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At least 595 records · Page 33Linked to original sources
PubMed2006

Cone photoreceptor function loss-3, a novel mouse model of achromatopsia due to a mutation in Gnat2.

PURPOSE: To report a novel mouse model of achromatopsia with a cpfl3 mutation found in the ALS/LtJ strain. METHODS: The effects of a cpfl3 mutation were documented using fundus photography, electroretinography (ERG), and histopathology. Genetic analysis was performed using linkage studies and PCR gene identification. RESULTS: Homozygous cpfl3 mice had poor cone-mediated responses on ERG at 3 weeks that became undetectable by 9 months. Rod-mediated waveforms were initially normal, but declined with age. Microscopy of the retinas revealed progressive vacuolization of the photoreceptor outer segments. Immunocytochemistry with cone-specific markers showed progressive loss of labeling for alpha-transducin, but the cone outer segments in the oldest mice examined remained intact and positive with peanut agglutinin (PNA). The cpfl3 mapped to mouse chromosome 3 at the same location as human GNAT2, known to cause achromatopsia. Sequence analysis revealed a missense mutation due to a single base pair substitution in exon 6 in cpfl3. CONCLUSIONS: The Gnat2(cpfl3) mutation leads to cone dysfunction and the progressive loss of cone alpha-transducin immunolabeling. Despite a poor cone ERG signal and loss of cone alpha-transducin label, the cones survive at 14 weeks as demonstrated by PNA staining. This mouse model of achromatopsia will be useful in the study of the development, pathophysiology, and treatment of achromatopsia and other cone degenerations. The gene symbol for the cpfl3 mutation has been changed to Gnat2(cpfl3).

Animals↗
PubMed1985

cGMP phosphodiesterase in rod and cone outer segments of the retina.

Immunochemical, chromatographic, and sodium dodecyl sulfate gel electrophoresis studies suggest that immunologically related but distinct cyclic GMP phosphodiesterases are present in rod and cone outer segments of the retina. Immunocytochemical studies demonstrated that one monoclonal antibody (ROS-1) recognized a determinant present in both rod and cone outer segments, while another monoclonal antibody (ROS-2) only recognized rod outer segments. At least two peaks of phosphodiesterase activity could be separated by high-performance anion-exchange chromatography of retinal extracts. Both peaks were recognized by ROS-1. None of the first peak and only 80% of the second broad peak of activity were recognized by ROS-2. High-performance liquid chromatography profiles from human fovea and several other types of cone-enriched retina showed that most of the activity was contained in the first peak, suggesting that this activity was derived from cone outer segments. Conversely, the phosphodiesterase in rod-enriched preparations migrated predominately in the second peak. Sodium dodecyl sulfate-gel electrophoresis indicated that this first peak contained a single large immunoreactive polypeptide (alpha') that migrated with the same mobility as a phosphorylase b standard and was distinct from the more rapidly migrating large immunoreactive polypeptides (alpha and beta) present in a broad second peak. The second peak could be further separated into a first part that contained a doublet of two immunoreactive polypeptides (alpha and beta) that migrated faster than phosphorylase b and a later part that contained only the most rapidly migrating polypeptide (beta). All of the peaks could be activated by histone or transducin:GTP, implying that all contained a small 11-kDa inhibitory subunit (gamma) of the enzyme. Since the larger (alpha') and smaller (beta) immunoreactive polypeptides could be completely separated from the alpha polypeptide and from each other, yet still retain the ability to be activated by histone or transducin, the data suggest that only a single species of polypeptide-inhibitor complex (e.g. alpha' gamma, alpha gamma, or beta gamma) was required for histone or transducin:GTP activation.

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

Heterogeneous N-acylation is a tissue- and species-specific posttranslational modification.

Heterogeneous N-terminal glycine acylation recently has been reported for two proteins involved in visual signal transduction. Similar N-acylations have typically involved only myristate; however, none of the previously examined proteins were isolated from retinas. To determine whether heterogeneous N-acylation is tissue-specific or protein sequence-specific, the N-terminal modifications of the catalytic subunit of cAMP-dependent protein kinase, partially purified from bovine retinas, heart, and brain tissues, were characterized. Using tandem mass spectrometry and liquid chromatography coupled directly to an electrospray mass spectrometer, we found only myristate at the N termini of catalytic subunits from brain and heart tissue, whereas the N termini of the retina-derived subunits were heterogeneously acylated in a manner similar to recoverin and transducin. Thus it appears that the nature of N-terminal glycine acylation is determined by the cell or tissue type in which it is located, and not by the sequence of the modified protein. We also examined the N-acylation of recoverin purified from human retinas, as well as transducin purified from frog retinas, to determine if heterogeneous acylation of retinal proteins is a uniquely bovine phenomenon. Interestingly, human recoverin was modified by the same family of fatty acids found on the bovine retinal proteins, while frog transducin was modified homogeneously not with myristate, but with a doubly unsaturated (C14:2) fatty acyl group.

