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.     

Chemical modification of bovine transducin: effect of fluorescein 5'-isothiocyanate labeling on activities of the transducin alpha subunit

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residues of bovine transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells. The incorporation of FITC showed a stoichiometry of approximately 1 mol of FITC/mol of transducin. The labeling was specific for the T alpha subunit. There was no significant incorporation on the T beta gamma subunit. The modification had no effect on the transducin-rhodopsin interaction or on the binding of guanosine 5'-(beta, gamma-imidotriphosphate) [Gpp(NH)p] to transducin in the presence of photolyzed rhodopsin. The dissociation of the FITC-transducin-Gpp(NH)p complex from rhodopsin membrane remained unchanged. However, the intrinsic GTPase activity of T alpha and its ability to activate the cGMP phosphodiesterase were diminished by FITC modification. The rate of FITC labeling of the transducin-Gpp(NH)p complex was about 3-fold slower than that of transducin. Limited tryptic digestion and peptide mapping were used to localize the FITC labeling site. The majority of the FITC label was on the 23-kilodalton fragment, and a minor amount was on the 9-kilodalton fragment of the T alpha subunit. These results indicate that FITC labeling does not alter the activation of transducin by photolyzed rhodopsin but does affect the GTP hydrolytic activity as well as the GTP-induced conformational change of T alpha, which ultimately leads to the activation of cGMP phosphodiesterase.     

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.     

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.     

Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation.

Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gamma S]), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gamma S] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP[gamma S] and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stabilization of an intermediate activation state for transducin by a fluorescent GTP analogue

The GTP-binding protein (G protein), transducin, serves as a key molecular switch in vertebrate vision through the tight regulation of its GTP-binding (activation)/GTP hydrolytic (deactivation) cycle by the photoreceptor rhodopsin. To better understand the structure-function characteristics of transducin activation, we have set out to identify spectroscopic probes that bind to the guanine nucleotide-binding site of this G protein and maintain its ability to interact with its specific cellular target/effector, the cyclic GMP phosphodiesterase (PDE). In this study, we describe the characterization of a fluorescently labeled GTP analogue, BODIPY-FL GTPgammaS (BOD-GTPgammaS), that binds to the alpha subunit of transducin (alpha(T)) in a rhodopsin- and Gbetagamma-dependent manner, similar to the binding of GTP or GTPgammaS, with an apparent dissociation constant of 100 nM. The rhodopsin-dependent binding of BOD-GTPgammaS to alpha(T) is slow, relative to the rate of binding of GTPgammaS, particularly under conditions where rhodopsin must act catalytically to stimulate the exchange of BOD-GTPgammaS for GDP on multiple alpha(T) subunits. This reflects a slower rate of dissociation of rhodopsin and Gbetagamma from alpha(T)-BOD-GTPgammaS complexes, relative to their rates of dissociation from alpha(T)-GTPgammaS. The binding of BOD-GTPgammaS occurs without a change in the intrinsic tryptophan fluorescence of alpha(T), indicating that only a subtle movement of the Switch 2 domain on alpha(T) accompanies the binding of this GTPgammaS analogue. Nevertheless, the BOD-GTPgammaS-bound alpha(T) subunit is able to bind with high affinity to the recombinant, purified gamma subunit of PDE (gamma(PDE)) labeled with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS (K(d) approximately 13 nM)), as well as bind to and stimulate the activity of PDE, albeit less efficiently compared to alpha(T)-GTPgammaS. Taken together, these findings suggest that the binding of BOD-GTPgammaS to transducin causes it to adopt a distinct conformation that appears to be intermediate between the inactive and fully active states of alpha(T), and this fluorescent nucleotide analogue can be used as a reporter group to characterize the interactions of alpha(T) in this conformational state with its biological target/effector.     

Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange

Photolyzed rhodopsin acts in a catalytic manner to mediate the exchange of GTP for GDP bound to transducin. We have analyzed the steady-state kinetics of this activation process in order to determine the molecular mechanism of interactions between rhodopsin, transducin, and guanine nucleotides. Initial velocities (Vo) of the exchange reaction catalyzed by rhodopsin were measured for various transducin concentrations at several fixed levels of the GTP analog, [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). The initial rate data analysis rigorously demonstrates that rhodopsin mediates the activation of transducin by a double-displacement catalytic mechanism. The Michaelis-Menten curves determined as a function of [transducin] reveal remarkable allosteric behavior; analysis of this data yields a Hill coefficient of 2. Lineweaver-Burk plots of Vo-1 versus [transducin]-1 display curvilinearity indicative of positive cooperativity and a series of parallel lines are generated by plotting Vo-1 as a function of [transducin]-2. The plots of Vo-1 versus [GTP gamma S]-1 show no evidence of allosterism and are a parallel series. Furthermore, the allosteric behavior observed in the activation of transducin is also witnessed in the rhodopsin-catalyzed guanine nucleotide exchange of the G protein's purified alpha subunit in the absence of the beta X gamma subunit complex. The latter observation implies that the molecular basis for allosterism in the activation process resides in the interactions between the photoreceptor and transducin's alpha subunit.     

