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PubMed1986

Ability of guanine nucleotide derivatives to bind and activate bovine transducin.

Several guanine nucleotide analogs, in one series of which a hydrogen on the 2-amino group is replaced with the p-n-butylphenyl group (BuPGNP derivatives), were used to probe the GTP binding domain of bovine transducin. The order of apparent binding affinities in a series of nucleoside 5'-triphosphates was GTP gamma S greater than GTP approximately BuPGTP greater than dGTP approximately ITP much greater than ATP, values which were 30-100 times higher than affinities of the corresponding 5'-diphosphates. A derivative bearing a 6-aminohexylamino group on the gamma-phosphate, BuPGTP X C6, had a 60-fold lower affinity compared to BuPGTP. In contrast, the p-n-butylphenyl substituent on the 2-amino group had little effect on the binding affinity relative to GTP. Substitutions at the 2-amino group had little effect on either the hydrolysis of the derivatives by the GTPase activity associated with the alpha-subunit of transducin or the activation of cGMP phosphodesterase. The results indicate that the GTP binding domain of transducin is similar in tertiary structure to the corresponding domain of EF-Tu. The 5'-phosphates of GTP are oriented in the binding site of transducin so that the bulky C6 group of BuPGTP X C6 dramatically interferes with binding. The 2-amino group on the guanine ring is probably located at the periphery of the binding site, with the p-n-butylphenyl substituent of BuPGTP facing outward and only weakly interacting with the protein. BuPGTP should be an excellent parent compound for development of novel probes of G-protein interactions with other cellular proteins involved in receptor signal transduction.

Animals↗
PubMedDate not supplied

[Adenylate kinase and GDP-kinase activity of rod outer segments in the frog retina. Possible functional role of the T beta subunit of transducin].

Pure frog retina rod outer segments (ROS) preparations (A280/A500 = 2,1-2,3) catalyze the synthesis of ATP from ADP in the presence of Mg2+. Adenylate kinase (AK) (ATP:AMP phosphotransferase, EC 2.7.4.3) specific activities for ROS preparations are within the range 2-4 mumole per hour for mg protein. The enzymatic activity of investigated preparations is due to intact, but not destroyed ROS. The component which possesses AK is found in water-soluble, but not in membranous ROS fractions and seems to be a part of the predominant ROS plasma protein--GTP-binding complex of transducin. It has been shown, that this component is the T beta subunit of transducin and its enzymatic activity is controlled by other subunits of the transducin complex. The obtained results indicate that GDP kinase (ATP:GDP phosphotransferase, EC 2.7.4.6) activity of transducin depends on the work of both of T beta and T alpha subunits. It has been shown that in the ROS preparations synthesis of the ATP from ADP and GDP phosphorylation are stimulated by a lowering of Ca2+ concentration (less than 10(-5)-10(-7) M). T beta component is suggested to play the role of phosphotransferase which phosphorylates GDP associated with the T alpha subunits and it leads to formation of a complex T alpha X GTP which is an activator of vertebrate retina ROS phosphodiesterase.

Adenosine Diphosphate↗
PubMed1983

Characterization of transducin from bovine retinal rod outer segments. I. Separation and reconstitution of the subunits.

Transducin, a guanine nucleotide regulatory protein found in the bovine retinal rod outer segment, mediates the signal coupling between rhodopsin and a cyclic GMP phosphodiesterase. Previous studies have demonstrated that photolyzed rhodopsin catalyzed the exchange of GTP for GDP bound to transducin. The transducin-GTP complex, in turn, activates the phosphodiesterase. Purified transducin (T) has been resolved by omega-amino octylagarose chromatography into two functional subunits: T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). The guanine nucleotide binding site is on the T alpha subunit. Neither the T alpha nor the T beta gamma subunit showed significant GTPase activity, Gpp(NH)p-GDP exchange, and ability to bind to rhodopsin when assayed in the presence of reconstituted membranes containing photolyzed rhodopsin. However, all the above activities were restored if the two subunits were recombined. Analysis of the reconstituted GTPase activity as a function of the T alpha subunit concentration revealed a linear relationship. On the other hand, GTPase activity rapidly saturated at T beta gamma concentration much lower than the T alpha concentration, indicating that the two subunits were dissociated during GTP hydrolysis. These findings strongly suggest that the T beta gamma subunit is an activator of the GTPase activity. Its mode of action is to enable the T alpha subunit to interact with rhodopsin effectively.

