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Vadim Y Arshavsky - One of the best experts on this subject based on the ideXlab platform.
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Mechanistic basis for the failure of cone Transducin to translocate: why cones are never blinded by light.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2010Co-Authors: Ekaterina S. Lobanova, Nikolai P Skiba, Rolf Herrmann, Stella Finkelstein, Boris Reidel, Wen-tao Deng, Ellen R. Weiss, William W. Hauswirth, Vadim Y ArshavskyAbstract:The remarkable ability of our vision to function under ever-changing conditions of ambient illumination is mediated by multiple molecular mechanisms regulating the light sensitivity of rods and cones. One such mechanism involves massive translocation of signaling proteins, including the G-protein Transducin, into and out of the light-sensitive photoreceptor outer segment compartment. Transducin translocation extends the operating range of rods, but in cones Transducin never translocates, which is puzzling because cones typically function in much brighter light than rods. Using genetically manipulated mice in which the rates of Transducin activation and inactivation were altered, we demonstrate that, like in rods, Transducin translocation in cones can be triggered when Transducin activation exceeds a critical level, essentially saturating the photoresponse. However, this level is never achieved in wild-type cones: their superior ability to tightly control the rates of Transducin activation and inactivation, responsible for avoiding saturation by light, also accounts for the prevention of Transducin translocation at any light intensity.
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Electrostatic and lipid anchor contributions to the interaction of Transducin with membranes: mechanistic implications for activation and translocation.
The Journal of biological chemistry, 2008Co-Authors: Mickey Kosloff, Vadim Y Arshavsky, Emil Alexov, Barry HonigAbstract:The heterotrimeric G protein Transducin is a key component of the vertebrate phototransduction cascade. Transducin is peripherally attached to membranes of the rod outer segment, where it interacts with other proteins at the membrane-cytosol interface. However, upon sustained activation by light, the dissociated Gtα and Gβ1γ1 subunits of Transducin translocate from the outer segment to other parts of the rod cell. Here we used a computational approach to analyze the interaction strength of Transducin and its subunits with acidic lipid bilayers, as well as the range of orientations that they are allowed to occupy on the membrane surface. Our results suggest that the combined constraints of electrostatics and lipid anchors substantially limit the rotational degrees of freedom of the membrane-bound Transducin heterotrimer. This may contribute to a faster Transducin activation rate by accelerating Transducin-rhodopsin complex formation. Notably, the membrane interactions of the dissociated Transducin subunits are very different from those of the heterotrimer. As shown previously, Gβ1γ1 experiences significant attractive interactions with negatively charged membranes, whereas our new results suggest that Gtα is electrostatically repelled by such membranes. We suggest that this repulsion could facilitate the membrane dissociation and intracellular translocation of Gtα. Moreover, based on similarities in sequence and electrostatic properties, we propose that the properties described for Transducin are common to its homologs within the Gi subfamily. In a broader view, this work exemplifies how the activity-dependent association and dissociation of a G protein can change both the affinity for membranes and the range of allowed orientations, thereby modulating G protein function.
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Transducin γ subunit sets expression levels of α and β subunits and is crucial for rod viability
The Journal of Neuroscience, 2008Co-Authors: Ekaterina S. Lobanova, Rolf Herrmann, Stella Finkelstein, Katherine J. Strissel, Norman Michaud, Yen Ming Chen, Christopher Kessler, Lynn H Trieu, Marie E Burns, Vadim Y ArshavskyAbstract:Transducin is a prototypic heterotrimeric G-protein mediating visual signaling in vertebrate photoreceptor cells. Despite its central role in phototransduction, little is known about the mechanisms that regulate its expression and maintain approximately stoichiometric levels of the α- and βγ-subunits. Here we demonstrate that the knock-out of Transducin γ-subunit leads to a major downregulation of both α- and β-subunit proteins, despite nearly normal levels of the corresponding transcripts, and fairly rapid photoreceptor degeneration. Significant fractions of the remaining α- and β-subunits were mislocalized from the light-sensitive outer segment compartment of the rod. Yet, the tiny amount of the α-subunit present in the outer segments of knock-out rods was sufficient to support light signaling, although with a markedly reduced sensitivity. These data indicate that the γ-subunit controls the expression level of the entire Transducin heterotrimer and that heterotrimer formation is essential for normal Transducin localization. They further suggest that the production of Transducin β-subunit without its constitutive γ-subunit partner sufficiently stresses the cellular biosynthetic and/or chaperone machinery to induce cell death.
