Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP.

Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of the gamma-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase.

The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane.     

Visual transduction in rod outer segments

The visual transduction cascade of the retinal rod outer segment responds to light by decreasing membrane current. This ion channel is controlled by cyclic GMP which is, in turn, controlled by its synthesis and degradation by guanylate cyclase and phosphodiesterase, respectively. When light bleaches rhodopsin there is an induced exchange of GTP for GDP bound to the alpha subunit of the retinal G-protein, transducin (T). The T alpha.GTP then removes the inhibitory constraint of a small inhibitory subunit (PDE gamma) on the retinal cGMP phosphodiesterase (PDE). This results in activation of the PDE and in hydrolysis of cGMP. Recently both low and high affinity binding sites have been identified for PDE gamma on the PDE alpha/beta catalytic subunits. The discovery of two PDE gamma subunits, each with different binding affinities, suggests that a tightly regulated shut-off mechanism may be present.     

The regulation of the cyclic GMP phosphodiesterase by the GDP-bound form of the alpha subunit of transducin

The functional interactions of the retinal G protein, transducin, with the cyclic GMP phosphodiesterase (PDE) have been examined using the different purified subunit components of transducin and the native and trypsin-treated forms of the effector enzyme. The limited trypsin treatment of the PDE removes the low molecular weight gamma subunit (Mr approximately 14,000) of the enzyme, yielding a catalytic moiety comprised of the two larger molecular subunits (alpha, Mr approximately 85,000-90,000; beta, Mr approximately 85,000-90,000), which is insensitive to the addition of either the pure alpha T.GTP gamma S species or the pure beta gamma T subunit complex. However, the addition of the pure alpha T.GDP species to the trypsin-treated PDE (tPDE) results in a significant (90-100%) inhibition of the enzyme activity. This inhibition can be reversed by excess beta gamma T, suggesting that the holotransducin molecule does not (functionally) interact with the tPDE. However, the inhibition by alpha T.GDP is not reversed by the alpha T.GTP gamma S complex, over a range of [alpha T.GTP gamma S] which elicits a marked stimulation of the native enzyme activity, suggesting that the activated alpha T species does not effectively bind to the tPDE. The alpha T.GDP complex also is capable of inhibiting the alpha T.GTP gamma S-stimulated cyclic GMP hydrolysis by the native PDE. This inhibition can be reversed by excess alpha T.GTP gamma S, as well as by beta gamma T, indicating that the binding site for the activated alpha T species is in close proximity and/or overlaps the binding site for the alpha T.GDP complex on the enzyme. Overall, these results are consistent with a scheme where (a) both the small and larger molecular weight subunits of PDE participate in alpha T-PDE interactions, (b) the activation of PDE by the alpha T.GTP gamma S (or alpha T.GTP) species does not result in the complete dissociation of the gamma subunit from the enzyme, and (c) the deactivation of this signal transduction system results from a direct interaction between the alpha T.GDP species and the catalytic moiety of the effector enzyme.     

Interaction between cGMP-phosphodiesterase and transducin alpha-subunit in retinal rods. A cross-linking study.

Cross-linking of the different subunits of the retinal cGMP-phosphodiesterase (PDE) with its activator G alpha GTP gamma S (alpha subunit of the retinal G-protein transducin with GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) bound) has been investigated using purified proteins, with a N-hydroxysuccinimide homobifunctional cross-linker, bis(sulfosuccinimidyl)suberate (BS3) and its cleavable analog 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). Interaction of purified G-protein and PDE is achieved in the presence of lecithin vesicles, at protein concentrations sufficient for full PDE activation. Protein subunits linked with DTSSP are separated by cleavage of the disulfide bridge and identified by electrophoresis. Complexes of PDE alpha (PDE beta) with 1 and 2 molecules of activator G alpha GTP gamma S are observed, providing direct evidence for an interaction or at least a close proximity between 2 molecules of activator G alpha and each of the catalytic PDE subunits in the activated state of PDE. The results also reveal symmetrical roles of PDE alpha and PDE beta, with the existence of one site for PDE gamma and one site for G alpha on each catalytic subunit.     

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.     

Guanine nucleotides and magnesium dependence of the association states of the subunits of transducin

When GTP gamma S is bound to transducin (T), the two subunits T alpha X GTP gamma S and T beta gamma dissociate, independently of the ionic environment. When GDP is bound, these subunits are associated as a monomeric T alpha X GDP-T beta gamma complex of 75 kDa when the ionic environment is comparable to that of the cytoplasm, but they dissociate in the presence of 10-100 mM Mg2+ or Ca2+. Using this property, the subunits could be separated and purified by a rapid one-step procedure on an ion-exchange column (FPLC), and their molecular masses were verified by neutron small angle scattering. The physiological relevances of the dissociating effect of Mg2+ are discussed.     

Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate

The visual excitation system of the retinal rod outer segments and the hormone-sensitive adenylate cyclase complex are regulated through guanine nucleotide-binding proteins, transducin in the former and inhibitory and stimulatory regulatory components, Gi and Gs, in the latter. These proteins are functionally and structurally similar; all are heterotrimers composed of alpha, beta, and gamma subunits and exhibit guanosine triphosphatase activity stimulated by light-activated rhodopsin or the agonist-receptor complex. Adenylate cyclase can be stimulated by vanadate, which, like NaF, probably acts through Gs. Effects of vanadate on the function of a guanine nucleotide-binding protein were investigated in a reconstituted model system consisting of purified transducin subunits (T alpha, T beta gamma) and rhodopsin in phosphatidylcholine vesicles. Vanadate (decameric) inhibited [3H]GTP binding to T alpha and noncompetitively inhibited GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP but did not affect the rate of hydrolysis of bound GTP (single turnover rate), indicating that vanadate did not interfere with the intrinsic GTPase activity of T alpha. Binding of T alpha to rhodopsin and the ADP-ribosylation of T alpha by pertussis toxin, both of which are enhanced in the presence of T beta gamma, were inhibited by vanadate. These findings are consistent with the conclusion that vanadate can cause the dissociation of T alpha from T beta gamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation could result from a similar effect of vanadate on Gs.     

Mechanism of inhibition of transducin GTPase activity by fluoride and aluminum

Transducin, the guanyl nucleotide-binding protein of the retinal light-activated cGMP phosphodiesterase system, is structurally and functionally similar to the inhibitory and stimulatory guanyl nucleotide-binding proteins, Gi and Gs, of the adenylate cyclase complex. All are heterotrimers composed of alpha, beta, and gamma subunits. Gs and Gi can be activated by NaF with AlCl3 as well as by agonists acting through specific receptors. The effects of NaF and AlCl3 on transducin were investigated in a reconstituted system consisting of the purified subunits of transducin (T alpha, T beta, gamma) and rhodopsin. NaF noncompetitively inhibited the GTPase activity of T alpha in a concentration- and time-dependent manner. Inhibition by NaF was enhanced synergistically by AlCl3 which alone only slightly inhibited GTPase activity. None of the other anions tested reproduced the effect of fluoride. Fluoride inhibited [3H]guanosine 5'-(beta, gamma-imido)triphosphate binding to T alpha and release of bound GDP. The ADP-ribosylation of T alpha by pertussis toxin and binding of T alpha to rhodopsin, both of which are enhanced in the presence of T beta gamma, were inhibited by NaF and AlCl3. These findings are consistent with the hypothesis that fluoride enhances the dissociation of T alpha from T beta gamma, resulting in the inhibition of GTP-GDP exchange, and therefore, GTP hydrolysis.     

Reconstitution of the vertebrate visual cascade using recombinant heterotrimeric transducin purified from Sf9 cells

For reconstitution studies with rhodopsin and cGMP phosphodiesterase (PDE), all three subunits of heterotrimeric transducin (T alpha beta gamma) were simultaneously expressed in Sf9 cells at high levels using a baculovirus expression system and purified to homogeneity. Light-activated rhodopsin catalyzed the loading of purified recombinant T alpha with GTP gamma S. In vitro reconstitution of rhodopsin, recombinant transducin, and PDE in detergent solution resulted in cGMP hydrolysis upon illumination, demonstrating that recombinant transducin was able to activate PDE. The rate of cGMP hydrolysis by PDE as a function of GTP gamma S-loaded recombinant transducin (T(*)) concentration gave a Hill coefficient of approximately 2, suggesting that the activation of PDE by T(*) was cooperatively regulated. Furthermore, the kinetic rate constants for the activation of PDE by T(*) suggested that only the complex of PDE with two T(*) molecules, PDE. T(2)(*), was significantly catalytically active under the conditions of the assay. We conclude that the model of essential coactivation best describes the activation of PDE by T(*) in a reconstituted vertebrate visual cascade using recombinant heterotrimeric transducin.     

