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PubMed1989

Similarities between G-proteins in visual cells of Sepia and cattle.

In contrast to antisera against native transducin a polyclonal antiserum raised against heat-denatured bovine transducin crossreacts with the G-protein from Sepia visual cells. This antiserum recognizes a 44 kDa (G alpha) and a 36 kDa (G beta) protein band from Sepia photosensory membrane preparation. Furthermore we purified the antibody-binding G-protein from Sepia by binding it to light-activated rhodopsin of Sepia and GTP-induced extraction, similar to the purification of bovine transducin. This G-protein is probably involved in the phototransduction process. The purified Sepia G-protein did bind to vertebrate photosensoric membrane upon illumination, but was not eluted by GTP-containing buffer solution. After extensive bleaching, the G-protein became soluble.

Animals↗
PubMed1990

Interaction of small G proteins with photoexcited rhodopsin.

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [32P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed [32P]ADP-ribosylation of the alpha-subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.

ADP Ribose Transferases↗
PubMed1994

Molecular cloning of G proteins and phosphodiesterases from rat taste cells.

To identify and characterize those proteins involved in taste transduction, we cloned G proteins and phosphodiesterases from rat taste tissue. Using degenerate primers corresponding to conserved regions of G protein alpha subunits, the polymerase chain reaction was used to amplify and clone eight distinct cDNAs: alpha i-2, alpha i-3, alpha 12, alpha 14, a(s), alpha t-rod, alpha t-cone and alpha gustducin. alpha i-3, alpha 14, alpha s, and alpha t-rod are more highly expressed in taste tissue than in the surrounding nonsensory tissue. alpha gustducin is only expressed in taste cells. Rod transducin had previously been found only in the rod cells of the retina, where it converts light stimulation of rhodopsin into activation of cGMP phosphodiesterase. The primary sequence of alpha gustducin shows striking similarities to rod transducin in the receptor interaction domain and the phosphodiesterase activation site. We propose that gustducin and transducin regulate phosphodiesterase activity in taste cells and that this may promote bitter transduction and inhibit sweet transduction. Consistent with this proposal, we cloned two types of cAMP PDE from taste tissue: dnc-1 and PDE-3.

Amino Acid Sequence↗
PubMed2002

Functionally rodless mice: transgenic models for the investigation of cone function in retinal disease and therapy.

Two genetically engineered strains of mice were used to characterize murine cone function electroretinographically, without interference of rod-driven responses: (1) mice with a deletion of the gene for the rod transducin alpha-subunit (transducin alpha-/-), and (2) mice with rod arrestin deleted (arrestin -/-). In the first three months of age, both strains have a normal complement of rods and normal rod structure, but transducin alpha-/- mice have no rod-driven responses to light, while rod-driven activity of arrestin -/- mice can be suppressed by a single intense flash for hours. In response to intense flashes the electroretinograms of these strains of mice showed a readily identifiable, pure-cone a-wave of approximately 10 microV saturating amplitude. A 530 nm background that saturates rod responses of wild type mice was found to desensitize the b-wave responses of mice of both transgenic lines, whether the b-waves were driven by photons captured by M- or UV-cone pigments. The desensitizing effect of the 530 nm background on UV-pigment driven responses provides new evidence in support of the hypothesis of functional co-expression of the M-pigment in cones expressing primarily the UV-pigment.

Animals↗
PubMed1997

Cloning and analysis of the cDNA encoding the rod G-protein transduction alpha, beta1 and gamma1 subunits from the canine retina.

The canine (Canis familiaris) retinal rod transducin (G(T)) alpha, beta1 and gamma1 subunits were sequenced. Cloning of the cDNAs was accomplished by polymerase chain reaction (PCR) using degenerate and wild type retinal cDNA libraries as templates. The deduced amino acid sequences were highly similar to rod transducins from other species: G(T alpha) differed by 5 amino acids from the corresponding human sequence, whereas beta1 and gamma1 were identical to human sequences. The coding sequence of rod transducin was evaluated as a possible cause for the recessively inherited retinal rod-cone degeneration: there were no nucleotide differences between the wild type and retinal degenerate strains.

Amino Acid Sequence↗
PubMed1997

Photochemical and biochemical properties of chicken blue-sensitive cone visual pigment.