Animals↗
PubMed1984

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.

Adenosine Diphosphate Ribose↗
PubMed1993

Regulation of G protein function by an effector in GTP-dependent signal transduction. An inhibitory subunit of cGMP phosphodiesterase inhibits GTP hydrolysis by transducin in vertebrate rod photoreceptors.

The regulation of cGMP phosphodiesterase in vertebrate rod photoreceptors is a typical G protein-dependent signal transduction mechanism. The interaction of P gamma, an inhibitory subunit of cGMP phosphodiesterase, with transducin alpha subunit (T alpha) is essential for the activation of cGMP phosphodiesterase. It has been shown that, in a homogenized preparation of frog (Rana catesbeiana) rods, P gamma interacts with GTP.T alpha and remains tightly bound to GDP.T alpha after GTP hydrolysis on T alpha. Association of this complex with beta gamma subunits of transducin (T beta gamma) triggers the release of P gamma from the complex and the subsequent inactivation of cGMP phosphodiesterase. In a system reconstituted with purified components, both GTP- and GDP-bound forms of T alpha were found to interact with P gamma. Under these conditions, P gamma inhibited GTP hydrolysis by transducin in a noncompetitive manner with a Ki of 92 nM. Binding of an hydrolysis-resistant GTP analog to T alpha was also inhibited by P gamma. These inhibitions of transducin function were resulted from the inhibition of both hydrolysis of GTP bound to T alpha and interaction of GDP.T alpha with membrane-bound T beta gamma. However, after GDP.T alpha reassociated with membrane-bound T beta gamma, the inhibitory effect of P gamma on the binding of an hydrolysis-resistant GTP analog to T alpha was greatly diminished, suggesting that the GTP/GDP exchange on T alpha was not inhibited by P gamma. These data indicate that the T alpha function is altered during complexing with P gamma. G protein functions may be modified by interacting with an effector in the G protein-dependent signal transduction.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Saccharomyces cerevisiae cdc15 mutants arrested at a late stage in anaphase are rescued by Xenopus cDNAs encoding N-ras or a protein with beta-transducin repeats.

We have constructed a Xenopus oocyte cDNA library in a Saccharomyces cerevisiae expression vector and used this library to isolate genes that can function in yeast cells to suppress the temperature sensitive [corrected] defect of the cdc15 mutation. Two maternally expressed Xenopus cDNAs which fulfill these conditions have been isolated. One of these clones encodes Xenopus N-ras. In contrast to the yeast RAS genes, Xenopus N-ras rescues the cdc15 mutation. Moreover, overexpression of Xenopus N-ras in S. cerevisiae does not activate the RAS-cyclic AMP (cAMP) pathway; rather, it results in decreased levels of intracellular cAMP in both mutant cdc15 and wild-type cells. Furthermore, we show that lowering cAMP levels is sufficient to allow cells with a nonfunctional Cdc15 protein to complete the mitotic cycle. These results suggest that a key step of the cell cycle is dependent upon a phosphorylation event catalyzed by cAMP-dependent protein kinase. The second clone, beta TrCP (beta-transducin repeat-containing protein), encodes a protein of 518 amino acids that shows significant homology to the beta subunits of G proteins in its C-terminal half. In this region, beta Trcp is composed of seven beta-transducin repeats. beta TrCP is not a functional homolog of S. cerevisiae CDC20, a cell cycle gene that also contains beta-transducin repeats and suppresses the cdc15 mutation.

Anaphase↗
PubMed1994

The photoreceptor G-protein transducin (Gt) is a substrate for ubiquitin-dependent proteolysis.

Bovine photoreceptor (rod outer segment) proteins were selectively degraded by an ATP- and ubiquitin-dependent mechanism in rabbit reticulocyte lysate and human retinal pigment epithelial cell supernatant. Proteolysis of 37, 40, 58, 68 and approximately 100 kDa proteins was accompanied by the formation of high mass (> 150 kDa) species (putative ubiquitin-protein conjugates. To identity degraded substrates, the photoreceptor GTP-binding protein, transducin (Gt alpha beta gamma), was purified by GTP elution. Degradation of transducin (> or = 50% loss of each subunit) by retinal pigment epithelial cell supernatant was ubiquitin-dependent (EC50 = 1.2 microM). Formation of high mass transducin species was coincident with degradation. These data identify a selective proteolytic mechanism that may regulate the photoreceptor GTP-binding protein.

