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PubMed1983

The regulatory components of adenylate cyclase and transducin. A family of structurally homologous guanine nucleotide-binding proteins.

G/F and transducin are guanine nucleotide-binding regulatory proteins that mediate activation of adenylate cyclase and of a rod outer segment cyclic GMP-specific phosphodiesterase, respectively. The substrate for islet-activating protein is a third guanine nucleotide-binding protein that is postulated to mediate inhibition of adenylate cyclase. The extent of structural homology among subunits of all three proteins was examined by analyses of amino acid compositions and electrophoretic patterns of proteolytic peptides. The lower molecular weight subunits (beta subunits; Mr = 35,000) of these proteins have identical amino acid compositions and yield similar peptides upon proteolysis with Staphylococcus aureus V8 protease and elastase. The higher molecular weight subunits (alpha subunits; Mr = 39,000, 41,000, and 45,000) are also similar to each other in these respects. Similarity between the subunits of transducin and the islet-activating protein (IAP) substrate is especially evident. Substantial differences do, however, exist between the lower and higher molecular weight subunits within each protein. In addition, evidence was obtained that the 41,000-Da subunit of the IAP substrate is not derived from the 45,000-Da subunit of G/F. It is concluded that transducin, the IAP substrate, and G/F represent a family of structurally homologous guanine nucleotide-binding regulatory proteins.

Adenylate Cyclase Toxin↗

PubMed1994

Role of histidine 35 of the S1 subunit of pertussis toxin in the ADP-ribosylation of transducin.

Molecular modeling of the S1 subunit (S1) of pertussis toxin with other ADP-ribosylating bacterial exotoxins predicted that histidine 35 (His-35) would residue within the active site of S1. Recombinant derivatives of S1 (rS1 and the C180 peptide) which contained either a H35Q or H35P mutation were analyzed to determine the role of His-35 in ADP-ribosylation. C180 peptide is a recombinant peptide composed of the amino-terminal 180 amino acids of S1. Under linear velocity conditions, C180H35Q possessed 2% of wild type C180 peptide activity and C180H35P possessed no detectable activity in the ADP-ribosylation of transducin. The H35Q mutation did not change the affinity of recombinant peptides for NAD or two targets for ADP-ribosylation, transducin, or alpha i3C20, but did lower the kcat in the NAD glycohydrolase and ADP-ribosyltransferase reactions. Neither the H35Q nor H35P mutation reduced the ability of recombinant proteins to be photocross-linked with NAD which was consistent with the His-35 mutations not reducing the affinity for NAD. These data indicate that His-35 does not reduce the affinity of S1 for NAD or transducin, but functions as a catalytic residue in the ADP-ribosylation reaction possibly in a hydrogen bonding capacity.

Adenosine Diphosphate Ribose↗

PubMed1995

Improved purification of transducin subunits from bovine retinal rod outer segments.

Transducin serves as a mediator between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase, in the visual process. Transducin is a protein composed of three polypeptides: T alpha, T beta, and T gamma, and acts as two functional units, the alpha-subunit and the beta gamma-complex. In the present study, I describe an efficient and fast method of purifying T alpha and T beta gamma using chromatography on a blue agarose column connected in tandem with an omega-amino octylagarose column. The recombination of T alpha and T beta gamma reconstitutes the functional heterotrimeric holoprotein, as demonstrated by the recovery of three native properties of transducin: 1) its capacity to exchange guanine nucleotide, 2) its GTP hydrolytic activity, and 3) the ADP-ribosylation of T alpha catalysed by pertussis toxin.

Animals↗

PubMed2000

rTLE3, a newly identified transducin-like enhancer of split, is induced by depolarization in brain.

The transducin-like enhancers of split are a family of mammalian proteins that share sequence homology with the Drosophila protein Groucho. Using representational difference analysis, we isolated the cDNA for a previously unidentified gene, rTLE3 (rat transducin-like enhancer of split 3), as a sequence induced by depolarization and forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. rTLE3 encodes the protein rTLE3, a 764-amino acid orthologue of mouse and human TLE3. R-esp2, the gene encoding the closest related rat protein, is not induced by any of the four treatments in PC12 cells. rTLE3 and R-esp2 have different patterns of expression in the adult rat CNS and other tissues. After systemic administration of kainic acid, rTLE3 is induced specifically in the dentate gyrus of the hippocampus. We propose that members of the transducin-like enhancer of split family of proteins may have distinct functions in the mature CNS, in addition to their functions during development.