Through low-temperature spectroscopy and G-protein (transducin) activating experiments, we have investigated molecular properties of chicken blue, the cone visual pigment present in chicken blue-sensitive cones, and compared them with those of the other cone visual pigments, chicken green and chicken red (iodopsin), and rod visual pigment rhodopsin. Irradiation of chicken blue at -196 degrees C results in formation of a batho intermediate which then converts to BL, lumi, meta I, meta II, and meta III intermediates with the transition temperatures of -160, -110, -40, -20, and -10 degrees C. Batho intermediate exhibits an unique absorption spectrum having vibrational fine structure, suggesting that the chromophore of batho intermediate is in a C6-C7 conformation more restricted than those of chicken blue and its isopigment. As reflected by the difference in maxima of the original pigments, the absorption maxima of batho, BL, and lumi intermediates of chicken blue are located at wavelengths considerably shorter than those of the respective intermediates of chicken green, red and rhodopsin, but the maxima of meta I, meta II, and meta III are similar to those of the other visual pigments. These facts indicate that during the lumi-to-meta I transition, retinal chromophore changes its original position relative to the amino acid residues which regulate the maxima of original pigments through electrostatic interactions. Using time-resolved low-temperature spectroscopy, the decay rates of meta II and meta III intermediates of chicken blue are estimated to be similar to those of chicken red and green, but considerably faster than those of rhodopsin. Efficiency in activating transducin by the irradiated chicken blue is greatly diminished as the time before its addition to the reaction mixture containing transducin and GTP increases, while that by irradiated rhodopsin is not. The time profile is almost identical with those observed in chicken red and green. Thus, the faster decay of enzymatically active state is common in cone visual pigments, independent of their spectral sensitivity.

Animals↗
PubMed1998

Role of the C9 methyl group in rhodopsin activation: characterization of mutant opsins with the artificial chromophore 11-cis-9-demethylretinal.

Activation of the visual pigment rhodopsin involves both steric and electrostatic interactions between the chromophore and opsin within the retinal-binding site. Removal of the C9 methyl group of 11-cis-retinal inhibits light-dependent activation of the G protein, transducin, suggesting a direct steric contact. More recently, we have shown that steric interactions lead to receptor activation when Gly121 in the middle of transmembrane helix 3 is replaced by larger hydrophobic residues. In order to understand in more detail the role of the C9 methyl group of retinal in the structure and function of rhodopsin, we first studied the properties of recombinant 9-dm-Rho (opsin reconstituted with 11-cis-9-demethylretinal). The 9-dm-Rho pigment displayed a blue-shifted lambdamax, increased hydroxylamine reactivity, and decreased ability to activate transducin. These properties are consistent with the hypothesis that the C9 methyl group is a crucial structural anchor for the correct docking of the chromophore in its binding site. Next, we investigated the possible interaction between Gly121 of opsin and the C9 methyl group of retinal by characterizing recombinant pigments produced by combining mutant opsins (G121A, -V, -I, -L, and -W) with 11-cis-9-demethylretinal. Mutant opsins G121I, -L, and -W failed to bind the chromophore. However, the double mutant G121L/F261A bound 11-cis-9-demethylretinal to form a stable pigment with a lambdamax of 451 nm. When activity was assayed in membranes, the reduction in transducin activation by 9-dm-Rho caused by the lack of a C9 methyl group on the chromophore could be partially restored by replacing Gly121 with a bulky residue (leucine, isoleucine, or tryptophan). These results support a model of receptor activation that involves steric interaction between the C9 methyl group of the chromophore and the opsin in the vicinity of Gly121 on transmembrane helix 3.

Isomerism↗
PubMed1993

Heterozygous missense mutation in the rhodopsin gene as a cause of congenital stationary night blindness.

A number of mutations in the rhodopsin gene have been shown to cause both dominant and recessive retinitis pigmentosa. Here we describe another phenotype associated with a defect in this gene. We discovered a patient with congenital stationary night blindness who carries the missense mutation Ala292Glu. When coupled with 11-cis-retinal in vitro, Ala292Glu rhodopsin is able to activate transducin in a light-dependent manner like wild-type rhodopsin. However, without a chromophore, Ala292Glu opsin anomalously activates transducin. We speculate that the rod dysfunction in this patient is due to an abnormal, continuous activation of transducin by mutant opsin molecules in photoreceptor outer segments.

Adult↗
PubMed2002

Evidence for two apoptotic pathways in light-induced retinal degeneration.