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Transducin Translocation in Rods Is Triggered by Saturation of the GTPase-Activating Complex
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2007Co-Authors: Ekaterina S. Lobanova, Nikolai P Skiba, Chingkang Chen, Stella Finkelstein, Hongman Song, Maxim Sokolov, Stephen H. Tsang, Vadim Y ArshavskyAbstract:Light causes massive translocation of G-protein Transducin from the light-sensitive outer segment compartment of the rod photoreceptor cell. Remarkably, significant translocation is observed only when the light intensity exceeds a critical threshold level. We addressed the nature of this threshold using a series of mutant mice and found that the threshold can be shifted to either a lower or higher light intensity, dependent on whether the ability of the GTPase-activating complex to inactivate GTP-bound Transducin is decreased or increased. We also demonstrated that the threshold is not dependent on cellular signaling downstream from Transducin. Finally, we showed that the extent of Transducin α subunit translocation is affected by the hydrophobicity of its acyl modification. This implies that interactions with membranes impose a limitation on Transducin translocation. Our data suggest that Transducin translocation is triggered when the cell exhausts its capacity to activate Transducin GTPase, and a portion of Transducin remains active for a sufficient time to dissociate from membranes and to escape from the outer segment. Overall, the threshold marks the switch of the rod from the highly light-sensitive mode of operation required under limited lighting conditions to the less-sensitive energy-saving mode beneficial in bright light, when vision is dominated by cones.
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Phosducin facilitates light-driven Transducin translocation in rod photoreceptors. Evidence from the phosducin knockout mouse.
The Journal of biological chemistry, 2004Co-Authors: Maxim Sokolov, Katherine J. Strissel, Ilya Leskov, Norman Michaud, Viktor I. Govardovskii, Vadim Y ArshavskyAbstract:Abstract Phosducin is a photoreceptor-specific protein known to interact with the βγ subunits of G proteins. In pursuit of the function of phosducin, we tested the hypothesis that it regulates the light-driven translocation of G protein Transducin from the outer segments of rod photoreceptors to other compartments of the rod cell. Transducin translocation has been previously shown to contribute to rod adaptation to bright illumination, yet the molecular mechanisms underlying the translocation phenomenon remain unknown. In this study we provide two major lines of evidence in support of the role of phosducin in Transducin translocation. First, we have demonstrated that Transducin βγ subunits interact with phosducin along their entire intracellular translocation route, as evident from their co-precipitation in serial tangential sections from light-adapted but not dark-adapted retinas. Second, we generated a phosducin knockout mouse and found that the degree of light-driven Transducin translocation in the rods of these mice was significantly reduced as compared with that observed in the rods of wild type animals. In knockout animals the translocation of Transducin βγ subunits was affected to a larger degree than the translocation of the α subunit. We also found that the amount of phosducin in rods is sufficient to interact with practically all of the Transducin present in these cells and that the subcellular distribution of phosducin is consistent with that of a soluble protein evenly distributed throughout the entire rod cytoplasm. Together, these data indicate that phosducin binding to Transducin βγ subunits facilitates Transducin translocation. We suggest that the mechanism of phosducin action is based on the reduction of Transducin affinity to the membranes of rod outer segments, achieved by keeping the Transducin βγ subunits apart from the α subunit. This increased solubility of Transducin would make it more susceptible to translocation from the outer segments.
