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PubMed1989

Inhibition of transducin activation and guanosine triphosphatase activity by aluminum ion.

Aluminum ion perturbs the activity of a number of physiologically important enzymes, including members of a family of guanine nucleotide-binding proteins (G-proteins). G-proteins couple cellular receptor proteins to a variety of effector enzymes (including adenylate cyclase, phospholipase C, and the rod photoreceptor phosphodiesterase). We show herein that subnanomolar concentrations of free aluminum ion, produced in a carefully defined and kinetically stable manner through the buffering of total aluminum at 0.1-1.0 mM with calculated ratios of chelating agents, inhibit both the receptor-mediated activation and the self-inactivating GTPase activity of the rod photoreceptor G-protein, Gv. In the presence of 4 X 10(-10) M free aluminum ion, GTPase activity is inhibited from about 25-60% as the magnesium ion concentration is reduced from 10(-3) to about 5 X 10(-5) M. The principal effect of aluminum ion upon Gv is to inhibit receptor catalyzed nucleotide exchange. Binding of the GTP analog 5'-guanylyl imidodiphosphate can be reduced by as much as 90% by aluminum ion following subsaturating rhodopsin stimulation. Aluminum ion can produce either competitive or mixed noncompetitive inhibition of rhodopsin-catalyzed Gv activation and GTPase activity, as a function of whether Gv undergoes single (competitive), or multiple (mixed noncompetitive) nucleotide exchanges. The rod photoreceptor phosphodiesterase is only slightly inhibited by similar aluminum ion activities. Light- and Gv-coupled phosphodiesterase activation exhibits both a lower maximum rate of cyclic guanosine monophosphate hydrolysis and a slower inactivation in the presence of aluminum ion activities from about 10(-12) - 10(-10) M. These data suggest that intracellular free aluminum ion concentrations in the subnanomolar range could markedly affect the ability of cells to transduce extracellular signals. Interestingly, the combination of Al3+ and F- to produce the fluoro-aluminate species (AlFx) also inhibits the GTPase of G-proteins, although the mechanism of inhibition (e.g. binding to the G-protein.Mg2+.GDP complex) is totally distinct from that observed for free Al3+ and the overall effect on signal transduction (e.g. enhanced signal amplification) is in complete opposition to that observed for free Al3+.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

A monoclonal antibody against the rod outer segment guanyl nucleotide-binding protein, transducin, blocks the stimulatory and inhibitory G proteins of adenylate cyclase.

GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, tranducin (Gt), might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the alpha subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified alpha subunits of other G proteins (Gi and Gs, regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc- variant of S49 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc- variant and the S49 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1985

The G-protein of retinal rod outer segments (transducin). Mechanism of interaction with rhodopsin and nucleotides.

The mechanism of interaction of the G-protein of retinal rods with rhodopsin and with nucleotides has been investigated using two independent techniques, light-scattering and direct binding measurements with labeled nucleotides. Binding of photoexcited rhodopsin (R*) and nucleotides are shown to be antagonist, and three conformations of the G-protein are described, each of which is proposed to be related to a different level of light-scattering, as follows: (a) the "dark" state, stable in the absence of photoexcited rhodopsin, in which the nucleotide site is poorly accessible and has a high affinity (dissociation constants, 0.1 microM for GDP and 0.01 microM for GppNHp); (b) the R*-bound state in which the nucleotide site is rapidly accessible with a lower affinity (dissociation constants, about 20 microM for GDP and GTP; 20-100 microM for GppNHp). Binding of R* to the G-protein therefore enables rapid binding or exchange of the nucleotide; this in turn reduces the affinity of the G-protein for R* (dissociation constants, 0.2 microM for G-protein with GDP bound and 2-10 microM for G-protein with GppNHp bound, compared to 1 nM in absence of bound nucleotide); and (c) the third state, the activator of the phosphodiesterase. In the presence of GTP, an additional irreversible and fast step, which is proposed to be the dissociation of alpha-GTP from beta gamma, is shown to occur; a steady state equilibrium is obtained, and the dissociation constant measured between GTP and this third state of the G-protein in the presence of R* is an apparent constant which depends on the rate of transconformation between the first two states and on the rate of GTP hydrolysis. The minimum value of this apparent dissociation constant for GTP (0.05-0.1 (microM) is obtained at high levels of illumination. Finally, some results (number of nucleotide sites and saturation of the rate of the light-scattering signal) suggest an oligomeric association of the G-protein.

Animals↗
PubMed1994

Molecular cloning and biological activity of a novel developmentally regulated gene encoding a protein with beta-transducin-like structure.