Excessive phototransduction signaling is thought to be involved in light-induced and inherited retinal degeneration. 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. Bright light induces apoptosis that is independent of transducin and accompanied by induction of the transcription factor AP-1. By contrast, low light induces an apoptotic pathway that requires transducin. We also provide evidence that additional genetic factors regulate sensitivity to light-induced damage. Our use of defined mouse mutants resolves some of the complexity underlying the mechanisms that regulate susceptibility to retinal degeneration.

Animals↗
PubMed1995

The photoactivatable NAD+ analogue [32P]2-azido-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein G alpha t.

Recently, we reported the synthesis and use of [32P]2-azido-NAD+ as a probe to study the structural organization of G-proteins. Pertussis toxin was used to 'tether' [32P]2-azido-ADP-ribose of [32P]2-azido-NAD+ to Cys347 of the alpha subunit of the G-protein Gt. Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of alpha t with neighbouring intra- and inter-molecular domains of holo-transducin. The radiolabel from [32P]2-azido-ADP-ribose was then transferred to the 'acceptor' domain by cleaving the thioglycosidic bond between Cys347 and [32P]2-azido-ADP- ribose with mercuric acetate. ADP-ribosylation followed by photocross-linking of holo-transducin indicated intramolecular interactions of the C-terminal domain with other alpha t domains and intermolecular interactions with holotransducin alpha and gamma subunits. The radiolabelled peptides, which were radiolabelled because of the transfer of the photoactive moiety, were identified by utilizing 2-(2'-nitrophenylsulphenyl)-3-methyl-3'- bromoindolenine ('BNPS-skatole') and CNBr. The results indicate that the C-terminus of alpha t interacts with both N-terminal and C-terminal domains within the alpha t molecular. Mapping the interacting sites between cross-linked alpha dimers and alpha trimers indicates that the C-terminal domain of alpha t is involved in the formation of alpha t homopolymers in solution. In addition, our studies place the beta gamma subunit in close proximity to Cys347 of alpha t, as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the gamma subunit, which was further localized to the C-terminal half of gamma t. The studies presented here identify the C-terminal intra- and inter-molecular interactions of the alpha subunit of holo-transducin.

Adenosine Diphosphate↗
PubMed1996

Protein kinase C in rod outer segments: effects of phosphorylation of the phosphodiesterase inhibitory subunit.

The inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE alpha beta gamma 2) in rod outer segments (ROS) realizes its regulatory role in phototransduction by inhibition of PDE alpha beta catalytic activity. The photoreceptor G-protein, transducin, serves as a transducer from the receptor (rhodopsin) to the effector (PDE) and eliminates the inhibitory effect of PDE gamma by direct interaction with PDE gamma. Our previous study [Udovichenko, Cunnick, Gonzalez and Takemoto (1994) J: Biol. Chem. 269, 9850-9856] has shown that PDE gamma is a substrate for protein kinase C (PKC) from ROS and that phosphorylation by PKC increases the ability of PDE gamma to inhibit PDE alpha beta catalytic activity. Here we report that transducin is less effective in activation of PDE alpha beta (gamma p)2 (a complex of PDE alpha beta with phosphorylated PDE gamma, PDE gamma p) than PDE alpha beta gamma 2. PDE gamma p also increases the rate constant of GTP hydrolysis of transducin (from 0.16 S-1 for non-phosphorylated PDE gamma to 0.21 s-1 for PDE gamma p). These data suggest that phosphorylation of the inhibitory subunit of PDE by PKC may regulate the visual transduction cascade by decreasing the photoresponse.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1994

cGMP binding sites on photoreceptor phosphodiesterase: role in feedback regulation of visual transduction.