Nikolai O Artemyev - One of the best experts on this subject based on the ideXlab platform.
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Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Anurima Majumder, Kimberly K. Boyd, Vasily Kerov, Johan Pahlberg, Saravanan Kolandaivelu, Visvanathan Ramamurthy, Alapakkam P Sampath, Nikolai O ArtemyevAbstract:In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein Transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced Transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that Transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca2+ current, suggesting interactions of Transducin with the synaptic machinery.
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Unique Transducins expressed in long and short photoreceptors of lamprey Petromyzon marinus.
Vision Research, 2008Co-Authors: Hakim Muradov, Kimberly K. Boyd, Vasily Kerov, Nikolai O ArtemyevAbstract:Lampreys represent the most primitive vertebrate class of jawless fish and serve as an evolutionary model of the vertebrate visual system. Transducin-α (Gαt) subunits were investigated in lamprey Petromyzon marinus in order to understand the molecular origins of rod and cone photoreceptor G proteins. Two Gαt subunits, GαtL and GαtS, were identified in the P. marinus retina. GαtL is equally distant from cone and rod G proteins and is expressed in the lamprey’s long photoreceptors. The short photoreceptor GαtS is a rod-like Transducin-α that retains several unique features of cone Transducins. Thus, the duplication of the ancestral Transducin gene giving rise to rod Transducins has already occurred in the last common ancestor of the jawed and jawless vertebrates.
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mutation r238e in Transducin alpha yields a gtpase and effector deficient but not dominant negative g protein alpha subunit
Molecular Vision, 2006Co-Authors: Brandy Barren, Michael Natochin, Nikolai O ArtemyevAbstract:PURPOSE Certain forms of inherited and light-induced retinal degenerations are believed to involve excessive phototransduction signaling. A dominant-negative mutant of the visual G-protein, Transducin, would represent a major tool in designing potential therapeutical strategies for this group of visual diseases. We thought to further investigate a novel mutant of the Transducin-alpha subunit, R238E, that was recently reported to be a dominant-negative inhibitor of the rhodopsin/Transducin/PDE visual system. METHODS The R238E substitution was introduced into a tranducin-like chimeric Gtalpha*-subunit. The nucleotide-bound state of the Gtalpha*R238E mutant was assessed using the trypsin-protection assay. The ability of the Gtalpha*R238E mutant to interact with Gtbetagamma, couple to photoexcited rhodopsin (R*), and undergo R*-stimulated guanine nucleotide exchange was examined by a GTPgammaS binding assay. The GTPase activity of the mutant Gtalpha* and its interaction with RGS proteins was characterized in the steady-state and single turnover measurements of GTP hydrolysis. A binding assay utilizing the fluorescently-labeled gamma-subunit of PDE6 (Pgamma) was employed to monitor the effector function of Gtalpha*R238E. RESULTS The Gtalpha*R238E mutant bound GDP and was capable of the AlF4--induced activational conformational change. The capacity of Gtalpha*R238E to couple to R* in the presence of Gtbetagamma was similar to that of Gtalpha*. However, the mutant GTPase activity was markedly impaired. This defect was further exacerbated by the diminished interactions of Gtalpha*R238E with the GAP proteins, RGS9 and RGS16. Another consequence of the mutation was the reduction in Gtalpha*R238E's affinity for Pgamma. CONCLUSIONS Transducin mutant Gtalpha*R238E exists in a nucleotide-bound state and is fully capable of activational coupling to R*. This mutation results in a significant impairment of Gtalpha*'s ability to hydrolyze GTP and interact with the inhibitory subunit of PDE6. This phenotype is entirely inconsistent with that of a dominant-negative inhibitor as recently reported.
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Rhodopsin determinants for Transducin activation: a gain-of-function approach.
