C-beta isoforms and K channels as well as interactions with G protein alpha-subunits did not reveal striking differences except for beta(1)(1), the beta complex found in retinal rods, which showed lower potencies in its actions(19, 20, 21) . Receptor phosphorylation by betaARK was stimulated more potently by (2)-containing dimers and a preparation of beta-subunits from bovine brain than by complexes with (3) or by beta(1)(1)(22) .

The proteins of the retinal signal transduction cascade are homologous but distinct from those of receptor-mediated signaling(23) . They are restricted in their expression to the retina and the developmentally related pineal gland, and they show specificity toward each other when compared with the proteins of other receptor systems. This applies also to the G protein beta-subunits, with beta(1)(1) being a retina-specific combination. Phosducin, on the other hand, is highly expressed in the retina and pineal gland, but also in other tissues(8) . This suggests that it may show less specificity for the other components of the retinal signaling system than has been found for other members of this system. In fact, phosducin has been shown to inhibit the GTPase-activity not only of G(t) but also of G(o), G(i), and G(s) preparations(8, 9) . Since the beta-subunits appear to be the primary interaction partners for phosducin, the aim of the present investigation was to examine the specificity of interactions between defined G protein beta combinations and phosducin.


EXPERIMENTAL PROCEDURES

Expression and Purification of G Protein beta-Subunits

The generation of recombinant baculoviruses for the G protein subunits beta(1), beta(2), (2), and (3) has been described earlier(22) . For large scale preparations of recombinant beta-subunits, 200 ml of suspension cultures of Sf9 cells (2 times 10^6 cells/ml) were co-infected with the recombinant baculoviruses with a multiplicity of infection of 5 or 10 for the beta- and the -subunits, respectively. The expression of the beta- and -subunits was monitored with Western blots using antibodies recognizing conserved epitopes of the respective subunits. The blots were developed with peroxidase-coupled goat-anti-rabbit IgG and visualized using ECL reagents (Amersham Corp.). The cells were harvested 70 h after infection, and the beta complexes were purified from the membranes by solubilization with cholate and chromatography over C7 heptylamine-Sepharose and DEAE-Sephacel columns as described previously(22) . The purified beta complexes were quantitated according to Bradford (24) and also from Coomassie-stained SDS-polyacrylamide gels. Purified beta complexes were concentrated to about 15 pmol/µl and were stored at -80 °C with 5% glycerol.

G protein beta-subunits and alpha(o) from bovine brain and transducin-beta-subunits from bovine retina were purified according to established procedures(25, 26) . In order to reduce the contamination of alpha(o) with beta-subunits to a minimum, the usual purification process was extended by a further chromatography step on a C7 heptylamine-Sepharose column.

Phosducin was expressed in Escherichia coli and purified following the procedure of Bauer et al.(8) .

ADP-ribosylation of alpha(o)

The activity of the recombinant beta-subunits was measured by their ability to support the ADP-ribosylation of alpha(o) by pertussis toxin. The assay was performed as described by Hekman et al.(27) with minor modifications. 2 pmol of alpha(o) (40 nM) were used as the substrate in an assay volume of 50 µl containing 600 ng of pertussis toxin (Sigma) and increasing concentrations (2-60 nM) of beta-subunits. For inhibition of the ADP-ribosylation, 0.8 pmol of alpha(o) (16 nM) was incubated with 0.8 pmol of beta-subunits (16 nM), and phosducin was present at concentrations of 8-240 nM (0.4-12 pmol).

GTPase Activity of alpha(o)

The GTPase activity of alpha(o) was determined as described elsewhere(8, 28) . Reaction mixtures (100 µl) contained 0.1 pmol of alpha(o) and 0.1-2.4 pmol of beta complexes (1-24 nM). The effects of phosducin were examined at a constant concentration of beta-subunits (24 nM) and 24-720 nM phosducin.

Phosphorylation of Rhodopsin

Urea-treated rod outer segments containing >95% rhodopsin (29) were phosphorylated by purified recombinant betaARK1 essentially as described earlier(30) . The reaction mixtures (60 µl) contained 50 µM [-P]ATP (Amersham Corp.), 10 pmol (167 nM) rhodopsin, 0.3 pmol (5 nM) of betaARK, 3 pmol (50 nM) of beta-subunits, and 1.5-60 pmol (25-1000 nM) of phosducin. The incubation was carried out at 30 °C for 8 min under bright white light. The reaction was stopped, and the samples were analyzed by electrophoresis, autoradiography, and Cerenkov counting of the rhodopsin bands.

