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.     

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.     

Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go alpha with rhodopsin, anti-Go alpha polyclonal antibodies, and a monoclonal antibody against transducin alpha subunit and Gi alpha

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of guanyl nucleotides and rhodopsin on ADP-ribosylation of the inhibitory GTP-binding component of adenylate cyclase by pertussis toxin

Hormonal inhibition of adenylate cyclase is mediated by a guanyl nucleotide binding protein, Gi, which is composed of alpha, beta, and gamma subunits (Gi alpha, G beta gamma). Pertussis toxin blocks hormonal inhibition by catalyzing the ADP-ribosylation of Gi alpha. With purified Gi subunits, but without nucleotides, it was observed that toxin-catalyzed ADP-ribosylation of Gi alpha was negligible in the absence of G beta gamma; ATP, previously shown to increase ADP-ribosylation in membranes, enhanced the ADP-ribosylation of Gi alpha in the absence, more than in the presence, of G beta gamma. Prior studies (Kanaho, Y., Tsai, S.-C., Adamik, R., Hewlett, E.L., Moss, J., and Vaughan, M. (1984) J. Biol. Chem. 259, 7378-7381) had demonstrated that rhodopsin, the retinal photon receptor protein, can replace inhibitory hormone receptors, and stimulate the hydrolysis of GTP by Gi alpha in the presence of G beta gamma. Photolyzed rhodopsin, but not the inactive, dark protein, inhibited ADP-ribosylation of Gi alpha in the presence of G beta gamma. ADP-ribosylation of Gi alpha, in the presence of G beta gamma and photolyzed (but not dark) rhodopsin was increased by guanosine 5'-O-(2-thiodiphosphate) or GDP, but not by (beta, gamma-methylene)guanosine triphosphate or guanosine 5'-O-(3-thiotriphosphate). Presumably, photolyzed rhodopsin and nucleoside triphosphate analogues activate Gi, whereas with dark rhodopsin and nucleoside diphosphates Gi is in the inactive state. The latter appears to be the preferred substrate for pertussis toxin. These observations are consistent with other evidence that rhodopsin and inhibitory hormone receptors are functionally similar.     

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.     

Characterization of transducin from bovine retinal rod outer segments. II. Evidence for distinct binding sites and conformational changes revealed by limited proteolysis with trypsin

The first stage of amplification in the cyclic GMP cascade in bovine retinal rod is carried out by transducin, a guanine nucleotide regulatory protein consisting of two functional subunits, T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). Limited trypsin digestion of the T beta gamma subunit converted the beta polypeptide to two stable fragments (Mr approximately 26,000 and approximately 14,000). The GTPase and Gpp(NH)p binding activities were not significantly affected by the cleavage. Trypsin digestion of the T alpha subunit initially removed a small segment from the polypeptide terminus and resulted in the formation of a single 38,000-Da fragment. When this fragment was recombined with the intact T beta gamma subunit in the presence of membranes containing photolyzed rhodopsin, the reconstituted transducin exhibited greatly reduced GTPase and Gpp(NH)p binding activities. The loss in activities was due to the inability of the cleaved T alpha to bind to the photolyzed rhodopsin. Prolonged digestion converted the 38,000-Da fragment to a transient 32,000-Da fragment and then to two stable 23,000-Da and 12,000-Da fragments. The cleavage of the 32,000-Da fragment, however, can be blocked by bound Gpp(NH)p. The 32,000-Da fragment contains the Gpp(NH)p binding site and retains the ability to activate phosphodiesterase. These results indicate that the guanine nucleotide binding and rhodopsin binding sites are located in topologically distinct regions of the T alpha subunit and proved evidence that a large conformational transition of the molecule occurs upon the conversion of the bound GDP to GTP.     

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.     

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)     

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.     

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.     

Production of antibodies against rhodopsin after immunization with beta gamma-subunits of transducin: evidence for interaction of beta gamma-subunits of guanosine 5'-triphosphate binding proteins with receptor

The light-detecting system of retinal rod outer segments is regulated by a guanyl nucleotide binding (G) protein, transducin, which is composed of alpha-, beta-, and gamma-subunits. Transducin couples rhodopsin to the intracellular effector enzyme, a cGMP phosphodiesterase. The beta gamma complex (T beta gamma) is required for the alpha-subunit (T alpha) to interact effectively with the photon receptor rhodopsin. It is not clear, however, whether T beta gamma binds directly to rhodopsin or promotes T alpha binding to rhodopsin only by binding to T alpha. We have found that serum from rabbits immunized with T beta gamma contained a population of antibodies that were reactive against rhodopsin. These antibodies could be separated from T beta gamma antibodies by absorbing the latter on immobilized transducin. Binding of purified rhodopsin antibodies was inhibited by T beta gamma, suggesting that the rhodopsin antibodies and T beta gamma bound to the same site on rhodopsin. We propose that the rhodopsin antibodies act both as antiidiotypic antibodies against the idiotypic T beta gamma antibodies and as antibodies against rhodopsin. This hypothesis is consistent with the conclusion that T beta gamma interacts directly with the receptor. It is probable that in an analogous way, G beta gamma interacts directly with receptors of the adenylate cyclase system.     