Adenosine Triphosphate↗
PubMed1994

Muscarinic K+ channels are activated by beta gamma subunits and inhibited by the GDP-bound form of alpha subunit of transducin.

It was reported that transducin beta gamma subunits (T beta gamma) inhibited cardiac muscarinic K+ (KACh) channels activated through GK. This result has been used to support the proposal that GK activates this channel through its alpha subunits. Here, we have reexamined the effects of transducin subunits on KACh channels. T beta gamma applied to the internal side of the inside-out patch membrane activated KACh channels in a concentration-dependent manner (EC50 approximately 1 microM). In contrast, the GDP-bound form of transducin alpha subunits (50 nM-3 microM), but not T beta gamma (1 nM-1 microM), irreversibly inhibited KACh channel openings induced either by GTP plus acetylcholine or by GTP gamma S (guanosine 5'-3-O-(thio)triphosphate). This inhibition was antagonized by exogenously applied T beta gamma (600 nM-3 microM) or brain G protein beta gamma subunits (10-30 nM). These data, opposite to the previous report, indicate that GK activates KACh channels through its beta gamma subunits.

Animals↗
PubMed1999

Light activation of phosphatidylethanolamine N-methyltransferase in rod outer segments and its modulation by association states of transducin.

Phosphatidylethanolamine N-Methyltransferase (PE N-MTase) is the enzyme responsible for the synthesis of phosphatidylcholine from phosphatidylethanolamine by successive transfer of methyl groups. This enzyme is present in bovine rod outer segments (ROS) and it is the only pathway for the synthesis of phosphatidylcholine in the outer segment of rod photoreceptor cells. In dark-adapted ROS membranes PE N-MTase activity is stimulated by 100% when ROS membranes are incubated under light condition. To determine whether the retinal G protein, transducin (Gt), intervenes in the regulation of PE N-MTase in these membranes, the effects of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and guanosine 5'-O-(2-thiodiphosphate (GDPbetaS) on the enzyme activity were examined. In dark, GTPgammaS which induces dissociation of Gt, stimulates the enzyme activity mimicking the stimulation by light. On the contrary, GDPbetaS stabilizes the inactive state of Gt, inhibiting the stimulation by light of PE N-MTase without affecting basal activities. In addition, adenosine 5'-diphosphate (ADP)-ribosylation by cholera and pertussis toxin was studied. ADP-ribosylation of ROS membrane with pertussis toxin, which stabilizes transducin in its inactive state, prevents the light-induced increase in PE N-MTase activity. On the contrary ADP-ribosylation with cholera toxin stimulates the enzyme activity. Our findings therefore suggest that light-stimulated effect of PE N-MTase activity is transducin-mediated.

Adenosine Diphosphate↗
PubMed1988

Light and GTP dependence of transducin solubility in retinal rods. Further analysis by near infra-red light scattering.

The physical origin and functional significance of the near infra-red light scattering changes observable upon flash illumination of diluted suspensions of magnetically oriented, permeabilised frog retinal rods has been reinvestigated with particular attention paid to the degree with which transducin remains attached to the membrane. In the absence of GTP, the so called "binding" signal is shown to include two components of distinctive origins, widely different kinetics, and whose relative amplitudes depend on the dilution of the suspension and resulting detachment of transducin from the disc membrane. The fast component is a consequence of the fast interaction between photoexcited rhodopsin (R*) and the transducin remaining on the membrane. Its kinetics monitors a structural modification of the discs caused by a change in electrostatic interaction between closely packed membranes upon the formation of R*-T complexes. The slow component monitors the slow rebinding to the membrane and possible subsequent interaction with excess R* of T-GDP which, in spite of its low solubility, had eluted into solution given the high dilution of the permeated rods. In the presence of GTP, the so called "dissociation" signal includes a fast, anisotropic "release" component that specifically monitors the release into the interdiscal space of T alpha-GTP formed from the membrane-bound pool, and a slower isotropic "loss" component monitoring the leakage from the permeated rod of the excess T alpha-GTP which did not interact with the cGMP phosphodiesterase. The amplitudes of both components depend exclusively on the membrane bound T-GDP pool. The kinetics of the "loss" component is limited by the size and degree of permeation of the rod fragments, rather than by the dissociation rate of T alpha-GTP from the membrane.

