Disease relevance of Gnat1

• Bright light induces retinal degeneration by a transducin-independent mechanism [1].
• In transducin, pertussis toxin catalyzes the ADP-ribosylation of a cysteine residue, whereas choleragen (cholera toxin) modifies an arginine moiety [2].
• Baculovirus expression yields a membrane protein with all functional characteristics of a rod visual pigment (lambda(max) = 497 +/- 2 nm; pK(a) of meta I/meta II equilibrium = 6.5; rapid activation of transducin in the light) [3].

High impact information on Gnat1

• A genetic block of transducin signaling completely rescued degeneration irrespective of an elevated level of retinyl ester [4].
• Using knockout mice with defects in rhodopsin shut-off and transducin signaling, we show that two different pathways of photoreceptor-cell apoptosis are induced by light [5].
• Bright light induces apoptosis that is independent of transducin and accompanied by induction of the transcription factor AP-1 [5].
• Crystal structure at 2.4 angstroms resolution of the complex of transducin betagamma and its regulator, phosducin [6].
• The homologous segments correspond mostly to the regions thought to be involved in the guanine nucleotide binding and GTPase activity of ras proteins and to the ADP-ribosylation sites of the transducin alpha-subunit [7].

Biological context of Gnat1

• The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood [8].
• These results indicate that most if not all rods use a single transducin type in phototransduction [9].
• A genomic clone spanning the mouse rod transducin alpha subunit (Tr alpha) gene has been isolated by screening a mouse genomic library with a bovine Tr alpha cDNA clone [10].
• These introns are in the same locations as introns in human Gi alpha genes, that encode G proteins closely related to transducin [10].
• The gene for the alpha-subunit of retinal rod transducin is on mouse chromosome 9 [11].

Anatomical context of Gnat1

• Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation [8].
• Transducin activation state controls its light-dependent translocation in rod photoreceptors [8].
• 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 [9].
• We have used a cDNA clone for the alpha-subunit of bovine rod transducin (T alpha 1) to map the corresponding gene, Gnat-1, to mouse chromosome 9 with a panel of Chinese hamster-mouse somatic cell hybrid DNAs [11].
• The AS/6 antibody was made against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of transducin, and the LE/3 antibody was made against the peptide corresponding to amino acids 160-169 of a guanine nucleotide binding protein (Gi-2-alpha) cloned from a mouse macrophage cell line [12].

Associations of Gnat1 with chemical compounds

• Furthermore, the GPR domain of LGN potently inhibits receptor-mediated guanine nucleotide exchange and steady-state GTPase activity of transducin [13].
• This slow current was blocked by l-cis diltiazem, indicating that it was produced by ion flux through the cyclic nucleotide-gated channels of the outer segment; however, it could not have been produced by the normal transduction cascade, since it was recorded from rods lacking transducin [14].
• 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 [15].
• The rat D4 dopamine receptor couples to cone transducin (Galphat2) to inhibit forskolin-stimulated cAMP accumulation [16].
• However, increased light-independent activation of transducin (due to bleached opsin) could be demonstrated after the addition of exogenous 11-cis retinal [17].

Physical interactions of Gnat1

• First, we have demonstrated that transducin beta gamma 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 [18].
• To our knowledge, this is the first report demonstrating that the D4 dopamine receptor functionally couples to a specific G-protein and that a non-opsin-like receptor can couple with a transducin subunit [16].
• Immobilized PDE gamma binds transducin alpha subunit charged with GTP, PDE alpha and beta subunits, and, unexpectedly, arrestin (S-antigen) [19].
• Here we demonstrate that transducin colocalizes with the Ca(2+)-binding protein centrin 1 in a specific domain of this cilium [20].
• Several other G proteins of known functions have been purified: Gi, which couples inhibitory receptors to adenylate cyclase, and transducin which couples photoexcited rhodopsin to cyclic GMP phosphodiesterase [21].

Regulatory relationships of Gnat1

• Both RGS9-/- and Gbeta5-/- responses were consistent with another factor weakly regulating GTP hydrolysis by transducin in a manner proportional to the inward current [22].
• In a reconstituted biochemical system, the reconstituted melanopsin was capable of activating transducin, the G-protein of rod photoreceptors, in a light-dependent manner [23].
• Transducin translocation in rods is triggered by saturation of the GTPase-activating complex [24].
• Sequestration of Gbetagamma subunits by transducin suppressed the opioid-induced JNK activity [25].
• Nevertheless, the mutant opsin constitutively activates transducin in vitro and causes photoreceptor degeneration in vivo, possibly by continuously activating the phototransduction cascade, analogous to constant exposure to environmental light [26].

Other interactions of Gnat1

• 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 [18].
• An illumination threshold for the Gtalpha movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST [8].
• In this study, double CaBP4/rod alpha-transducin knockout (Cabp4(-/-)Gnat1(-/-)) mice lacking the rod-mediated component of electrophysiologic responses were generated and analyzed to investigate the role of CaBP4 in cones [27].
• We have probed these mechanisms by examining light-dependent localizations of the transducin-alpha subunit (Gtalpha)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtalphaQ200L [8].
• Interaction of transducin-alpha with LGN, a G-protein modulator expressed in photoreceptor cells [13].

Analytical, diagnostic and therapeutic context of Gnat1

• Immunohistochemistry showed that the alpha-transducin transgene was expressed in about two-thirds of the alpha-gustducin lineage of taste receptor cells [15].
• Using a low stringency polymerase chain reaction cloning method, we found an additional Ptx-sensitive G-protein cone transducin (Galphat2) expressed in the MN9D cells [16].
• Northern blot analysis of different tissues revealed a transcript of about 2.5 kb, which is expressed only in the fish eye and not in other tissues from adult fish, supporting its identification as transducin [28].
• Coimmunoprecipitation, centrifugation, centrin overlay, size exclusion chromatography, and kinetic light-scattering experiments indicate that Ca(2+)-activated centrin 1 binds with high affinity and specificity to transducin [20].
• The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding [29].

References