In the developing mammalian central nervous system, neural precursor cells show a tightly regulated inverse relationship between cell proliferation and differentiation. The molecular mechanisms which control the inter-relationship between these two events are poorly understood. To approach this problem, we previously identified several novel genes which are most prominently expressed in the early embryonic brain. Further cloning and sequencing of one such gene, Nedd1, revealed that it can encode a protein with a M(r) of 71,000, the amino-terminal half of which shares significant structural similarity with the beta-subunit of heterotrimeric GTP-binding proteins. Nedd1 mRNA is strongly expressed in early embryonic brain, but it can be detected at low levels in a number of adult tissues as well as cell lines and is up-regulated in an embryonal carcinoma cell line upon retinoic acid-induced differentiation. Ectopic expression of Nedd1 gene by means of eukaryotic vectors in various cell lines resulted in varying degrees of growth suppression. The strongest effects were evident in differentiation-competent neuroblastoma-derived cell lines. Our results suggest that the Nedd1 gene may play a role in the differentiation-coupled growth arrest in neuronal cells.

Amino Acid Sequence↗
PubMed1988

Structural and functional relationships of guanosine triphosphate binding proteins.

Information available at present documents the existence of three well-defined classes of guanine nucleotide binding proteins functioning as signal transducers: Gs and Gi which stimulate and inhibit adenylate cyclase, respectively, and transducin which transmits and amplifies the signal from light-activated rhodopsin to cGMP-dependent phosphodiesterase in ROS membranes. Go is a fourth member of this family. Its function is the least known among GTP binding signal transducing proteins. The family of G proteins has a number of properties in common. All are heterotrimers consisting of three subunits, alpha, beta, and gamma. Each of the subunits may be heterogeneous depending on species and tissue of origin and may be posttranslationally modified covalently. The alpha subunits vary in size from 39 to 52 kDa. The sequences for Gs alpha and transducin alpha have 42% overall homology and those of Gi alpha and Gs alpha 43%, whereas those of Gi alpha and transducin alpha have a higher degree (68%) of homology. All alpha subunits bind guanine nucleotides and are ADP-ribosylated by either pertussis toxin (Gi, transducin, Go) or cholera toxin (Gs, Gi, transducin). Thus, transducin and Gi, which have the highest degree of sequence homology, are also ADP-ribosylated by both toxins. The beta subunits have molecular weights of 36 and 35 kDa, respectively. While Gs, Gi, and Go contain a mixture of both, transducin contains only the larger (36-kDa) beta-polypeptide. The relationship of the 36- and the 35-kDa beta subunits is not defined. Although the complete sequence of the 36-kDa beta subunit of transducin has been deduced from the cDNA sequence, complete sequences of other beta subunits are not yet available so that detailed comparisons cannot be made at present. However, the proteolytic profiles of each class of the beta subunits of different G proteins are indistinguishable. The gamma subunit of bovine transducin has been completely sequenced. It has a Mr of 8400. Again complete sequences of other gamma subunits are not yet available. While the gamma subunits of Gs, Gi, and Go have identical electrophoretic mobility in SDS gels, they differ significantly in this respect from the gamma subunit of transducin. Moreover, crossover experiments point to functional differences between gamma subunits from G protein and transducin complexes. In addition, a role for beta, gamma in anchoring guanine nucleotide binding proteins to membranes has been postulated.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1985

Interspecies conservation of retinal guanosine 5'-triphosphatase. Characterization by photoaffinity labelling and tryptic-peptide mapping.

Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5'-triphosphatase referred to as 'transducin'. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidoguanosine [gamma-32P]triphosphate demonstrated that the 37000-Mr components of the cow, pig and chick and the 75000-Mr component of the frog were major GTP-binding components. In addition, peptide maps of radioiodinated tryptic peptides indicate that the frog 75000-Mr protein is highly related to the pig transducin. These results demonstrate evolutionary conservation of retinal transducin and the presence of a higher-Mr, but nonetheless highly conserved form, of transducin in the frog. The relationship of this component to the recently reported rod-outer-segment inhibitor protein [Yamazaki, Stein, Chernoff & Bitensky (1983) J. Biol. Chem. 258, 8188-8194] is discussed.

Animals↗
PubMed1988

The photoreceptor-specific 33 kDa phosphoprotein of mammalian retina: generation of monospecific antibodies and localization by immunocytochemistry.