Amino Acid Sequence↗

PubMed2003

Proteomic analysis of transducin beta-subunit structural heterogeneity.

Partially purified transducin was resolved using two-dimensional gel electrophoresis (2-DE). Peptide mass fingerprinting of several different spots believed to correspond to the 37 kDa beta-subunit of transducin (T(beta)) was performed. Spots were excised and proteolyzed using modified trypsin. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was performed on the peptide mixture resulting from each spot. As many as six spots with different pI, ranging from 5.2 to 6.1, were observed when separated using 2-DE. MALDI peptide mass fingerprinting determined with high probability that all of the spots were the same gene product, guanine nucleotide-binding protein G(I)/G(S)/G(T) beta-subunit 1 (GNB1; T(beta1)). This suggested that post-translational modification was responsible for the differences in pI. Phosphorylation experiments showed that at least one T(beta1) spot was phosphorylated in vitro with [gamma-(32)P]ATP by an endogenous kinase. Treatment of T(beta) with alkaline phosphatase caused a large change in the spot pattern of T(beta), suggesting that phosphorylated T(beta) is a substrate for alkaline phosphatase. We conclude that T(beta1) constitutes over 99% of the T(beta) expressed in bovine rod outer segments and displays structural heterogeneity that is due to post-translational modification. We also conclude that some, but not all, of the heterogeneity observed is due to phosphorylation of Tb1.

Animals↗

PubMed1991

Carboxyl methylation and farnesylation of transducin gamma-subunit synergistically enhance its coupling with metarhodopsin II.

A heterotrimeric G-protein in vertebrate photoreceptor cells is called transducin (T alpha beta gamma), whose gamma-subunit is a mixture of two components, T gamma-1 and T gamma-2. T gamma-2 is S-farnesylated and partly carboxyl methylated at the C-terminal cysteine residue, whereas T gamma-1 lacks the modified cysteine residue. To elucidate the physiological significance of the double modifications in T gamma, we established a simple chromatographic procedure to isolate T gamma-1, methylated T gamma-2 and non-methylated T gamma-2 on a reversed phase column. Taking advantage of the high and reproducible yield of T gamma from the column, we analyzed the composition of T gamma subspecies in the T alpha-T beta gamma complex which did not bind with transducin-depleted rod outer segment membranes containing metarhodopsin II. The binding of T alpha-T beta gamma with the membranes was shown to require the S-farnesylated cysteine residue of T gamma, whose methylation further enhanced the binding. This synergistic effect was not evident when T alpha was either absent or converted to the GTP-bound form which is known to dissociate from T beta gamma. Thus we concluded that a formation of the ternary complex, T alpha-T beta gamma-metarhodopsin II, is enhanced by the farnesylation and methylation of T gamma. This suggests that the double modifications provide most efficient signal transduction in photoreceptor cells.

Amino Acid Sequence↗

PubMed1986

Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin.

The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2000

Effect of light on phosphatidate phosphohydrolase activity of retina rod outer segments: the role of transducin.

The aim of the present paper is to evaluate the modulation of phosphatidate phosphohydrolase (PAPase) and diacylglyceride lipase (DGL) activities in bovine rod outer segment (ROS) under dark and light conditions and to evaluate the role of transducin (T) in this phenomenon. In dark-adapted ROS membranes exposed to light, PAPase activity is inhibited by 20% with respect to the activity found under dark conditions. To determine whether the retinal G protein, T, participates in the regulation of PAPase activity in these membranes, the effects of GTPgammaS and GDPbetaS on enzyme activity were examined. Under dark conditions in the presence of GTPgammaS, which stabilizes T in its active form (Talpha + Tbetagamma), enzyme activity was inhibited and approached control values under light conditions. GDPbetaS, on the other hand, which stabilizes the inactive state of T (Talphabetagamma), stimulated PAPase activity by 36% with respect to control light conditions. ADP-ribosylation by cholera and pertussis toxin was also studied. In ADP-rybosilated ROS membranes with pertussis toxin under dark conditions, PAPase activity was 36% higher than the activity found under control light conditions. ADP-ribosylation by CTx, on the other hand, inhibited PAPase activity by 22%, with respect to dark control conditions, mimicking light effect. The effects of GTPgammaS and GDPbetaS and conditions of ADP-ribosylation by PTx and CTx on DGL activity were similar to those of PAPase activities. Based on NEM sensitivity we have also demonstrated that the PAPase present in ROS is the PAP 2 isoform. Our findings therefore suggest that light inhibition of PAP 2 in ROS is a transducin-mediated mechanism.