A central step in vertebrate visual transduction is the rapid drop in cGMP levels that causes cGMP-gated ion channels in the photoreceptor cell membrane to close. It has long been a puzzle that the cGMP phosphodiesterase (PDE) whose activation causes this decrease contains not only catalytic sites for cGMP hydrolysis but also noncatalytic cGMP binding sites. Recent work has shown that occupancy of these noncatalytic sites slows the rate of PDE inactivation. We report here that PDE activation induced by activated transduction lowers the cGMP binding affinity for noncatalytic sites on PDE and accelerates the dissociation of cGMP from these sites. These sites can exist in three states: high affinity (Kd = 60 nM) for the nonactivated PDE, intermediate affinity (Kd approximately 180 nM) when the enzyme is activated in a complex with transducin, and low affinity (Kd > 1 microM) when transducin physically removes the inhibitory subunits of PDE from the PDE catalytic subunits. Activation of PDE by transducin causes a 10-fold increase in the rate of cGMP dissociation from one of the two noncatalytic sites; physical removal of the inhibitory subunits from the PDE catalytic subunits further accelerates the cGMP dissociation rate from both sites > 50-fold. Because PDE molecules lacking bound cGMP inactivate more rapidly, this suggests that a prolonged cGMP decrease may act as a negative feedback regulator to generate the faster, smaller photoresponses characteristic of light-adapted photoreceptors.

Animals↗
PubMed1997

Modulation of opsin apoprotein activity by retinal. Dark activity of rhodopsin formed at low temperature.

The bovine opsin apoprotein activates transducin, although at a much reduced level than light-activated rhodopsin (Surya, A., Foster, K., and Knox, B. (1995) J. Biol. Chem. 270, 5024-5031). The ability of retinal to modulate opsin apoprotein activity was investigated using a guanyl nucleotide exchange assay on transducin. 11-cis-Retinal reacted with opsin at 22 degrees C to (a) reform pigment having maximal absorbance at 500 nm and (b) reduce opsin activity by >80%. Pigment formation also occurred at 0 degrees C with a t1/2 of 260 min. However, unlike rhodopsin formed at 22 degrees C (R22), the rhodopsin formed at 0 degrees C (R0) activated transducin with the same half-saturating concentration as opsin in an exhaustive binding assay. Thus, the formation of a protonated Schiff base associated with 500 nm absorbance does not by itself lead to the inactivation of opsin. The R0 conformation was partially inactivated by incubation at 22 degrees C (t1/2 = 61 +/- 9 min), suggesting that it may be an intermediate conformation in the regeneration of rhodopsin.

Animals↗
PubMed1998

Mutational analysis of the Asn residue essential for RGS protein binding to G-proteins.

Members of the RGS family serve as GTPase-activating proteins (GAPs) for heterotrimeric G-proteins and negatively regulate signaling via G-protein-coupled receptors. The recently resolved crystal structure of RGS4 bound to Gialpha1 suggests two potential mechanisms for the GAP activity of RGS proteins as follows: stabilization of the Gialpha1 switch regions by RGS4 and the catalytic action of RGS4 residue Asn128. To elucidate a role of the Asn residue for RGS GAP function, we have investigated effects of the synthetic peptide corresponding to the Galpha binding domain of human retinal RGS (hRGSr) containing the key Asn at position 131, and we have carried out mutational analysis of Asn131. Synthetic peptide hRGSr-(123-140) retained its ability to bind the AlF4--complexed transducin alpha-subunit, Gtalpha.AlF4-, but failed to elicit stimulation of Gtalpha GTPase activity. Wild-type hRGSr stimulated Gtalpha GTPase activity by approximately 10-fold with an EC50 value of 100 nM. Mutant hRGSr proteins with substitutions of Asn131 by Ser and Gln had a significantly reduced affinity for Gtalpha but were capable of substantial stimulation of Gtalpha GTPase activity, 80 and 60% of Vmax, respectively. Mutants hRGSr-Leu131, hRGSr-Ala131, and hRGSr-Asp131 were able to accelerate Gtalpha GTPase activity only at very high concentrations (>10 microM) which appears to correlate with a further decrease of their affinity for transducin. Two mutants, hRGSr-His131 and hRGSr-Delta131, had no detectable binding to transducin. Mutational analysis of Asn131 suggests that the stabilization of the G-protein switch regions rather than catalytic action of the Asn residue is a key component for the RGS GAP action.

Amino Acid Sequence↗
PubMed1998

Onset of feedback reactions underlying vertebrate rod photoreceptor light adaptation.