The Journal of biological chemistry, 2003Co-Authors: Michael Natochin, Karim G. Gasimov, Mustapha Moussaif, Nikolai O ArtemyevAbstract:Three cytoplasmic loops in the G protein-coupled receptor rhodopsin, C2, C3, and C4, have been implicated as key sites for binding and activation of the visual G protein Transducin. Non-helical portions of the C2- and C3-loops and the cytoplasmic helix-8 from the C4 loop were targeted for a "gain-of-function" mutagenesis to identify rhodopsin residues critical for Transducin activation. Mutant opsins with residues 140-148 (C2-loop), 229-244 (C3-loop), or 310-320 (C4-loop) substituted by poly-Ala sequences of equivalent lengths served as templates for mutagenesis. The template mutants with poly-Ala substitutions in the C2- and C3-loops formed the 500-nm absorbing pigments but failed to activate Transducin. Reverse substitutions of the Ala residues by rhodopsin residues have been generated in each of the templates. Significant ( approximately 50%) restoration of the rhodopsin/Transducin coupling was achieved with re-introduction of residues Cys140/Lys141 and Arg147/Phe148 into the C2 template. The reverse substitutions of the C3-loop residues Thr229/Val230 and Ser240/Thr242/Thr243/Gln244 produced a pigment with a full capacity for Transducin activation. The C4 template mutant was unable to bind 11-cis-retinal, and the presence of Asn310/Lys311 was required for correct folding of the protein. Subsequent mutagenesis of the C4-loop revealed the role of Phe313 and Met317. On the background of Asn310/Lys311, the inclusion of Phe313 and Met317 produced a mutant pigment with the potency of Transducin activation equal to that of the wild-type rhodopsin. Overall, our data support the role of the three cytoplasmic loops of rhodopsin and suggest that residues adjacent to the transmembrane helices are most important for Transducin activation.
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Regulation of Transducin GTPase Activity by Human Retinal RGS
The Journal of biological chemistry, 1997Co-Authors: Michael Natochin, Alexey E. Granovsky, Nikolai O ArtemyevAbstract:Abstract The intrinsic GTPase activity of Transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory γ-subunit of PDE (Pγ), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by Transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of Transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the Transducin α-subunit, Gtα GDPAlF 4 −, while its binding to GtαGTPγS or GtαGDP was weak. hRGSr and Pγ did not compete for the interaction with Gtα GDPAlF 4 −. Affinity of the Pγ-Gtα GDPAlF 4 −interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of Gtα GDPAlF 4 −binding to Pγ labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PγBC). Binding of hRGSr to Gtα GDPAlF 4 −complexed with PγBC resulted in a maximal ∼40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for Gtα GDPAlF 4 −(K d 35 nm). In a single turnover assay, hRGSr accelerated GTPase activity of Transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s−1. Addition of Pγ to the reconstituted system reduced the GTPase level accelerated by hRGSr (k cat 0.085 s−1). The GTPase activity of Transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 μm rhodopsin these rates exceeded 0.7 s−1. Our data suggest that effects of hRGSr on Transducin’s GTPase activity are attenuated by Pγ but independent of a putative membrane GTPase activating protein factor. The rate of Transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.
Jian Xing - One of the best experts on this subject based on the ideXlab platform.
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Differences in the pharmacological activation of visual opsins.
Visual Neuroscience, 2006Co-Authors: Tomoki Isayama, Masahiro Kono, Rosalie K. Crouch, Ying Chen, Willem J. Degrip, Jian Xing, Clint L. MakinoAbstract:Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of “pigment adaptation” with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, β-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different Transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of β-ionone. To test the two hypotheses, we applied β-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone Transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of Transducin, whereas all cones contain another. β-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod Transducin upon the addition of β-ionone. Thus opsin structure determines the identity of β-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.
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Differences in the pharmacological activation of visual opsins.