Phosducin Binding Assay

Wells of microtiter plates were coated with 300 ng of beta-subunits for at least 4 h at 4 °C in 100 µl of 20 mM Hepes, 20 mM NaCl, 0.1 mM EDTA, pH 7.6, and 0.05% cholate (=incubation buffer). The wells were then washed several times with the same ice-cold buffer supplemented with 0.05% Tween 20 (=wash buffer). After blocking with 3% bovine serum albumin in wash buffer, 0.1-20 µg of phosducin (30 nM to 6 µM) were incubated in the wells at 4 °C for 2 h in 100 µl of incubation buffer plus 5 mM MgCl(2). The wells were then washed and blocked as above. Bound phosducin was determined by addition of affinity-purified rabbit anti-phosducin antibodies for 1 h at room temperature. After incubation with peroxidase-coupled goat-anti-rabbit IgG a color reaction was evoked with o-phenylenediamine dihydrochloride (Sigma) and stopped with 50 µl of 3 M sulfuric acid, and absorption was measured at 490 nm.


RESULTS

In order to investigate effects of beta-subunits of defined composition, the proteins were produced in Sf9 cells by co-infection with recombinant baculoviruses directing the expression of defined beta- and -subunits. The resultant beta complexes were then purified to >95% purity by chromatography on C7 heptylamine-Sepharose and DEAE-Sephacel as described previously(22) . The beta complexes used in this study were beta(1)(2), beta(1)(3), beta(2)(2), and beta(2)(3). Mock preparations of cells infected with wild-type baculoviruses were prepared in the same manner for control purposes. Transducin-beta (beta(1)(1)) and a bovine brain beta preparation containing multiple beta- as well as -subunits were prepared by established procedures to serve as controls.

All beta preparations were able to support pertussis toxin-catalyzed ADP-ribosylation of alpha(o) (Fig. 1A). There were no major differences between the different recombinant beta complexes. The two native preparations, transducin-beta and the mixed preparation of beta-subunits from bovine brain, were more effective at lower concentrations than the recombinant preparations; at higher concentrations this difference disappeared for transducin-beta and became small for the bovine brain beta preparation.


Figure 1: Pertussis toxin-mediated ADP-ribosylation of alpha(o). Stimulation by beta-dimers (panel A) and inhibition by phosducin (panel B). A, alpha(o) (2 pmol, 40 nM) purified from bovine brain was ADP-ribosylated in the presence of pertussis toxin with increasing amounts of different beta combinations (2-60 nM). beta stands for transducin-beta, beta for a mixture of beta-subunits from bovine brain, CON for a mock preparation from uninfected Sf9 cells. Nonlinear curve fitting to the Hill equation gave the following E(max) and EC values: beta(1)(2), 0.52 ± 0.04 mol/mol, 20.9 ± 3.8 nM; beta(2)(2) 0.65 ± 0.09 mol/mol, 25.4 ± 7.7 nM; beta(1)(3), 0.48 ± 0.04 mol/mol, 19.3 ± 3.8 nM; beta(2)(3), 0.58 ± 0.08 mol/mol, 33. 0 ± 9.3 nM; beta(B), 0.68 ± 0.04 mol/mol, 13.7 ± 2.2 nM; beta(T), 0.41 ± 0.02 mol/mol, 5.8 ± 1.1 nM. Data are means ± S.E. of three experiments. B, 0.8 pmol (16 nM) alpha(o) was incubated with 0.8 pmol (16 nM) beta-subunits in the presence of various concentrations of phosducin (8 to 240 nM). The effects evoked by the individual beta-dimers alone were set to 100%. Nonlinear curve fitting to the Hill equation gave the following I(max) and IC values: beta(1)(2), 46 ± 9%, 32 ± 21 nM; beta(2)(2), 49 ± 11%, 49 ± 41 nM; beta(1)(3), 51 ± 6%, 40 ± 15 nM; beta(2)(3), 58 ± 11%, 26 ± 21 nM; beta(B), 45 ± 5.5%, 34 ± 13 nM; beta(T), 79 ± 13%, 34 ± 19 nM. Data are means ± S.E. of three experiments.