Characterization of transducin from bovine retinal rod outer segments. Use of monoclonal antibodies to probe the structure and function of the subunit

A panel of monoclonal antibodies has been developed against the T alpha, T beta and T gamma subunits of bovine transducin. Two anti-T alpha antibodies from this panel (TF15 and TF16) and a third one (4A) against frog T alpha (Witt, P. L., Hamm, H. E., and Bownds, M. D. (1984) J. Gen. Physiol. 84, 251-263) were characterized. Each of these monoclonal antibodies recognizes a different region of T alpha and has a specific effect on the function of transducin. The binding of TF15 is reversibly enhanced by treating T alpha with either 1 M guanidinium chloride or, to a smaller extent, by the removal of bound guanine nucleotide. Its epitope is located in a 12-kDa tryptic fragment containing the binding site for the guanine moiety of GTP. Taken together, these results support previous observations that the conformation of T alpha is modulated by the occupancy of the guanine nucleotide binding site. In contrast to TF15, TF16 recognizes only the native form of T alpha. Its epitope resides within the central portion of the T alpha molecule. While T alpha-bound TF16 does not inhibit either pertussis toxin-catalyzed ADP-ribosylation, rhodopsin binding, or transducin subunit interaction, it blocks both the light-activated uptake of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the GTP-dependent elution of transducin from photolyzed rhodopsin. These effects are unlikely to be caused by the occupation of the guanine nucleotide binding site by TF16 because this antibody quantitatively precipitates T alpha-GTP gamma S. We propose that bound TF16 locks T alpha in a conformation that prevents the entrance of guanine nucleotide and favors T beta gamma association. In contrast to TF16, the epitope of 4A was mapped to the amino-terminal region of T alpha. This monoclonal antibody blocks pertussis toxin-catalyzed ADP-ribosylation, GTP gamma S uptake, and T alpha-T beta gamma association. Moreover, the binding site for 4A becomes inaccessible when transducin binds to photolyzed rhodopsin. These results suggest that the inhibitory effects of 4A are due to a simultaneous steric blockage of both the interaction of T alpha with T beta gamma and their binding to photolyzed rhodopsin. The results obtained from these studies are correlated with the structure and function of T alpha.     

Rhodopsin/transducin interactions. I. Characterization of the binding of the transducin-beta gamma subunit complex to rhodopsin using fluorescence spectroscopy.

In this work we have used fluorescence spectroscopic approaches to examine the binding of the beta gamma T subunit complex of transducin to the photoreceptor, rhodopsin. To do this, we have covalently labeled the beta gamma T subunit complex with the environmentally sensitive fluorescent cysteine reagent 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). By using the MIANS moiety as a fluorescent reporter group, we were able to monitor directly the binding of the MIANS-beta gamma T complex to light-activated rhodopsin, which was reconstituted into phosphatidylcholine vesicles, through an enhancement (30-50%) in the MIANS fluorescence. Phosphatidylcholine vesicles, alone, elicited only minor changes in the MIANS-beta gamma T fluorescence (i.e. less than 10% enhancement), whereas the addition of rhodopsin in the absence of lipid vesicles and in minimal detergent fully mimicked the effects of reconstituted rhodopsin and caused a significant enhancement of the MIANS fluorescence. The interactions between the MIANS-beta gamma T complex and rhodopsin also resulted in a quenching of the rhodopsin tryptophan fluorescence (approximately 30%), which most likely reflected resonance energy transfer between the tryptophan residues and the MIANS moieties. The binding of the MIANS-beta gamma T species to the alpha T subunit was accompanied by an enhancement of the MIANS fluorescence (30-50%) and a slight blue shift of the emission maximum, as described previously (Phillips, W. J., and Cerione, R. A. (1991) J. Biol. Chem. 266, 11017-11024). However, the alpha T-induced enhancement of the MIANS-beta gamma T fluorescence was not additive with the enhancement elicited by rhodopsin. Conditions which resulted in the activation of the alpha T subunit reversed the alpha T-induced enhancement of the MIANS emission, whereas the rhodopsin-induced enhancement persisted, thereby suggesting that the rhodopsin-beta gamma T complex can remain intact throughout the G protein activation event. Studies with synthetic peptides representing different regions of the cytoplasmic domain of rhodopsin demonstrated that a portion of the putative carboxyl-terminal tail (amino acid residues 310-324) was capable of eliciting changes in the MIANS-beta gamma T fluorescence as well as inhibiting the MIANS-beta gamma T-induced quenching of the rhodopsin tryptophan fluorescence. These results suggest that this region of the rhodopsin molecule may constitute a portion of the binding domain for the beta gamma T complex.     

Lack of association of Ca(2+)-calmodulin with the beta gamma-subunits of the photo-receptor G protein (transducin).