Light adaptation in vertebrate photoreceptors is thought to be mediated through a number of biochemical feedback reactions that reduce the sensitivity of the photoreceptor and accelerate the kinetics of the photoresponse. Ca2+ plays a major role in this process by regulating several components of the phototransduction cascade. Guanylate cyclase and rhodopsin kinase are suggested to be the major sites regulated by Ca2+. Recently, it was proposed that cGMP may be another messenger of light adaptation since it is able to regulate the rate of transducin GTPase and thus the lifetime of activated cGMP phosphodiesterase. Here we report measurements of the rates at which the changes in Ca2+ and cGMP are followed by the changes in the rates of corresponding enzymatic reactions in frog rod outer segments. Our data indicate that there is a temporal hierarchy among reactions that underlie light adaptation. Guanylate cyclase activity and rhodopsin phosphorylation respond to changes in Ca2+ very rapidly, on a subsecond time scale. This enables them to accelerate the falling phase of the flash response and to modulate flash sensitivity during continuous illumination. To the contrary, the acceleration of transducin GTPase, even after significant reduction in cGMP, occurs over several tens of seconds. It is substantially delayed by the slow dissociation of cGMP from the noncatalytic sites for cGMP binding located on cGMP phosphodiesterase. Therefore, cGMP-dependent regulation of transducin GTPase is likely to occur only during prolonged bright illumination.

Adaptation, Ocular↗
PubMed1998

Role for the target enzyme in deactivation of photoreceptor G protein in vivo.

Heterotrimeric guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are deactivated by hydrolysis of the GTP that they bind when activated by transmembrane receptors. Transducin, the G protein that relays visual excitation from rhodopsin to the cyclic guanosine 3',5'-monophosphate phosphodiesterase (PDE) in retinal photoreceptors, must be deactivated for the light response to recover. A point mutation in the gamma subunit of PDE impaired transducin-PDE interactions and slowed the recovery rate of the flash response in transgenic mouse rods. These results indicate that the normal deactivation of transducin in vivo requires the G protein to interact with its target enzyme.

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

Calcium-dependent assembly of centrin-G-protein complex in photoreceptor cells.

Photoexcitation of rhodopsin activates a heterotrimeric G-protein cascade leading to cyclic GMP hydrolysis in vertebrate photoreceptors. Light-induced exchanges of the visual G-protein transducin between the outer and inner segment of rod photoreceptors occur through the narrow connecting cilium. Here we demonstrate that transducin colocalizes with the Ca(2+)-binding protein centrin 1 in a specific domain of this cilium. 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. The assembly of centrin-G-protein complex is mediated by the betagamma-complex. The Ca(2+)-dependent assembly of a G protein with centrin is a novel aspect of the supply of signaling proteins in sensory cells and a potential link between molecular translocations and signal transduction in general.

Animals↗
PubMed2003

Acceleration of key reactions as a strategy to elucidate the rate-limiting chemistry underlying phototransduction inactivation.

PURPOSE: A reconstituted system was used to establish a strategy to determine the rate-limiting chemistry responsible for recovery of the dim-flash response in rod photoreceptors. METHODS: A general approach for identifying the rate-limiting step in a series of reactions is to evaluate the consequences of accelerating each step separately, while monitoring the rate of formation of the end product of the series. This strategy was applied to the reactions involved in quenching phototransduction in bovine rod outer segment (bROS) homogenates. The decay of photoactivated rhodopsin (R*) and inactivation of transducin by guanosine triphosphate (GTP) hydrolysis are the leading candidates for limiting the rate of phototransduction turn-off. These reactions were accelerated separately and together by adding hydroxylamine and/or the regulator of G-protein signaling-9 catalytic domain (RGS9d) while monitoring phosphodiesterase (PDE) activity triggered by a pulse of light in bROS homogenates. RESULTS: PDE activity in bROS homogenates triggered by a flash of light returned to its dark value with a rate constant of 0.087 +/- 0.002 seconds in this system. The rate of PDE recovery increased to 0.11 +/- 0.004 seconds when R* decay was accelerated with 10 to 50 mM hydroxylamine, suggesting that R* inactivation limits the rate of phototransduction turn-off under these conditions. Adding both hydroxylamine and RGS9d, a factor that accelerates transducin inactivation, increased the rate of PDE decay even further. RGS9d had no effect on PDE recovery kinetics unless quenching of R* was also accelerated. CONCLUSIONS: Under in vitro conditions in bROS homogenates, the quenching of R* normally limits the rate of phototransduction shut-off. If R* decay is accelerated, inactivation of transducin by GTP hydrolysis becomes rate limiting. This study offers a general approach that could be used to investigate the rate-limiting chemistry of phototransduction turn-off in vivo.

3',5'-Cyclic-GMP Phosphodiesterases↗