Visual neuroscience, 2006Co-Authors: Tomoki Isayama, Masahiro Kono, Rosalie K. Crouch, Ying Chen, Willem J. Degrip, Jian Xing, Clint L. MakinoAbstract:Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of "pigment adaptation" with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, beta-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different Transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of beta-ionone. To test the two hypotheses, we applied beta-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone Transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of Transducin, whereas all cones contain another. beta-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod Transducin upon the addition of beta-ionone. Thus opsin structure determines the identity of beta-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.
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Salamander rods and cones contain distinct Transducin alpha subunits.
Visual neuroscience, 2000Co-Authors: James C. Ryan, Rosalie K. Crouch, Sergey L. Znoiko, Jian XingAbstract:The mammalian retina is known to contain two distinct Transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct Transducins. In the present study, we report the cloning and cellular localization of two Transducin a subunits (G alpha t) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of G alpha t. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone Transducins. The salamander G alpha t-1 has 91.2-93.7% amino acid sequence identity to mammalian rod G alpha t subunits and 79.7-80.9% to mammalian cone Gats. The salamander G alpha t-2 has 86.2-87.9% sequence identity to mammalian cone G alpha ts and 78.9-80.9% to mammalian rod G alpha ts at the amino acid level. The G alpha t-1 cDNA encodes 350 amino acids while the G alpha t-2 cDNA encodes 354 residues, which is typical for rod and cone G alpha ts, respectively, and we thus identified the G alpha t- 1 as rod and G alpha t-2 as cone G alpha t. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone G alpha t subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod G alpha t and 2.6 kb for cone G alpha t. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod G alpha t is localized to rods, predominantly in the outer segments; similarly, cone G alpha t is localized to cone outer segments. The results confirm that the two sequences encode rod and cone Transducins and demonstrate that this lower vertebrate contains two distinct Transducins that are localized specifically to rod and cone photoreceptors.
Robert F. Margolskee - One of the best experts on this subject based on the ideXlab platform.
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Partial Rescue of Taste Responses of α-Gustducin Null Mice by Transgenic Expression of α-Transducin
Chemical senses, 2002Co-Authors: Vicktoria Danilova, Robert F. Margolskee, Shiying Zou, Göran Hellekant, Marianna Max, Sami DamakAbstract:The transduction of responses to bitter and sweet compounds utilizes guanine nucleotide binding proteins (G proteins) and their coupled receptors. Alpha-gustducin, a Transducin-like G protein alpha-subunit, and rod alpha-Transducin are expressed in taste receptor cells. Alpha-gustducin knockout mice have profoundly diminished behavioral and electrophysiological responses to many bitter and sweet compounds, although these mice retain residual responses to these compounds. Alpha-gustducin and rod alpha-Transducin are biochemically indistinguishable in their in vitro interactions with retinal phosphodiesterase, rhodopsin and G protein betagamma-subunits. To determine if alpha-Transducin can function in taste receptor cells and to compare the function of alpha-gustducin versus alpha-Transducin in taste transduction in vivo, we generated transgenic mice that express alpha-Transducin under the control of the alpha-gustducin promoter in the alpha-gustducin null background. Immunohistochemistry showed that the alpha-Transducin transgene was expressed in about two-thirds of the alpha-gustducin lineage of taste receptor cells. Two-bottle preference tests showed that transgenic expression of rod alpha-Transducin partly rescued responses to denatonium benzoate, sucrose and the artificial sweetener SC45647, but not to quinine sulfate. Gustatory nerve recordings showed a partial rescue by the transgene of the response to sucrose, SC45647 and quinine, but not to denatonium. These results demonstrate that alpha-Transducin can function in taste receptor cells and transduce some taste cell responses. Our results also suggest that alpha-Transducin and alpha-gustducin may differ, at least in part, in their function in these cells, although this conclusion must be qualified because of the limited fidelity of the transgene expression.