Phosducin inhibited these enhancing effects of all beta complexes on pertussis toxin-catalyzed ADP-ribosylation of alpha(o). Fig. 1B shows the phosducin-mediated inhibition of ADP-ribosylation in the presence of a constant, submaximally effective concentration (16 nM) of the various beta complexes. In this assay, only transducin-beta showed a distinct inhibition curve; its effects were antagonized to a greater degree than those of other beta complexes. This occurred even though the enhancing effects of 16 nM transducin-beta on alpha(o)-ADP-ribosylation were not larger than those of the recombinant beta complexes and even smaller than those of the bovine brain beta preparation (Fig. 1, A and B). Among the recombinant beta complexes there were only very small differences in their inhibition by phosducin, with a slight tendency toward better inhibition by (3)-containing complexes versus (2)-containing complexes. These minor differences were not statistically significant.

Interactions between the beta complexes and alpha(o) were also investigated by measuring the enhancement of the intrinsic GTPase activity of alpha(o) which is exerted by beta-subunits in the presence of high concentrations of Mg(31) . All beta preparations were able to enhance the GTPase activity of alpha(o) (Fig. 2A). Again, there were small differences between the different recombinant beta complexes; those containing the beta(1)-subunit appeared approx40% more effective than those containing the beta(2)-subunit. Of the native preparations, the brain preparation of beta-subunit was very effective, whereas transducin-beta was the least effective of all combinations. None of the combinations reached saturation within the concentration range employed in these experiments; this was particularly the case for the most potent combinations, beta(1)(2) and the bovine brain beta preparation.


Figure 2: Stimulation of the GTPase activity of alpha(o) by beta-subunits (panel A) and its inhibition by phosducin (panel B). A, the GTPase of alpha(o) (1 nM) was stimulated in the presence of high Mg (10 mM) with increasing concentrations of different beta-dimers (1 to 25 nM). Nonlinear curve fitting to the Hill equation gave the following E(max) and EC values: beta(1)(2), 614 ± 44%, 29.9 ± 3.4 nM; beta(2)(2), 347 ± 107%, 24.6 ± 12.8 nM, beta(1)(3), 338 ± 18%, 15.8 ± 1.7 nM; beta(2)(3), 331 ± 27%, 24 ± 3.3 nM; beta(B), 751 ± 163%, 47 ± 14 nM; beta(T), 235 ± 52%, 16.5 ± 6.9 nM. Data are means ± S.E. of five experiments. B, the stimulatory effect of 24 nM of the different beta complexes on 1 nM alpha(o) (set to 100%) was antagonized with various concentrations of phosducin (24 to 720 nM). I(max) and IC values were calculated using nonlinear curve fitting according to the Hill equation: beta(1)(2), 65 ± 3.6%, 31 ± 7.7 nM; beta(2)(2), 60 ± 4.2%, 46 ± 13.1 nM; beta(1)(3), 58 ± 8.8%, 26 ± 19 nM; beta(2)(3), 71 ± 1.6%, 48 ± 4.4 nM; beta(B), 47 ± 4.8%, 165 ± 49 nM; beta(T), 87 ± 11.6%, 44 ± 2.4 nM. Data are means ± S.E. of six experiments.



Again, a submaximally effective concentration of beta-subunits was chosen to examine the inhibitory effects of phosducin (Fig. 2B). As was the case with the ADP-ribosylation assays, phosducin was most efficacious in inhibiting the effects of transducin-beta. There was >40% inhibition at the lowest concentration of phosducin, at which the concentrations of phosducin and beta-subunits were identical (24 nM), and the inhibition was almost complete at higher concentrations of phosducin. Transducin-beta was followed by the various recombinant beta complexes, which were all inhibited in a very similar manner by phosducin; maximal inhibition amounted to approx60% in all cases, and the IC values were not significantly different. The effects of the bovine brain beta preparation, in contrast, were not well antagonized by phosducin; there was almost no effect at the lowest concentration of phosducin, and even at the highest concentration of phosducin (720 nM) the inhibition was only about 30%. These data suggest the possibility that the bovine brain beta preparation might contain some beta complexes which are not well recognized by phosducin.

In order to study this hypothesis further, we investigated another effect of G protein beta complexes, the enhancement of the phosphorylation of G protein-coupled receptors by the beta-adrenergic receptor kinase 1 (betaARK1). We had reported earlier that there are marked differences between the ability of defined beta complexes in enhancing receptor phosphorylation by betaARK1(22) . Using rhodopsin (approx830 nM) as the substrate and a submaximally effective concentration of beta-subunits (50 nM), the combination beta(2)(2) caused the largest enhancement, followed by the modestly effective combination beta(1)(2) and the bovine brain beta preparation, while beta(1)(3) and beta(2)(3) as well as transducin-beta had only very small effects (Fig. 3A).