Activation mechanism of retinal rod cyclic GMP phosphodiesterase probed by fluorescein-labeled inhibitory subunit

The cyclic GMP phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is kept inactive in the dark by its gamma subunits and is activated following illumination by the GTP form of the alpha subunit of transducin (T alpha-GTP). Recent studies have shown that the stoichiometry of the inhibited holoenzyme is alpha beta gamma 2. T alpha-GTP and gamma act reciprocally. We have investigated the activation mechanism using fluorescein-labeled gamma subunit (gamma F) as a probe. gamma F containing a single covalently attached fluorescein was prepared by reaction of PDE with 5-(iodoacetamido)fluorescein and purification by reversed-phase high-pressure liquid chromatography (HPLC). gamma F, like native gamma, inhibits the catalytic activity of trypsin-activated PDE and transducin-activated PDE. Inhibition by gamma F was overcome by further addition of T alpha-GTP. gamma F binds very weakly to ROS membranes stripped of PDE and other peripheral membrane proteins. gamma F added to ROS membranes became incorporated into a component that could be extracted with a low ionic strength buffer. HPLC gel filtration showed that gamma F became part of the PDE holoenzyme. Incorporation occurred in less than 1 min in the presence of light and GTP, but much more slowly (t1/2 approximately 500 s) in the absence of GTP. This result indicates that transducin activates PDE by binding to the holoenzyme and accelerating the dissociation of gamma from the inhibitory sites. The binding of gamma F to trypsin-activated PDE alpha beta was monitored by steady-state emission anisotropy measurements and compared with PDE activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance energy transfer as a direct monitor of GTP-binding protein-effector interactions: activated alpha-transducin binding to the cGMP phosphodiesterase in the bovine phototransduction cascade.

Resonance energy-transfer approaches have been used to directly monitor the interactions of the GTP gamma S-bound alpha subunit of transducin (alpha T GTP gamma S) with the retinal cyclic GMP phosphodiesterase (PDE). The PDE was labeled with 5-(iodoacetamido) fluorescein (IAF-PDE) and served as the fluorescence donor in these experiments while the alpha T GTP gamma S was labeled with eosin-5-isothiocyanate (EITC-alpha T GTP gamma S) and served as the energy acceptor. The EITC-alpha T GTP gamma S species was able to quench a significant percentage of the IAF-PDE fluorescence (typically greater than or equal to 30%) due to resonance energy transfer between the IAF and EITC moieties. The quenching by the EITC-alpha T GTP gamma S species was dose-dependent, saturable (Kd = 21 nM), and specific for the GTP gamma S-bound form of the alpha T subunit. Limited trypsin treatment of the IAF-PDE, which selectively removes a fluorescein-labeled gamma subunit (gamma PDE), completely eliminates the quenching of the IAF fluorescence by the EITC-alpha T GTP gamma S complex. Although the EITC-alpha T GTP gamma S complex competes with the unlabeled alpha T GTP gamma S for a binding site on the IAF-PDE, as well as for a site on the native PDE, it is not able to stimulate PDE activity. Thus, the modification of a single EITC-reactive residue on the alpha T GTP gamma S complex prevents this subunit from eliciting a key activation event within the retinal effector enzyme.     

The intrinsic fluorescence of the alpha subunit of transducin. Measurement of receptor-dependent guanine nucleotide exchange

We have made use of the enhancement of the intrinsic fluorescence of the alpha subunit of transducin (alpha T), which accompanies guanine nucleotide exchange, to follow the reconstituted interactions between pure rhodopsin and pure transducin in phospholipid vesicles. When the pure alpha T.GDP complex is added to lipid vesicles containing rhodopsin and the beta gamma T complex, a light- and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-dependent enhancement of the fluorescence emission of alpha T is observed. When GTP is substituted for GTP gamma S, a similar enhancement of the intrinsic fluorescence of alpha T occurs; however, this enhancement is transient and precedes a fluorescence decay which is complete in 2-5 min. The fact that the fluorescence decay is specifically induced by GTP and is not observed either with nonhydrolyzable GTP analogs or with NaF (plus AlCl3) indicates that the decay represents GTP hydrolysis in alpha T. The dose-response profiles for the effects of the beta gamma T complex on the rate and extent of the GTP gamma S-stimulated fluorescence enhancement of alpha T have also been examined. The addition of relatively low levels of beta gamma T to these reconstituted systems can promote the GTP gamma S-stimulated enhancement of the fluorescence of multiple alpha T subunits with half-maximal enhancement occurring at alpha T:beta gamma T ratios of 150:1. These findings are consistent with earlier suggestions (Fung, B. K.-K. (1983) J. Biol. Chem. 258, 10495-10502) that the beta gamma T subunit dissociates from alpha T as a result of the GDP-GTP exchange reaction and thus can act catalytically to promote the activation of a number of inactive alpha T species. However, the dependence of the rate of the GTP gamma S-stimulated fluorescence enhancement on beta gamma T is complex and cannot be explained adequately by simple models where alpha T-beta gamma T interactions (or rhodopsin-transducin interactions) are rate-limiting for the rhodopsin-stimulated activation of the alpha T subunits. Overall, the results reported here demonstrate that fluorescence spectroscopy can be used to monitor directly a receptor-catalyzed activation-deactivation cycle of a GTP-binding protein within a lipid milieu.     