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phototransduction in transgenic mice after targeted deletion of the rod Transducin α subunit
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Peter D Calvert, N V Krasnoperova, Arkady Lyubarsky, T Isayama, M Nicolo, Bela Kosaras, G Wong, K S Gannon, Robert F. MargolskeeAbstract:Retinal photoreceptors use the heterotrimeric G protein Transducin to couple rhodopsin to a biochemical cascade that underlies the electrical photoresponse. Several isoforms of each Transducin subunit are present in the retina. Although rods and cones seem to contain distinct Transducin subunits, it is not known whether phototransduction in a given cell type depends strictly on a single form of each subunit. To approach this question, we have deleted the gene for the rod Transducin α-subunit in mice. In hemizygous knockout mice, there was a small reduction in retinal Transducin α-subunit content but retinal morphology and the physiology of single rods were largely normal. In homozygous knockout mice, a mild retinal degeneration occurred with age. Rod-driven components were absent from the electroretinogram, whereas cone-driven components were retained. Every photoreceptor examined by single-cell recording failed to respond to flashes, with one exception. The solitary responsive cell was insensitive, as expected for a cone, but had a rod-like spectral sensitivity and flash response kinetics that were slow, even for rods. These results indicate that most if not all rods use a single Transducin type in phototransduction.
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blocking taste receptor activation of gustducin inhibits gustatory responses to bitter compounds
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Ding Ming, Yuzo Ninomiya, Robert F. MargolskeeAbstract:Gustducin, a Transducin-like guanine nucleotide-binding regulatory protein (G protein), and Transducin are expressed in taste receptor cells where they are thought to mediate taste transduction. Gustducin and Transducin are activated in the presence of bovine taste membranes by several compounds that humans perceive to be bitter. We have monitored this activation with an in vitro assay to identify compounds that inhibited taste receptor activation of Transducin by bitter tastants: AMP and chemically related compounds inhibited in vitro responses to several bitter compounds (e.g., denatonium, quinine, strychnine, and atropine). AMP also inhibited behavioral and electrophysiological responses of mice to bitter tastants, but not to NaCl, HCl, or sucrose. GMP, although chemically similar to AMP, inhibited neither the bitter-responsive taste receptor activation of Transducin nor the gustatory responses of mice to bitter compounds. AMP and certain related compounds may bind to bitter-responsive taste receptors or interfere with receptor-G protein coupling to serve as naturally occurring taste modifiers.
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Characterization and solubilization of bitter-responsive receptors that couple to gustducin
Proceedings of the National Academy of Sciences, 1998Co-Authors: Ding Ming, Luis Ruiz-avila, Robert F. MargolskeeAbstract:The tastes of many bitter and sweet compounds are thought to be transduced via guanine nucleotide binding protein (G-protein)-coupled receptors, although the biochemical nature of these receptors is poorly understood at present. Gustducin, a taste-specific G-protein closely related to the Transducins, is a key component in Transducing the responses to compounds that humans equate with bitter and sweet. Rod Transducin, which is also expressed in taste receptor cells, can be activated by the bitter compound denatonium in the presence of bovine taste membranes. In this paper, we show that gustducin is expressed in bovine taste tissue and that both gustducin and Transducin, in the presence of bovine taste membranes, can be activated specifically by several bitter compounds, including denatonium, quinine, and strychnine. We also demonstrate that the activation in response to denatonium of gustducin by presumptive bitter-responsive receptors present in taste membranes depends on an interaction with the C terminus of gustducin and requires G-protein βγ subunits to provide the receptor-interacting heterotrimer. The taste receptor–gustducin interaction can be competitively inhibited by peptides derived from the sites of interaction of rhodopsin and Transducin. Finally, as the initial step toward purifying taste receptors, we have solubilized this bitter-responsive taste receptor and maintained its biological activity.