Figure 3: Enhancement of betaARK-mediated phosphorylation of rhodopsin by different beta-subunits (panel A) and inhibition by phosducin (panels B and C). A, rod outer segments (50 pmol) were phosphorylated by 5 nM purified betaARK1 and 50 nM of different beta-dimers in a reaction volume of 60 µl. The concentration of 50 nM beta-subunits was chosen because it had a submaximal effect on rhodopsin phosphorylation. Data are means ± S.E. of three experiments. B and C, inhibition of the stimulatory effect of beta-subunits on betaARK-mediated rhodopsin phosphorylation by phosducin. Rhodopsin was phosphorylated with betaARK1 and 50 nM beta-dimers in the presence of increasing concentrations of purified phosducin (0.025 to 1 µM). The data shown in panels B and C are identical, except that the values in panel C were normalized to the values in the absence of phosducin. Nonlinear curve fitting to the Hill equation gave the following IC values: beta(1)(2), 123 ± 9 nM; beta(2)(2), 104 ± 47 nM; beta(B) 780 ± 280 nM. Data are means ± S.E. of three experiments.



The enhancing effects of beta-subunits on betaARK1-mediated rhodopsin phosphorylation were inhibited by phosducin (Fig. 3B). The presence of high concentrations of phosducin (1000 nM, i.e. 20-fold above beta) essentially abolished the effects of all beta combinations. The enhancing effects of several beta combinations (beta(1)(3), beta(2)(3), and transducin-beta) were too small to allow a reliable determination of the inhibitory effects of phosducin. For the remaining combinations, it can be seen that phosducin inhibited the effects of the two recombinant beta combinations, i.e. beta(2)(2) and beta(1)(2), more potently than those of the bovine brain beta preparation (Fig. 3C). The EC values were 105 and 123 nM for beta(2)(2) and beta(1)(2), respectively, and 780 nM for the bovine brain beta preparation. In contrast, the maximal extent of inhibition appeared to be similar for the three beta preparations.

Finally, we developed an enzyme-linked immunosorbent assay procedure to directly determine the binding of phosducin to the various beta complexes. The various beta-dimers were immobilized in wells of microtiter plates and phosducin was then incubated in the wells to allow binding to the immobilized beta-subunits. Phosducin bound to all beta complexes in a saturable manner with similar characteristics (Fig. 4). Contrary to the data shown above, there were no differences in the affinity of phosducin for the different beta combinations. There were minor differences in the maximal binding, but most notably transducin-beta and the bovine brain beta preparation had equal binding capacity for phosducin. Thus, phosducin appeared to show no selectivity in the direct interaction with beta-subunits, whereas in functional assays the effects of transducin-beta were antagonized more efficiently.


Figure 4: Direct binding of phosducin to immobilized beta-dimers. Different beta combinations (300 ng/well) were bound to 96-well microtiter plates. Various concentrations of purified phosducin (0.1 to 20 µg) were added in reaction volumes of 100 µl and bound phosducin was determined with affinity-purified anti-phosducin antibodies followed by a color reaction. E(max) and EC values were determined with the help of non-linear curve fitting to the Hill equation: beta(1)(2), 0.58 ± 0.05 A, 0.61 ± 0.20 µM; beta(2)(2), 0.70 ± 0.08 A, 0.73 ± 0.26 µM; beta(1)(3), 0.72 ± 0.06 A, 0.55 ± 0.16 µM; beta(2)(3), 0.77 ± 0.10 A, 1.05 ± 0.41 µM; beta(B), 0.82 ± 0.06 A, 0.44 ± 0.11 µM; beta(T), 0.82 ± 0.08 A, 0.51 ± 0.17 µM. Data are means ± S.E. of four experiments.