Animals↗
PubMed1985

Partial sequence homologies between cytoskeletal proteins, c-myc, Rous sarcoma virus and adenovirus proteins, transducin, and beta- and gamma-crystallins.

Computer based sequence comparisons indicate partial sequence homology between human c-myc, Rous sarcoma virus, adenovirus 7, and simian sarcoma virus proteins and the cytoskeletal proteins d9smin, keratin and vimentin. In addition, sections of the oncogene proteins showed partial but significant homology to alpha and gamma subunits of transducin. gamma-II and beta-BP crystallins showed partial but significant homology to the cytoskeletal proteins keratin, vimentin, desmin, alpha and beta-tubulin, and to adenovirus 7 and simian sarcoma virus transforming gene proteins. Beta-BP crystallin showed partial but significant homology to Rous sarcoma virus protein, and to alpha and gamma subunits of transducin. Both crystallins showed partial sequence homology to the GTP-binding protein elongation factor TU from Escherichia coli. These sequence homologies suggest a link between the mechanisms of normal lens cell differentiation, involving modifications to the cytoskeleton and subsequent changes to the pattern of protein synthesis, and mechanisms of neoplastic transformation. Furthermore, the transducin-like region on beta-crystallin may be important for its interaction with lens membranes and the maintenance of short-range order for lens transparency.

Adenoviridae↗
PubMed1987

Isolation of a major human placental substrate for the epidermal growth factor (urogastrone) receptor kinase: immunological cross-reactivity with transducin and sequence homology with lipocortin.

Using as a starting material either a detergent extract or a protein fraction eluted from membranes with ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, we have isolated from human placental membranes a major substrate for the epidermal growth factor (urogastrone) receptor kinase (EGF kinase). The substrate was isolated both in an intact form, having a molecular mass of approximately 38-kDa (p38), and in a 35-kDa form (p35) representing a proteolytic cleavage product of p38. Both p38 and p35 cross-reacted with antibodies directed against bovine retinal transducin, but did not cross-react with antibodies directed against the 35-kDa beta subunit of human placental G-protein. Antisera directed against the placental EGF kinase substrate failed to react with either bovine or human placental src kinase substrate, p36. Conversely, antisera directed against p36 reacted only poorly with placental p38 or p35. Although p38 had a blocked amino terminus that precluded sequence analysis, p35 yielded an N-terminal sequence that was identical with residues 13-36 of human lipocortin. Our data clearly distinguish p38 from the previously described intestinal calcium binding protein calpactin I or p36 that is also a tyrosine kinase substrate, and our work points to a close relationship (if not identity) between p35 and a 35-kDa EGF receptor kinase substrate previously characterized in A431 cells. We conclude that p38 and p35, which very likely represent human placental lipocortin, may share only limited epitope homology with transducin alpha subunit; however, the possibility that p38, along with intestinal p36 and with a family of related calcium binding proteins, may, like transducin, play a role in receptor-mediated transmembrane signaling is discussed.

Amino Acid Sequence↗
PubMed1990

Retinal accumulation of the phosducin/T beta gamma and transducin complexes in developing normal mice and in mice and dogs with inherited retinal degeneration.

Rod photoreceptors of mammalian retinas contain a 33-kDa phosphoprotein, phosducin, which complexes with the beta, gamma-subunits of transducin (T beta gamma). The level of phosducin phosphorylation is modulated by light, suggesting that the phosducin/T beta gamma complex has a pivotal role in light-regulated events that occur in photoreceptors. We have investigated, in developing mouse retinas, the age at which the complex is first detected and the subsequent accumulation of the phosducin/T beta gamma complex during postnatal life. Western blot analysis detected immunoreactivity both for phosducin and T beta in retinal homogenates of 3-day-old mice. Thereafter, the level of immunoreactivity for both proteins increased steadily, to reach adult levels in the next 2 postnatal weeks. Gel filtration analysis of extracts from immature mouse retina showed that phosducin and T beta co-eluted, like the phosducin/T beta gamma complex of adult retina, as a 77-kDa complex, indicating that the phosducin/T beta gamma complex is formed when photoreceptors first synthesize the components of the complex. While the levels of the phosducin/T beta gamma complex increased steadily during the first 2 postnatal weeks, the subunits of transducin complex, T alpha together with additional amounts of T beta gamma, only started to appear around the 7-9th postnatal day, and the level of transducin complex increased sharply at 11-14 days to reach adult levels that are similar to those of phosducin/T beta gamma complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence.