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coupling of bitter receptor to phosphodiesterase through Transducin in taste receptor cells
Nature, 1995Co-Authors: Luis Ruizavila, Susan K Mclaughlin, David Wildman, Peter J Mckinnon, Alain Robichon, Nancy Spickofsky, Robert F. MargolskeeAbstract:THE rod and cone Transducing are specific G proteins originally thought to be present only in photoreceptor cells of the vertebrate retina1–4. Transducins convert light stimulation of photoreceptor opsins into activation of cyclic GMP phosphodiesterase (reviewed in refs. 5-7). A Transducin-like G protein, gustducin, has been identified and cloned from rat taste cells8. We report here that rod Transducin is also present in vertebrate taste cells, where it specifically activates a phosphodiesterase isolated from taste tissue. Furthermore, the bitter compound denatonium in the presence of taste-cell membranes activates Transducin but not Gi. A peptide that competitively inhibits rhodopsin activation of Transducin9 also blocks taste-cell membrane activation of Transducin, arguing for the involvement of a seven-transmembrane-helix G-protein-coupled receptor. These results suggest that rod Transducin tranduces bitter taste by coupling taste receptor(s) to taste-cell phosphodiesterase. Phosphodiesterase-mediated degradation of cyclic nucleotides may lead to taste-cell depolarization through the recently identified cyclic-nucleotide-suppressible conductance10.
Clint L. Makino - One of the best experts on this subject based on the ideXlab platform.
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Differences in the pharmacological activation of visual opsins.
Visual Neuroscience, 2006Co-Authors: Tomoki Isayama, Masahiro Kono, Rosalie K. Crouch, Ying Chen, Willem J. Degrip, Jian Xing, Clint L. MakinoAbstract:Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of “pigment adaptation” with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, β-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different Transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of β-ionone. To test the two hypotheses, we applied β-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone Transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of Transducin, whereas all cones contain another. β-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod Transducin upon the addition of β-ionone. Thus opsin structure determines the identity of β-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.
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Differences in the pharmacological activation of visual opsins.
Visual neuroscience, 2006Co-Authors: Tomoki Isayama, Masahiro Kono, Rosalie K. Crouch, Ying Chen, Willem J. Degrip, Jian Xing, Clint L. MakinoAbstract:Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of "pigment adaptation" with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, beta-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different Transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of beta-ionone. To test the two hypotheses, we applied beta-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone Transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of Transducin, whereas all cones contain another. beta-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod Transducin upon the addition of beta-ionone. Thus opsin structure determines the identity of beta-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.
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Downregulation of cGMP Phosphodiesterase Induced by Expression of GTPase-Deficient Cone Transducin in Mouse Rod Photoreceptors
Investigative ophthalmology & visual science, 1994Co-Authors: Carol J. Raport, Clint L. Makino, Melvin I Simon, Janis Lem, X Ching-kang Chen, Cindy L. Fitch, Ann Hobson, Denis Baylor, James B. HurleyAbstract:Purpose. Photoexcitation of vertebrate retinal rod photoreceptors stimulates GTP binding to the Transducin a subunit. Like other GTP-binding proteins, Transducin restores itself to an inactive form by hydrolyzing its bound GTP. The role of GTP hydrolysis in phototransduction was investigated. Methods. A mutant form of cone Transducin a deficient in its ability to hydrolyze bound GTP was expressed in mouse rod photoreceptors. Results. Expression of the mutant cone Transducin a at levels threefold to sixfold higher than endogenous rod Transducin a led to a specific depletion of the Transducin target, cGMP phosphodiesterase, and a decrease in the cGMP level. Suction electrode recordings revealed abnormally prolonged flash responses, decreased maximal response amplitudes, and a shift in the stimulus-response relation to higher flash strengths. Conclusions. Rods expressing high levels of GTPase-deficient cone Transducin a have reduced levels of phosphodiesterase catalytic subunits and cGMP. These changes are associated with prolonged flash responses, reduced dark current, and decreased sensitivity to light. Invest Ophthalmol Vis Sci. 1994;35:2932-2947.