DISCUSSION

G protein-mediated signaling systems comprise a series of proteins, including the receptors, the G protein subunits, the effectors and various partially cytosolic proteins. The steadily increasing number of isoforms for all of these proteins that are being discovered raises the question of protein-protein specificity. A fairly consistent pattern is the specificity of the proteins that are involved in phototransduction: rhodopsin, G(t), cGMP-phosphodiesterase, rhodopsin kinase, and arrestin are all expressed only in the retina (and in the developmentally related pineal gland). Furthermore, in in vitro assays they show marked specificity toward each other. For example, rhodopsin kinase is much better in phosphorylating rhodopsin than are beta-adrenergic receptors, whereas the reverse is true for beta-adrenergic receptor kinase(32) . Similarly, arrestin binds much better to rhodopsin than to beta-adrenergic receptors, while beta-arrestin prefers beta-adrenergic receptors(28) . At the level of the signaling chain itself (receptor/G protein/effector) the issue of specificity is less clear-cut. While a series of experiments with antisense oligonucleotides against individual G protein alpha-, beta- and -subunits have suggested a remarkable specificity for certain receptor/ion channel coupling (33, 34, 35, 36) , reconstitution experiments have shown only moderate specificity of receptor/G protein coupling and various degrees of specificity for regulation of effectors by G protein subunits(19, 20, 22, 37, 38) .

Phosducin differs from the other proteins of such signaling chains in its expression pattern: while it is very abundant in the retina and in the pineal gland, it is also expressed in many other tissues(8) . Since phosducin interacts primarily with the G protein beta-subunits, and since the retinal beta-dimer beta(1)(1) is not found in other tissues, the presence of phosducin in nonretinal tissues would make sense only if it exhibited little or no selectivity toward specific beta-subunits.

This question was addressed in the present study by investigating the interaction of phosducin with a number of defined G protein beta complexes in several functional assays as well as in a direct binding assay. For this purpose, defined beta complexes were produced in Sf9 cells with the help of recombinant baculoviruses. The baculovirus expression system has been used by several groups for producing functional G protein subunits in high quantities(19, 22, 39, 40) . For the synthesis of functional beta-subunits it is necessary to express both subunits simultaneously. Their functionality can be ascertained by their ability to support the ADP-ribosylation of G protein alpha-subunits by pertussis toxin. In our hands, all the combinations tested so far were able to enhance the ADP-ribosylation of alpha(o) equally well. We interpret this finding as evidence for equal functional activity of the different beta preparations. Similar observations have been made by others regarding the ADP-ribosylation of native alpha(o) and alpha(t), whereas for alpha produced in E. coli (which was not myristoylated) transducin-beta was less efficient and potent(19, 20, 37) .

Another functional test for beta complexes which likewise involves interaction with the alpha-subunits is their ability to alter the intrinsic GTPase activity of the alpha-subunit. In the case of alpha(o), beta-subunits activate the GTPase in the presence of high concentrations of Mg whereas in the presence of low concentrations of Mg (below 1 mM) they exert an inhibitory effect(31) . In our studies, we measured modest differences in the activity of the different beta-dimers on alpha(o), which were about 2-fold for the best (beta(1)(2)) versus the worst (transducin-beta) combination. The pattern of selectivity was somewhat different from the one found in the ADP-ribosylation assays, suggesting that there are different requirements for the beta-subunits in the two different types of experiments. Investigations by Ueda et al.(20) on the inhibition of alpha-subunit GTPase activity by beta-subunits have also failed to reveal significant selectivity with the exception of beta(1)(1), which was less potent in all assays.

A third, quite different, test for the functionality of the beta complexes is their ability to serve as membrane anchors for betaARK to enhance agonist-dependent receptor phosphorylation(3, 4) . In an earlier study we had found differences between defined beta complexes in their ability to enhance betaARK-mediated rhodopsin phosphorylation(22) . Similar differences were found here, with beta(2)(2) being most efficient and transducin-beta least.

Phosducin had inhibitory effects on all of these beta-mediated effects. Among the different beta combinations, the stimulatory effects of transducin-beta both on ADP-ribosylation and GTPase activity were more efficiently antagonized, while we did not detect any real differences between the recombinant dimers consisting of beta(1), beta(2), (2), and (3). Phosducin also inhibited the beta-mediated enhancement of rhodopsin phosphorylation. However, the divergent enhancing potential of the beta complexes allowed a characterization of this inhibition only in the case of beta(1)(2), beta(2)(2), and beta(B).

While stimulation of rhodopsin phosphorylation by beta(1)(2) or beta(2)(2) were equally well inhibited by phosducin, the effect on beta(B) was less pronounced and appeared to require higher concentrations of phosducin. Similarly, we also observed only a weak inhibition on the beta(B)-mediated stimulation of alpha(o)-GTPase. We speculate that the mixture of beta-subunits from bovine brain might contain beta complexes which are only poorly recognized by phosducin.