The distribution in mouse retina of a 33,000 Da phosphoprotein (33 kDa) that complexes with the beta/gamma subunits of transducin (T beta gamma) and undergoes light-induced dephosphorylation was determined by immunocytochemistry. An antiserum containing antibodies for the 33 kDa protein and beta-transducin of mouse and bovine retinas was generated against the purified 33 kDa-T beta gamma complex from bovine retina. The antiserum reacts with beta-transducin derived from either 33 kDa-T beta gamma complex or transducin complex (T alpha beta gamma), but not with the alpha- or gamma-transducin. It also reacts with both the phosphorylated and unphosphorylated form of the 33 kDa-T beta gamma complex. Antibodies, monospecific for the 33 kDa and beta-transducin subunits respectively, were purified from the antiserum by immunoadsorption and used in immunocytochemical analysis of the respective antigens. The 33 kDa protein was found to be associated exclusively with the photoreceptor cells of the retinas, with the most intense staining in the inner and outer segments' layers and lighter staining in the synaptic terminal layers. beta-Transducin also is found in the photoreceptors, but some T beta immunoreactivity exists within the inner plexiform layer. The specific localization of the 33 kDa protein together with its light-modulated phosphorylation suggest that the 33 kDa-T beta gamma complex is involved in light-regulated activities of the rod photoreceptor cells.

Animals↗
PubMed1988

Chromium(III) beta, gamma-bidentate guanine nucleotide complexes as probes of the GTP-activated cGMP cascade of retinal rod outer segments.

The exchange-inert Cr(III) beta, gamma-bidentate guanine nucleotide complexes Cr(III)GTP and Cr(III)Gpp(NH)p were used to probe the role of transducin in activating the retinal cGMP cascade. The Cr(III) nucleotide complexes were found to have lower binding affinity for transducin as compared to the Mg2+ complexes. However, the rate of hydrolysis of the transducin-bound Cr(III)GTP was similar to that of Mg(II)GTP. Cr(III)Gpp(NH)p activated the cGMP phosphodiesterase of photolyzed rod outer segment membranes up to 75% of the Mg(II)Gpp(NH)p level but lacked the ability to dissociated the transducin subunits from the rod outer segment membrane. This result implies that the activation of the phosphodiesterase by transducin-GTP complex is a membrane-associated event and the formation of a soluble complex of transducin-GTP with the inhibitory peptide of the phosphodiesterase may not be an obligatory step. Both the delta and lambda screw sense stereoisomers of Cr(III)Gpp(NH)p were capable of activating the cGMP cascade with no apparent stereoselectivity. The nature of the interaction of the metal ion and GTP at the nucleotide-binding site of transducin is discussed together with the results from previous studies using the phosphorothioate GTP analogues [Yamanaka, G., Eckstein, F., & Stryer, L. (1985) Biochemistry 24, 8094-8101] and is compared to the site found in homologous GTP-binding proteins such as elongation factor Tu [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36; la Cour, T.F.M., Nyborg, J., Thirup, S., & Clark, B.F.C. (1985) EMBO J. 4, 2385-2388]. The implications of the observed results on the molecular mechanism of visual signal transduction are discussed.

Animals↗
PubMed1991

Deactivation kinetics of the transduction cascade of vision.

The response of the retinal rod cell to a dim flash lasts less than a second. This phototransduction is mediated by a guanine nucleotide-binding (G) protein cascade in which rhodopsin is the receptor, transducin is the G-protein, and the cGMP-specific phosphodiesterase (PDE) is the effector. Photoexcited rhodopsin activates transducin which in turn activates PDE. For this underlying biochemistry to be kinetically compatible with the photoresponse, both transducin and PDE must be deactivated in subsecond times. We report here direct measurements of their deactivation kinetics. The rate of heat release when transducin and PDE hydrolyze, respectively, GTP and cGMP was measured using time-resolved microcalorimetry. With only GTP present, the heat pulse comes from the activation of transducin and its subsequent deactivation by endogenous GTP hydrolysis. The nonhydrolyzable analog guanine 5'-[gamma-thio]triphosphate was used to distinguish between these two processes: about 40% of the total heat is due to activation. From the time course of the deactivation heat, the active lifetime of transducin is less than 1 s at 22 degrees C. With both GTP and cGMP present, the highly amplified hydrolytic activity of the PDE is responsible for most of the heat produced; its rate of release is directly proportional to the amount of activated PDE. Measurements of this rate at low photoexcitation levels (e.g., 30 molecules of photoexcited rhodopsin per rod) provide much kinetic information about the cascade. Notably, deactivation of the PDE takes 0.6 s (at 23 degrees C) and absolutely requires GTP hydrolysis. This concurs with the subsecond lifetime of active transducin and means that, once GTP hydrolysis has occurred, the hitherto active PDE is quickly inhibited.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Functional expression of the taste specific G-protein, alpha-gustducin.