Adenosine Diphosphate Ribose↗

PubMed1993

Structure of the bovine transducin gamma subunit gene and analysis of promoter function in transgenic mice.

Transducin, the major photoreceptor guanine nucleotide-binding protein (G protein), is composed of three polypeptides: alpha, beta and gamma subunits. The transducin gamma subunit (T gamma) is expressed preferentially in photoreceptors. To study the control mechanisms for photoreceptor-specific expression of the T gamma gene, clones of the bovine T gamma gene were isolated from a bacteriophage genomic library, and the structure of the gene, including a portion of its 5'-flanking region, was characterized. The gene consists of three exons and two introns. The first intron is 91 base pairs (bp) long and is located in the region corresponding to the 5'-untranslated sequence of the T gamma mRNA. The second intron is 5.3 kilobases (kb) long and splits the protein-coding region centrally. A bovine Alu-type repetitive sequence and putative Ret-1 and AP-1 binding site sequences are located in the 5'-flanking region. To investigate promoter function, 1.4 kb of DNA from the 5'-flanking region was joined to the prokaryotic chloramphenicol acetyltransferase (CAT) gene, and the chimeric bovine T gamma gene was used to generate a line of transgenic mice. CAT activity was readily detected in the retinas of the transgenic mice, but was absent in brain, heart, kidney, liver, lung, spleen and other tissues. These results suggest that the 1.4 kb 5'-flanking region of the bovine T gamma gene contains conserved sequence elements that direct tissue-specific expression. Human T gamma cDNA clones were characterized, and a short homologous region of the human T gamma gene promotor was obtained by polymerase chain reaction (PCR) amplification for comparison with the bovine promoter.

Animals↗

PubMed1993

Identification of the alpha-subunits of rod and cone transducin in chicken photoreceptor cells.

The chicken retina has several types of cone photoreceptor cells, each of which contains a visual pigment, chicken red (iodopsin), green, blue or violet. Although biochemical and photochemical properties of these cone pigments have been well characterized, no information is available about the chicken photoreceptor G-protein, transducin, which couples with the visual pigment to convert a photon signal into a cellular response. To identify alpha-subunits of chicken rod and cone transducins (Tr alpha and Tc alpha, respectively), we produced two site-directed antibodies which discriminate between bovine Tr alpha and Tc alpha. Immunohistochemical studies on chicken retinas revealed that the antibody against bovine Tr alpha specifically stained the rod outer segments. On the other hand, the antibody against bovine Tc alpha uniformly stained the outer segments of the double cones and all types of single cones, while the single cones were immunohistochemically classified into three types by using a combination of antibodies against bovine rhodopsin and chicken iodopsin. Immuno-blot analyses demonstrated that the antibody against Tc alpha recognized a single band of chicken photoreceptor protein, whose molecular weight (42,000) was in good agreement with that of bovine Tc alpha (41,000). The antibody against Tr alpha recognized a protein having the same molecular weight as that of bovine Tr alpha (39,000). These observations strongly suggested that all types of chicken cone cells have a single common Tc alpha (42 kDa) structurally related to bovine Tc alpha, though each cone cell type has a distinct visual pigment.

Animals↗

PubMed1993

Characterization of the gene encoding human cone transducin alpha-subunit (GNAT2).