We previously reported that the beta gamma-subunit of transducin (T beta gamma) is composed of two components, T beta gamma-1 and T beta gamma-2 with distinctive gamma-subunits, T gamma-1 and T gamma-2, respectively. T beta gamma-2 enhances GTP binding to the alpha-subunit of transducin (T alpha) in the presence of a photobleaching intermediate of rhodopsin, while T beta gamma-1 is an inactive component with little enhancement ability (Fukada, Y., Ohguro, H., Saito, T., Yoshizawa, T., and Akino, T. (1989) J. Biol. Chem. 264: 5937-5943). To further elucidate the functional differences between T beta gamma-1 and T beta gamma-2, we examined the association of T beta gamma s with Ca(2+)-calmodulin, and the effect of Ca2+ on binding of GTP to T alpha in the presence of either T beta gamma-1 or T beta gamma-2. Ca2+ had no effect on the GTP binding activity of transducin and T beta gamma s could not associate with Ca(2+)-calmodulin, indicating that the relationship of T beta gamma with Ca(2+)-calmodulin of is different from that of the brain G protein.     

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.     

Characterization of transducin from bovine retinal rod outer segments. Participation of the amino-terminal region of T alpha in subunit interaction

The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.     

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.     

Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes.

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical 'helper subunits' T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.     

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.     

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.     

Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation

Transducin, a guanine nucleotide-binding protein consisting of two subunits (T alpha and T beta gamma), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T alpha subunit is an activator of the phosphodiesterase, and the function of the T beta gamma subunit is to physically link T alpha with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T alpha has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T alpha with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) and T beta gamma were present at concentrations equal to that of T alpha and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T alpha X Gpp(NH)p, T beta gamma, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T alpha coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 X 10(-3)/s, which was 22-fold slower than the rate for the unmodified transducin.     

Chemical modification of bovine transducin: probing the GTP-binding site with affinity analogues

The structure of the GTP-binding site of transducin, a signal-transducing G-protein involved in the visual excitation process, was studied by affinity labeling. Radioactive GTP analogues with reactive groups attached to different moieties of the GTP molecule were obtained and include 8-azido-GTP, P3-(4-azidoanilino)-P1-5'-GTP (AA-GTP), 5'-[p-(fluorosulfonyl)benzoyl]guanosine (FSBG), 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)-GTP (ANPAP-GTP), the 2',3'-dialdehyde derivative of GTP (oGTP), and a bifunctional cross-linking analogue, 8-azido-P3-(4-azidoanilino)-P1-5'-GTP (8-azido-AA-GTP). With the exception of FSBG, all of the analogues were found to bind to transducin specifically and serve as a cofactor to activate the retinal cGMP cascade or act as a competitive inhibitor for the GTPase activity of transducin. The labeling sites of these analogues were localized by tryptic peptide mapping. ANPAP-GTP and oGTP were unable to covalently modify transducin, suggesting that the 2'- and 3'-hydroxy groups on the ribose ring of GTP are not in direct contact with the protein. AA-GTP only labeled the T alpha subunit of transducin and was localized on the 21-kDa tryptic fragment of T alpha. This indicates that the phosphate moiety of the bound GTP is in direct contact with this peptide. On the other hand, 8-azido-GTP labeled both the T alpha and T beta gamma subunits of transducin. The labeling on T alpha was on the 12-kDa tryptic fragment, suggesting that the guanine ring binding site is composed of a different peptide fragment than the phosphate binding region. Treatment with the bifunctional analogue 8-azido-AA-GTP generated the cross-linked products of T alpha and T beta gamma. This observation implies that the guanine ring of the bound GTP on T alpha could be in close proximity with T beta gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of retinal transducin with guanosine triphosphate analogues: specificity of the gamma-phosphate binding region

The interaction of six hydrolysis-resistant analogues of GTP with transducin, the signal-coupling protein in vertebrate photoreceptors, was investigated. GppNHp and GppCH2p differ from GTP at the bridging position between the beta- and gamma-phosphate groups. The other analogues studied (GTP gamma F, GTP gamma OMe, GTP gamma OPh, and GTP gamma S) differ from GTP in containing a substituent on the gamma-phosphorus atom or at a nonbridging gamma-oxygen atom. Competition binding experiments were carried out by adding an analogue, [alpha-32P]GTP, and a catalytic amount of photoexcited rhodopsin (R) to transducin and measuring the amount of bound [gamma-32P]GTP. The order of effectiveness of these analogues in binding to transducin was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh greater than GTP gamma OMe greater than GppCH2p greater than GTP gamma F A second assay measured the effectiveness of GTP gamma S, GppNHp, and GppCH2p in eluting transducin from disc membranes containing R. The basis of this assay is that transducin is released from disc membranes when it is activated to the GTP form. The relative potency of these three analogues in converting transducin from a membrane-bound to a soluble form was 1000, 75, and 1, respectively. Stimulation of cGMP phosphodiesterase activity served as a third criterion of the interaction of these analogues with transducin. The order of effectiveness of these analogues in promoting the transducin-mediated activation of the phosphodiesterase was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh much greater than GppCH2p greater than GTP gamma OMe greater than GTP gamma F GTP gamma S was more than a 1000 times as potent as GTP gamma F in activating the phosphodiesterase.(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.