The cGMP phosphodiesterase-transducin complex of retinal rods. Membrane binding and subunits interactions.

cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is composed of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Native PDE alpha beta gamma 2 is peripherally bound to the membranes of ROS discs. We studied quantitatively its partition between soluble and membrane-bound fractions in ROS homogenates. In the presence of its activator, the alpha-subunit of transducin loaded with a triphosphate guanine nucleotide (T alpha*), PDE displayed a greatly enhanced membrane binding. Neither the purified PDE gamma.T alpha* complex, nor the PDE alpha beta and PDE alpha beta gamma forms of active PDE, showed a membrane binding comparable to that of PDE alpha beta gamma 2 in the presence of T alpha*. The T alpha*-activated PDE is therefore an undissociated complex tightly bound to the ROS membranes. Using limited proteolysis, we showed that the membrane anchoring of the whole complex implies not only PDE (mainly by the C terminus of PDE beta) but also both termini of T alpha*. The membrane binding of the purified PDE alpha beta species was also enhanced in the presence of T alpha*; a direct link would therefore exist between the activator and the catalytic subunits. From this work emerges a plausible structural model of the T alpha*-activated PDE, with its internal interactions and its sites of anchoring into the ROS membrane.     

Interaction of the gamma-subunit of retinal rod outer segment phosphodiesterase with transducin. Use of synthetic peptides as functional probes

There is considerable evidence which suggests that the gamma-subunit of cGMP phosphodiesterase (PDE gamma) is a multifunctional protein which may interact directly with both the catalytic subunits of PDE (PDE alpha beta) and the alpha-subunit of transducin (T alpha) (Whalen, M., and Bitensky, M. (1989) Biochem. J. 259, 13-19; Griswold-Prenner, I., Young, J. H., Yamane, H. K., and Fung, B. K.-K. (1988) Invest. Ophthalmol. & Visual Sci. 29, (Suppl.) 218). To determine the region of interaction between the multifunctional PDE gamma and T alpha, and to determine the significance of this interaction, peptides corresponding to various regions of PDE gamma were synthesized and tested for their ability to inhibit the GTPase activity of T alpha. One of these peptides, PDE gamma-3 (bovine amino acid residues 31-45), inhibited the GTPase activity of T alpha with an I50 of 450 microM. The peptide (PDE gamma-3) was found to inhibit the GTPase activity of T alpha by inducing the binding of transducin to the rod outer segment membrane and by altering the GTP/GDP exchange. Analogs of PDE gamma-3 were synthesized to determine the required structure of the PDE gamma-3 region needed for the interaction of PDE gamma with T alpha. The results of these studies indicated that the removal of the positively charged amino acids or any of the potential hydrogen-bonding amino acids increased the I50 for the inhibition of the GTPase activity of T alpha Substitution of the hydrophobic amino acids had no effect. These results indicate the hydrophilic interactions may be essential for the binding of PDE gamma to T alpha and for the inhibition of the GTPase activity of T alpha by PDE gamma. The observed effects of PDE gamma-3 on T alpha and on PDE suggest that PDE gamma is a multifunctional protein which may play more than one role in the deactivation of the retinal transduction cascade.     

Interactions between the subunits of transducin and cyclic GMP phosphodiesterase in Rana catesbiana rod photoreceptors

In bullfrog (Rana catesbiana) rods the activity of cyclic GMP (cGMP) phosphodiesterase was stimulated 10 times by washing disc membranes with an isotonic, GTP-containing buffer. This stimulation was maintained following hydrolysis of GTP and after removal of guanine nucleotides. At least 60-70% of the inhibitory gamma subunit of cGMP phosphodiesterase (P gamma) was physically released from membranes by these washing procedures. When cGMP phosphodiesterase was activated by a hydrolysis-resistant GTP analogue, P gamma was found in the supernatant complexed with the transducin alpha subunit (T alpha) using three chromatography systems. When GTP was used to activate cGMP phosphodiesterase, P gamma was also found in the supernatant complexed with GDP.T alpha. This complex was also isolated using the same three chromatography systems, indicating that P gamma remained tightly bound to T alpha even after bound GTP was hydrolyzed. Interaction with the beta,gamma subunits of transducin, which remained associated with disc membranes, was required for the release of P gamma from the GDP.T alpha complex, which resulted in the deactivation of active cGMP phosphodiesterase. We conclude that during activation of cGMP phosphodiesterase, P gamma is complexed with T alpha (both GTP and GDP forms) in the supernatant and that, following GTP hydrolysis, beta,gamma subunits of transducin are necessary for the release of P gamma from the complex and the resulting inactivation of cGMP phosphodiesterase in frog photoreceptors.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