The human cone transducin alpha-subunit (GNAT2) gene has been completely characterized. The human GNAT2 transcription unit is 9967-bp in length and consists of eight exons with seven introns. The eight exons are identical to the reported cDNA sequence (Lerea et al., 1989). Northern blot analysis of RNA from human retinas and a retinoblastoma cell line, WERI-RB1, reveals a 1.7-kb transcript for GNAT2. Multiple transcription initiation sites were mapped for human retina and WERI-RB1 RNA by primer extension and S1 nuclease protection assays. This gene has seven initiation sites spanning 31 bp. The sequence upstream of the GNAT2 gene shows a TATA box consensus sequence at -29, a CCAAT box consensus sequence at -58 (reverse orientation), and a sequence (CCATAT) similar to the CCAAT box consensus at -76. The GNAT2 upstream sequence shows no significant identity with the upstream region of the human rod transducin alpha-subunit gene (GNAT1) or with the upstream regions of the color visual pigment genes, indicating that the expression of GNAT2 may be regulated differently than these other rod- and cone-specific proteins.

Amino Acid Sequence↗

PubMed1999

Formation of helical protein assemblies of IgG and transducin on varied lipid tubules.

Helical protein arrays on lipid tubules are valuable assemblies for studying protein structure and protein-lipid interactions through electron microscopy and crystallography. We describe conditions for forming such arrays from two proteins, IgG and transducin, the photoreceptor G protein, using a variety of lipid surfaces. Anti-dinitrophenyl (DNP) IgG arrays formed on DNP-phosphatidylethanolamine (DNP-PE) mixed with either galactosyl-ceramide lipids or phosphatidylcholine (PC) display different pH sensitivities and dimensions, yet have similar helical symmetries. DNP-PE/PC mixtures formed small crystals and large well-ordered flattened tubules. The peripheral membrane protein transducin (G(t)) formed helical arrays either on a mixture of cationic and neutral lipids or on residual photoreceptor lipids. Despite differences in lipid composition, the G(t) arrays have similar structures and show similar sensitivity to activation and variations in ionic environment. G(t) activation causes the helical assemblies to collapse to small vesicles, a process resembling the vesiculation of activated dynamin-lipid tubules. In a preliminary three-dimensional reconstruction, the hapten-bound IgG appears to make two contacts to the central lipid tubule, presumably via the F(ab) domains. The ability to generate a three-dimensional reconstruction without tilts illustrates one advantage of helical structures for two-dimensional crystallography, especially for visualizing protein-lipid interactions.

Crystallization↗

PubMed1995

Exclusion of the cone-specific alpha-subunit of the transducin gene in Stargardt's disease.

Stargardt's disease is an autosomal recessive infantile macular degeneration of unknown origin whose gene has been recently mapped to chromosome 1p21-p13 by linkage analysis in eight multiplex families. Since the cone-specific alpha-subunit of the transducin gene (GNAT2) has been mapped to chromosome 1p13, we tested GNAT2 as the disease-causing gene in our series. Using a novel intragenic polymorphism, we show here that GNAT2 is most probably located centromeric to the genetic interval encompassing the disease gene (D1S424-D1S236, location score = 3.54). In addition, single-strand conformation polymorphism and sequence analyses of the eight exons of the GNAT2 gene was performed in our probands. No evidence of a deleterious base substitution was observed in any affected individual. Taken together, these results support the exclusion of GNAT2 as the causal disease gene of Stargardt's disease. come. The pathogenesis is unknown and the treatment is limited to ultraviolet ray protection with dark glasses. Stargardt's disease has been recently mapped to chromosome 1p21-p13 in the genetic interval defined by loci D1S424 and D1S236. In addition, our previous study has suggested that Stargardt's disease is genetically homogeneous (Kaplan et al. 1993). With respect to the physical mapping of the proteins specific to the retinal pigmentary epithelium or to cones we noted that the cone-specific alpha-subunit of the transducin gene (GNAT2) has been mapped to chromosome 1p13 (Wilkie et al. 1992). The human GNAT2 is 9967 bp in length and consists of eight exons with seven introns (Morris and Fong 1993). In the present study, we examined this gene as the candidate gene in eighteen unrelated patients affected with Stargardt's disease.

Base Sequence↗

PubMed1991

Biotinylation of transducin and G0 from bovine brain.