A structural model for the alpha-subunit of transducin. Implications of its role as a molecular switch in the visual signal transduction mechanism

Transducin is a GTP-binding protein which mediates the light activation signal from photolyzed rhodopsin to cGMP phosphodiesterase and is pivotal in the visual excitation process. Biochemical studies suggest that the T alpha subunit of transducin is composed of three functional domains, one for rhodopsin/T beta gamma interaction, another for guanine nucleotide binding, and a third for the activation of phosphodiesterase. The integration of the primary sequence of T alpha along with secondary structure, hydropathy and folding topology predictions, and a comparison with homologous proteins have led to the construction of a three-dimensional model of the T alpha subunit. A molecular mechanism which underlies the coupling action of T alpha is suggested on the basis of this model.     

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.     

Nucleotide exchange and cGMP phosphodiesterase activation by pertussis toxin inactivated transducin.

Transducin, the signal coupling protein of retinal rod photoreceptor cells, is one of a family of G proteins that can be inactivated by pertussis toxin. We have investigated the nature of this inactivation in order to determine (1) whether it requires the toxin-catalyzed transfer of ADP-ribose from NAD+ to cysteine-347 of the alpha subunit and (2) whether it involves locking the alpha subunit in the inactive conformation characteristic of its GDP-bound state, or is limited to disruption of binding to photoexcited rhodopsin (R*). Our results indicate that all observed effects of pertussis toxin treatment, including a shift in the electrophoretic mobility of transducin's alpha subunit and functional inactivation, require NAD+ and that the appearance of the shift parallels incorporation of ADP-ribose. We have also found that, apart from interactions with photoexcited rhodopsin, the functional properties of ADP-ribosylated transducin are essentially the same as those of unmodified transducin. Normal spontaneous nucleotide exchange kinetics and the ability to activate cGMP phosphodiesterase are preserved following quantitative ADP-ribosylation, as are the abilities to hydrolyze GTP, to bind to a dye affinity column, and to display enhanced fluorescence upon addition of Al3+ and F-. Thus, ADP-ribosylation merely blocks catalysis of transducin nucleotide exchange by R* and does not lock transducin in an inactive state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhodopsin/transducin interactions. II. Influence of the transducin-beta gamma subunit complex on the coupling of the transducin-alpha subunit to rhodopsin.

In these studies we have investigated the role of the beta gamma T subunit complex in promoting the rhodopsin-stimulated guanine nucleotide exchange reaction (i.e. the activation event) of the alpha T subunit. The results of these studies demonstrate that although the beta gamma T subunit complex increases the association of the alpha T subunit with lipid vesicles that lack the photoreceptor, the beta gamma T complex is not necessary for the binding of alpha T to lipid vesicles containing rhodopsin, provided sufficient amounts of rhodopsin are present. The rhodopsin-promoted GDP/guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) exchange reaction, within the rhodopsin-alpha T complex, then results in the dissociation of the alpha TGTP gamma S species from the rhodopsin-containing phospholipid vesicles. A second line of evidence for the occurrence of rhodopsin/alpha T interactions, in the absence of beta gamma T, comes from phosphorylation studies using the beta 1 isoform of protein kinase C. The phosphorylation of the alpha T subunit by protein kinase C is inhibited by beta gamma T, both in the absence and in the presence of rhodopsin, but is enhanced by rhodopsin in the absence of beta gamma T. These rhodopsin-alpha T complexes also appear to be capable of undergoing a rhodopsin-stimulated guanine nucleotide exchange event. When the guanine nucleotide exchange is allowed to occur prior to the addition of protein kinase C, the phosphorylation of the alpha T subunit is inhibited. Although beta gamma T is not absolutely required for the rhodopsin/alpha T interaction, it appears to increase the apparent affinity of the alpha T subunit for rhodopsin, both when rhodopsin was inserted into phosphatidylcholine vesicles and when soluble lipid-free preparations of rhodopsin were used. This results in a significant kinetic advantage for the rhodopsin-stimulated guanine nucleotide exchange event, such that the addition of beta gamma T causes a 10-fold promotion of the rhodopsin-stimulation [35S]GTP gamma S binding to alpha T after 1 min but provides less than a 20% promotion of the rhodopsin-stimulated binding after 1 h. The ability of beta gamma T to increase the association of alpha T with the lipid vesicle surface does not appear to contribute significantly to the ability of rhodopsin to couple functionally to alpha T subunits, and there appears to be no requirement for beta gamma T in the alpha T activation event, once the rhodopsin-alpha T complex has formed.     