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.

Amino Acid Sequence↗
PubMed1985

The tryptic and chymotryptic fragments of the beta-subunit of guanine nucleotide binding proteins in brain are identical to those of retinal transducin.

The 35-kDa beta-subunit of transducin purified from rod outer segment membranes is cleaved into 2 major fragments by trypsin, and 7 major fragments by chymotrypsin. Identical fragments are visualized by immunoblotting with transducin-beta specific antisera after proteolysis of rod outer segment membranes, purified brain guanine nucleotide binding proteins, and brain membranes. The results indicate that the beta-subunits of transducin and of brain guanine nucleotide binding proteins are not only similar structurally, but are also similarly oriented in membranes with respect to accessibility to proteolytic enzymes.

Animals↗
PubMed1988

Rapid transducin deactivation in intact stacks of bovine rod outer segment disks as studied by light scattering techniques. Arrestin requires additional soluble proteins for rapid quenching of rhodopsin catalytic activity.

In photoreceptors of the living retina both activation and deactivation of transducin must occur in less than 1 s. In ROS preparations used for in vitro studies, however, deactivation takes minutes. This is due to the fact that activated transducin is released into the free aqueous space, whereby GTPase activity and consequent deactivation of the protein are slowed down, and due to the dilution of soluble ROS proteins involved in the quenching of rhodopsin activity. In this paper, using a convenient, non-invasive light scattering assay, we demonstrate that in an intact stack of disks, where active transducin stays membrane associated and is rapidly deactivated, the activity of rhodopsin can also be quenched in the time range of seconds when soluble ROS proteins are supplemented. Arrestin, the 48 kDa protein of the photoreceptor, is one of the proteins required for rapid recovery, however, it requires the synergistic action of other soluble proteins (besides rhodopsin kinase) in order to exert its effect: When arrestin is included in the reaction mixture without the 'helper protein(s)', it cannot speed recovery, and when a mixture of soluble proteins is added which lacks arrestin, there is also no effect. The nature and identity of this (these) helper protein(s) are still unclear.

Animals↗
PubMed1989

Transducin GTPase provides for rapid quenching of the cGMP cascade in rod outer segments.

The role of transducin GTPase in rapid cGMP phosphodiesterase quenching was studied by simultaneous registration of GTP hydrolysis and phosphodiesterase activity in the same rod outer segments (ROS) preparation. The results thus obtained allow the conclusion that: (i) phosphodiesterase quenching coincides with transducin-bound GTP hydrolysis independently of ROS concentration; (ii) an increase in the ROS concentration results in the acceleration of cascade quenching due to the existence of a GTPase accelerating mechanism in ROS; (iii) approximation to physiological conditions (protein concentration, temperature) provides a transducin GTPase rate equal to 1-2 turnovers per second i.e., sufficiently high for satisfying the real rate of photoresponse reversion in dark-adapted rods.

Animals↗
PubMed1994

Altered guanine nucleoside triphosphate binding to transducin by cholera toxin-catalysed ADP-ribosylation.

The influence of cholera toxin (CTX)-catalysed ADP-ribosylation on binding of guanine nucleoside triphosphates to transducin was studied by measuring the binding of the GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), to illuminated bovine rod outer segment (ROS) membranes treated with or without CTX. Besides the well-documented inhibition of the transducin GTPase activity, CTX treatment inhibited binding of GTP[gamma S] to illuminated ROS membranes. This inhibition was due to an approximately two-fold lower apparent affinity for the nucleotide, while the density of binding sites was not altered. CTX decreased the association rate of GTP[gamma S] by a factor of about two. Competition experiments with GTP, guanosine 5'-[beta, gamma]iminotriphosphate or GDP showed that the apparent affinities for both guanine nucleoside triphosphates, but not for GDP, were lowered by about two-fold upon CTX treatment. In contrast to CTX, pertussis toxin treatment of ROS membranes reduced the density of binding sites available to GTP[gamma S], while the apparent affinity of the remaining sites was unchanged. It is concluded that ADP-ribosylation of transducin by CTX not only inhibits its GTPase activity but also decreases the affinity for guanine nucleoside triphosphates, data which suggest that the arginine moiety modified by CTX is involved in both binding and hydrolysis of GTP.

Adenosine Diphosphate Ribose↗