A method is described for modifying G proteins with biotin. With transducin, better results were obtained with the amino group-specific derivative BXNHS (biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester) as compared to the -SH-group specific reagent MBB (maleimidobutyrylbiocytin). Modification occurred under conditions preserving functional activity: Interaction of the biotinylated transducin with rod outer segment membranes was shown by its light-dependent association and by a GTP gamma S-binding assay. G0 from bovine brain was also biotinylated under conditions preserving its activity. Biotinyl-alpha 0 was shown to bind to a streptavidin Sepharose matrix. Biotinyl-G proteins, therefore, are proposed as tools for extracting proteins (receptors and effector systems), which interact under specific conditions with G proteins.

Animals↗

PubMed1989

Concentration effects on reactions in membranes: rhodopsin and transducin.

The reaction rate of two laterally-diffusing species in a biological membrane shows a maximum at some concentration of reactants, because an increase in the concentration of reactants tends to increase the reaction rate by the law of mass action but decreases the diffusion rate of the reactants. The activation of transducin by rhodopsin in the disk membrane of the rod outer segment is described in terms of a steady-state diffusion model with concentration-dependent diffusion coefficients. The optimum concentrations of reactants are obtained from contour plots of the reaction rate as a function of rhodopsin and transducin concentrations, and the sensitivity of the results to the assumed values of the variables is examined. To determine whether the observed concentrations are in fact those yielding the maximum reaction rate, several variables must be known more accurately.

Diffusion↗

PubMed1986

Sequence analysis of bovine retinal S-antigen. Relationships with alpha-transducin and G-proteins.

S-Antigen is a major soluble protein of the retina and pineal. It is capable of inducing experimental autoimmune uveitis (EAU) in laboratory animals and also seems to play an important role in the visual cycle. The results of partial cDNA sequence analysis reveal interesting homologies with alpha-transducin, a GTP-binding protein of retina and other purine nucleotide-binding proteins. In particular S-antigen shows over 50% identity to the proposed pertussis toxin ADP-ribosylation site of alpha-transducin. It also contains the Gly-X-X-X-X-Gly-Lys pattern common to phosphoryl binding sites. A possible relationship between S-antigen and purine nucleotide-binding proteins is discussed. There is also evidence for a repetitious beta-structure in the C-terminal half of S-antigen, with a monoclonal antibody epitope in a helical region at the C-terminus.

Amino Acid Sequence↗

PubMed1986

Beta-subunits of the human liver Gs/Gi signal-transducing proteins and those of bovine retinal rod cell transducin are identical.

The complete cDNA encoding the beta-subunit of the human liver signal transducing proteins Gs/Gi (beta G) has been cloned from a lambda gtll library using an oligonucleotide as a screening agent. The cDNA has 3088 nucleotides and an 11 nucleotide poly(A) tail, of which 280 nucleotides constitute the 5'-untranslated region, 1023 form the open reading frame (ORF) and its stop codon, and 1785 are the 3'-untranslated region with two AATAAA cleavage and polyadenylation signals separated by 1467 nucleotides. The ORF codes for a 340 amino acid polypeptide that is identical to that encoded by bovine retinal rod cell cDNA of the beta-subunit of transducin. Yet, it does so by using 87 different codons. Curiously, the 280 nucleotide 5' leader sequence obtained starts with an ATG that is part of another ORF encoding a putative peptide X of 75 amino acids (nucleotide 280 to 55). This work proves for the first time that the beta-subunits of all signal-transducing G-proteins, including transducin, are the same.

Amino Acid Sequence↗

PubMed1987

Modulation of retinal transducin and phosphodiesterase activities by synthetic peptides of the phosphodiesterase gamma-subunit.

Synthetic peptides corresponding to various regions of the light-activated guanosine 3',5'-cyclic monophosphate phosphodiesterase (PDE) gamma-subunit (PDE gamma) from bovine retinal rod outer segments were synthesized and tested for their ability to inhibit PDE activity, and GTPase activity of transducin. One of these peptides, corresponding to PDE gamma residues 31-45, inhibited PDE activity and GTPase activity in a dose-dependent manner. The GTPase activity was inhibited by PDE gamma-3 non-competitively. This region of the PDE gamma subunit may be involved in the direct interaction of transducin and PDE alpha beta with PDE gamma.

Animals↗