Whereas the functional assays suggested a preference of phosducin for transducin-beta, we found no real differences in affinity or maximal binding when we assayed phosducin binding to immobilized beta-subunits directly. Thus, the apparent selectivity of phosducin for transducin-beta in the functional assays may be caused by events which occur subsequent to the initial binding step. An alternative explanation is that transducin-beta has a better solubility than the other beta complexes and that the binding assay was done with immobilized beta-subunits while the ADP-ribosylation and GTPase assays were carried out in solution. In this context it is important to note that the affinities measured in the direct binding assay with immobilized beta-subunits were approx10-fold lower than those determined in the functional assays in solution. Likewise, the affinities of phosducin for beta(1)(2) and beta(2)(2) found in the ADP-ribosylation and GTPase assays were approx3-fold higher than those determined in the betaARK assay, where the beta-subunits were integrated in the rod outer segment membranes. Similar discrepancies have been noted earlier. For example, Heithier et al.(41) reported severalfold higher affinities of beta complexes for alpha(o) in detergent-containing solution than in lipid vesicles.

Since transducin-beta differs from beta(1)(2) or beta(1)(3) only in its -subunit, the small functional specificity of phosducin for transducin-beta must be due to the presence of (1). (1) contains a different isoprenylation, farnesyl instead of geranylgeranyl in all other G protein -subunits. The functional importance of this farnesyl, compared to other prenyl modifications for the interaction with receptors, has very recently been elucidated by Kisselev et al.(42) . Alternatively, the slightly different effects of (1) may be due to its rather different amino acid sequence: the amino acid identity of (1) with (2) or (3) is less than 40%, while the sequence identity between (2) and (3) is about 80%(18) .

Overall, however, the interactions of phosducin with the various beta complexes appear to be quite similar. This relative lack of specificity goes in line with the nonretinal expression of phosducin, since it enables an interaction of phosducin with multiple G proteins and thus an interference with multiple signaling pathways. It contrasts with the more pronounced selectivity of betaARK for certain beta complexes where beta(2)(2) was the preferred partner and transducin-beta ineffective (22) (see also Fig. 3A). This agrees with our hypothesis mentioned in the introduction that the beta-binding domains of betaARK and phosducin are dissimilar. While some homologies between the N terminus of phosducin and the C terminus of betaARK have been noted(14, 15) , phosducin contains neither a PH domain nor the beta-binding consensus sequence Gln/Asn-X-X-Glu/Asp-Arg/Lys proposed by Chen et al.(12) . Furthermore, phosducin-like protein (43) with its much shorter N terminus which shows no similarity at all to the C terminus of betaARK, also interacts with G protein beta-subunits(44) , supporting the differences in the beta-binding mechanisms between phosducin and betaARK.

While these data suggest that the binding sites on G protein beta-subunits for betaARK and phosducin might be different, they must clearly be overlapping, since phosducin and betaARK have been shown to compete for beta binding(10) . Furthermore, the binding sites for phosducin and for betaARK appear to overlap also with the binding site for the alpha-subunits, since both proteins interfere with coupling between the beta- and the alpha-subunits. Thus the beta-subunits appear to possess various distinct but overlapping binding sites for different proteins.

Reconstitution experiments employing defined G protein beta complexes have largely failed to reveal much specificity. This contrasts with antisense experiments which indicate that G proteins of very specific alpha-, beta-, and -composition are required for efficient coupling of certain receptors to ion channels(33, 34, 35, 36) . This raises the possibility that selectivity may be encoded in the cellular organization rather than in protein-protein interactions. Further studies are required to determine whether similar rules might also govern the interactions of phosducin with G protein beta-subunits.


FOOTNOTES

*
These studies were supported by grants from the Deutsche Forschungsgemeinschaft (SFB 176), the European Commission and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Boehringer Ingelheim Fonds.

To whom correspondence should be addressed: Institute of Pharmacology, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany. Tel.: 49-931-201 5400; Fax: 49-931-201 3539.

(^1)
The abbreviations used are: betaARK, beta-adrenergic receptor kinase; G(i), G(s), G(o), G(t), stimulatory, inhibitory, ``other,'' and transducin GTP-binding proteins; PH domain, pleckstrin-homology domain.


ACKNOWLEDGEMENTS

We thank Prof. G. Schultz, Berlin, for providing antisera against G protein beta- and -subunits.


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