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)     

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.     

Rhodopsin-stimulated activation-deactivation cycle of transducin: kinetics of the intrinsic fluorescence response of the alpha subunit

The intrinsic tryptophan fluorescence of the alpha subunit of transducin (alpha T) has been shown to be sensitive to the binding of guanine nucleotides, with the fluorescence being enhanced by as much as 2-fold upon the binding of GTP or nonhydrolyzable GTP analogues [cf. Phillips and Cerione (1988) J. Biol. Chem. 263, 15498-15505]. In this work, we have used these fluorescence changes to analyze the kinetics for the activation (GTP binding)-deactivation (GTPase) cycle of transducin in a well-defined reconstituted phospholipid vesicle system containing purified rhodopsin and the alpha T and beta gamma T subunits of the retinal GTP-binding protein. Both the rate and the extent of the GTP-induced fluorescence enhancement are dependent on [rhodopsin], while only the rate (and not the extent) of the GTP gamma S-induced enhancement is dependent on the levels of rhodopsin. Comparisons of the fluorescence enhancements elicited by GTP gamma S and GTP indicate that the GTP gamma S-induced enhancements directly reflect the GTP gamma S-binding event while the GTP-induced enhancements represent a composite of the GTP-binding and GTP hydrolysis events. At high [rhodopsin], the rates for GTP binding and GTPase are sufficiently different such that the GTP-induced enhancement essentially reflects GTP binding. A fluorescence decay, which always follows the GTP-induced enhancement, directly reflects the GTP hydrolytic event. The rate of the fluorescence decay matches the rate of [32P]Pi production due to [gamma-32P]GTP hydrolysis, and the decay is immediately reversed by rechallenging with GTP. The GTP-induced fluorescence changes (i.e., the enhancement and ensuing decay) could be fit to a simple model describing the activation-deactivation cycle of transducin. The results of this modeling suggest the following points: (1) the dependency of the activation-deactivation cycle on [rhodopsin] can be described by a simple dose response profile; (2) the rate of the rhodopsin-stimulated activation of multiple alpha T(GDP) molecules is dependent on [rhodopsin] and when [alpha T] greater than [rhodopsin], the activation of the total alpha T pool may be limited by the rate of dissociation of rhodopsin from the activated alpha T(GTP) species; and (3) under conditions of optimal rhodopsin-alpha T coupling (i.e., high [rhodopsin]), the cycle is limited by GTP hydrolysis with the rate of Pi release, or any ensuing conformational change, being at least as fast as the hydrolytic event.     

Heterogeneity of the retinal G-protein transducin from frog rod photoreceptors. Biochemical identification and characterization of new subunits.

Transducin, a retinal G-protein, has been shown to exist as heterotrimers of alpha (39,000), beta (36,000), and gamma (approximately 7,000) subunits. Blue Sepharose CL-6B column chromatography of a transducin preparation extracted with a metal-free, low salt buffer containing GTP showed three distinct alpha and two distinct beta gamma activities in frog (Rana catesbeiana) rod outer segment. The binding of a hydrolysis-resistant GTP analog in these alpha fractions was proportional to the amount of the M(r) 39,000 protein. The first alpha was eluted in a complex with an inhibitory subunit of cGMP phosphodiesterase, but alpha subunits in the second and the third fractions were not complexed with any proteins. Two-dimensional gel electrophoresis and characterization with regard to the interaction with the inhibitory subunit of cGMP phosphodiesterase suggested that the first and the second alpha s were the same protein; however, the third alpha showed different characters as follows. We designated alpha in the first two fractions as alpha 1, and alpha in the third fraction as alpha 2. Nonlinear regression analysis for the binding of a hydrolysis-resistant GTP analog to both alpha subunits revealed a single class of GTP binding sites with an apparent stoichiometry of 1 mol of GTP/mol of alpha. Compared with alpha 1, alpha 2 required larger amounts of rhodopsin and beta gamma for the binding of a hydrolysis-resistant GTP analog. alpha 2 also showed less binding with the inhibitory subunit of cGMP phosphodiesterase. Both alpha 1 and alpha 2 complexed with beta gamma or beta delta (described below) were substrates for pertussis toxin-dependent ADP-ribosylation. The protein profiles of two beta gamma fractions revealed that the main fraction was composed of a beta gamma complex; however, the second active fraction was composed of beta complexed with delta (M(r) 12,000). Compared with beta gamma, beta delta stimulated GTP binding to alpha 1 at approximately 10-fold higher concentration. Two-dimensional gel electrophoresis revealed five beta and two gamma isoforms in beta gamma. Only one beta isoform was present in beta delta. The diversity of transducin subunits may reflect different signaling pathways in visual signal transduction.