ADP-ribosylation of transducin by pertussis toxin

Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is [32P]ADP-ribosylated by pertussis toxin and [32P]NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1 molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and 32 kDa. The amino terminus of both 38- and 32-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The 32-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of [32P]ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased [32P]ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed [32P]ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma. The amino terminus of T alpha, while it does not contain the pertussis toxin ADP-ribosylation site, appeared critical to its reactivity.     

Stereochemistry of the guanyl nucleotide binding site of transducin probed by phosphorothioate analogues of GTP and GDP

The stereochemistry of the guanyl nucleotide binding site of transducin from bovine retinal rod outer segments was probed with phosphorothioate analogues of GTP and GDP. Transducin has markedly different affinities for the five thio analogues of GTP, as measured by their effectiveness in inhibiting GTPase activity, competing with GTP for entry into transducin, and displacing GDP bound to transducin. The order of binding affinities is GTP gamma S = (Sp)-GTP alpha S greater than (Rp)-GTP alpha S greater than (Sp)-GTP beta S much greater than (Rp)-GTP beta S. The affinity of transducin for GTP gamma S is greater than 10(4) higher than that for (Rp)-GTP beta S. These five analogues have the same relative potencies in eliciting the release of transducin from the membrane and in activating the phosphodiesterase. Transducin hydrolyzes (Sp)-GTP alpha S with a l/e time of 55 s, compared with 28 s for GTP. In contrast, (Rp)-GTP alpha S, like GTP gamma S, is not hydrolyzed on the time scale of several hours. The order of effectiveness of thio analogues of GDP in displacing bound GDP is (Sp)-GDP alpha S greater than GDP greater than (Rp)-GDP alpha S greater than GDP beta S. The affinity of transducin for (Sp)-GDP alpha S is about 10-fold higher than that for GDP beta S. Mg2+ is required for the binding of GTP and GDP to transducin. Cd2+ does not lead to a reversal of stereospecificity at either the alpha- or beta-phosphorus atom of GTP. These results lead to the following conclusions: The pro-R oxygen atom at the alpha-phosphorus of GTP does not bind Mg2+ but instead interacts with the protein. The pro-S oxygen at the alpha-phosphorus does not appear to be involved in a critical interaction with transducin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin.

Photoexcitation of retinal rod photoreceptor cells involves the activation of cGMP enzyme cascade in which sequential activation of rhodopsin, transducin, and the cGMP phosphodiesterase in the rod outer segment constitutes the signal amplification mechanism. Phosducin, a 33-kDa phosphoprotein, has been shown to form a tight complex with the T beta gamma subunit of transducin. In this study, we examined the interaction of phosducin-T beta gamma and the possible regulatory role of phosducin on the cGMP cascade. Addition of phosducin to photolyzed rod outer segment (ROS) membrane reduced the GTP hydrolysis activity of transducin as well as the subsequent activation of the cGMP phosphodiesterase. Phosducin also inhibited the pertussis toxin-catalyzed ADP-ribosylation of transducin, indicating that the interaction between the T alpha and T beta gamma subunits of transducin was interrupted upon binding of phosducin. The inhibitory effects of phosducin were reversed by the addition of exogenous T beta gamma. These results suggest that phosducin is capable of regulating the amount of T beta gamma available to interact with T alpha to form the active transducin complex and thereby functions as a negative regulator of the cGMP cascade. The phosducin-induced alteration of the subunit organization of transducin was examined by chemical cross-linking method using para-phenyl dimaleimide as cross-linker. It was found that the cross-linking among T alpha and T beta gamma was blocked in the presence of phosducin. This result implies that T beta gamma may undergo a conformational change upon phosducin binding which leads to the release of T alpha. Since phosducin is a soluble protein, the interaction with transducin only occurs when transducin is dissociated from ROS disc membrane. Indeed, phosducin failed to dissociate membrane-bound transducin and did not inhibit the initial cycle of transducin activation as measured by the presteady state GTP hydrolysis. However, phosducin interacts effectively with transducin released into solution after the initial activation and blocks the re-binding of T alpha. T beta gamma to ROS membrane by forming a tight complex with T beta gamma. This interaction may play an important role in regulating the turnover of the cGMP cascade in photoreceptor cells.