The taste-specific G-protein alpha-subunit, alpha-gustducin, was expressed using a baculovirus based system. alpha-Gustducin was demonstrated to be myristoylated and was also palmitoylated in insect larval cells. Recombinant alpha-gustducin was purified to homogeneity. Neither receptors nor effectors that interact with gustducin in taste are known. However, alpha-gustducin has a close structural similarity to the visual G-protein, alpha-transducin. Therefore alpha-gustducin was reconstituted with components of the visual system to determine the degree of its functional similarity with alpha-transducin. Despite the fact that the sequences of alpha-gustducin and alpha-transducin share only 80% identity with each other, the interactions and functions of these two proteins were quantitatively identical. These included the interaction with receptor, bovine rhodopsin, with effector, bovine retinal cyclic GMP-phosphodiesterase, and with bovine brain and retinal G-protein beta gamma-heterodimers; receptor-catalysed GDP-GTP exchange and the intrinsic GTPase activity of alpha-gustducin and alpha-transducin were also identical. Gi alpha which is 70% identical with alpha-transducin interacts with different receptor and effector proteins and has very different guanine-nucleotide binding properties. Therefore, the functional equivalence of alpha-gustducin and alpha-transducin suggest that taste buds are likely to contain receptor and effector proteins that share many properties with their retinal equivalents.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

An effector site that stimulates G-protein GTPase in photoreceptors.

Heterotrimeric G-proteins mediate between receptors and effectors, acting as molecular clocks. G-protein interactions with activated receptors catalyze the replacement of GDP bound to the alpha-subunit with GTP. alpha-Subunits then modulate the activity of downstream effectors until the bound GTP is hydrolyzed. In several signal transduction pathways, including the cGMP cascade of photoreceptor cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with corresponding effectors. In the phototransduction cascade the GTPase activity of photoreceptor G-protein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. Here we characterize the stimulation of transducin GTPase by a set of 23 mutant phosphodiesterase gamma-subunits (PDE gamma) containing single alanine substitutions within a stretch of the 25 C-terminal amino acid residues known to be primarily responsible for the GTPase regulation. The substitution of tryptophan at position 70 completely abolished the acceleration of GTP hydrolysis by transducin in a complex with this mutant. This mutation also resulted in a reduction of PDE gamma affinity for transducin, but did not affect PDE gamma interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDE gamma to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDE gamma in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2002

Noncatalytic domains of RGS9-1.Gbeta 5L play a decisive role in establishing its substrate specificity.

The complex between the photoreceptor-specific regulator of G protein signaling (RGS) protein, RGS9-1, and type 5 G protein beta-subunit, Gbeta5L, regulates the duration of the cellular response to light by stimulating the GTPase activity of G protein, transducin. An important property of RGS9-1.Gbeta5L is that it interacts specifically with transducin bound to its effector, cGMP phosphodiesterase, rather than with transducin alone. The minimal structure within the RGS9-1.Gbeta5L complex capable of activating transducin GTPase is the catalytic domain of RGS9. This domain itself is also able to discriminate between free and effector-bound transducin but to a lesser degree than RGS9-1.Gbeta5L. The goal of this study was to determine whether other, noncatalytic domains of RGS9-1.Gbeta5L enhance the intrinsic specificity of the catalytic domain or whether they set the specificity of RGS9-1.Gbeta5L regardless of the specificity of its catalytic domain. We found that a double L353E/R360P amino acid substitution reversed the specificity of the recombinant catalytic domain but did not reverse the specificity of RGS9-1.Gbeta5L. However, the degree of discrimination between free and effector-bound transducin was reduced. Therefore, noncatalytic domains of RGS9-1.Gbeta5L play a decisive role in establishing its substrate specificity, yet the high degree of this specificity observed under physiological conditions requires an additional contribution from the catalytic domain.

Animals↗
PubMed2005

A role for cytoskeletal elements in the light-driven translocation of proteins in rod photoreceptors.

PURPOSE: Light-driven protein translocation is responsible for the dramatic redistribution of some proteins in vertebrate rod photoreceptors. In this study, the involvement of microtubules and microfilaments in the light-driven translocation of arrestin and transducin was investigated. METHODS: Pharmacologic reagents were applied to native and transgenic Xenopus tadpoles, to disrupt the microtubules (thiabendazole) and microfilaments (cytochalasin D and latrunculin B) of the rod photoreceptors. Quantitative confocal imaging was used to assess the impact of these treatments on arrestin and transducin translocation. A series of transgenic tadpoles expressing arrestin truncations were also created to identify portions of arrestin that enable arrestin to translocate. RESULTS: Application of cytochalasin D or latrunculin B to disrupt the microfilament organization selectively slowed only transducin movement from the inner to the outer segments. Perturbation of the microtubule cytoskeleton with thiabendazole slowed the translocation of both arrestin and transducin, but only in moving from the outer to the inner segments. Transgenic Xenopus expressing fusions of green fluorescent protein (GFP) with portions of arrestin implicates the C terminus of arrestin as an important portion of the molecule for promoting translocation. This C-terminal region can be used independently to promote translocation of GFP in response to light. CONCLUSIONS: The results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motor-driven mechanism for complete movement of arrestin and transducin.

Actin Cytoskeleton↗