Acylation↗
PubMed1998

Rod and cone function in the Nougaret form of stationary night blindness.

BACKGROUND: Recently, a mutation (Gly38Asp) was identified in the alpha subunit of rod transducin in members of the Nougaret pedigree affected with dominantly inherited stationary night blindness. OBJECTIVE: To evaluate retinal function in patients with the Gly38Asp gene defect. DESIGN: Ocular examinations, including specialized measures of rod and cone function. SETTING: A clinical research facility in Boston, Mass. PATIENTS: A father (aged 48 years) and son (aged 25 years) with the Gly38Asp mutation. MAIN OUTCOME MEASURES: Psychophysical thresholds to white and narrowband lights and full-field electroretinographic (ERG) responses. RESULTS: Both patients showed dark-adapted thresholds to white light that were elevated approximately 2 log-units across the retina. Spectral sensitivity testing revealed thresholds that seemed to be governed mostly by rods. Although both patients' dark-adapted ERG responses to a dim blue flash were nondetectable, their dark-adapted ERGs to a white flash showed an a-wave with cone and rod components and a b-wave amplitude larger than what could have been generated by cone function alone. Rod ERGs to bright blue flashes had subnormal, but detectable, amplitudes that seemed to result from a profound reduction in sensitivity. The patients also showed loss of a cone subcomponent in the dark-adapted response to a red flash. The abnormal dark-adapted ERG responses of the patients could be simulated in the ERG responses of normal subjects tested with blue, white, and red flashes presented in the presence of a mesopic background. CONCLUSIONS: Although the Nougaret form of stationary night blindness has been cited as a prototype of absent rod function with normal cone function, our findings, based on the genealogically and genotypically documented descendants of Jean Nougaret, show that rod function is present, although subnormal, and that there is slight impairment of cone function. The data also suggest that these abnormalities can be simulated by light-adapting the normal retina, compatible with the proposal that the rod transducin encoded by the mutant gene is constitutively active and that the night blindness results from partial desensitization of rods caused by the constitutive activity.

Adult↗
PubMed1993

The G protein cascade of visual transduction: kinetics and regulation.

In retinal rods photoexcited rhodopsin (R*) catalyses the activation of transducin (T) by GTP, which in turn activates the cGMP phosphodiesterase (PDE). The ensuing decrease in cGMP concentration reduces the cell membrane's channel conductance. To account for the kinetics of the response to light, all underlying biochemical reactions must reach maximum speed and be turned off within a second. Kinetic analysis of transducin activation suggests that because of the fast lateral diffusion of T, the rate-limiting step is not the collision between R* and T but the entry of GTP after the release fo GDP from the R*-bound T alpha. T alpha-GTP dissociates from both R* and T beta gamma and diffuses through the cytoplasm to activate PDE. In suspensions of bovine rod outer segments, time-resolved microcalorimetry yields rates of approximately 1-2 s-1 for the GTPase of T alpha and the correlated deactivation of PDE. But for isolated T alpha-GTP the single turnover GTPase rate measured by a stopped-flow technique is only 0.05 s-1. To activate PDE, T alpha-GTP binds tightly to the PDE gamma subunit. In vitro the soluble T alpha-GTP.PDE gamma complex dissociates from activated PDE alpha beta. Thus PDE gamma might be the GTPase activator of T alpha, but no GTPase acceleration was observed in isolated T alpha-GTP.PDE gamma. The GTPase activation must depend on the interaction of T alpha-GTP.PDE gamma with membrane-bound PDE alpha beta.

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

Cytoskeleton participation in subcellular trafficking of signal transduction proteins in rod photoreceptor cells.

Light sensitivity and adaptation, general characteristics of rod photoreceptor cell vision, allow rods to modulate their response depending on the lighting environment to which they are exposed. In dim light, rods are maximally sensitive, whereas, in bright light, rods are essentially inactive. In the retinas of dark-adapted mice, arrestin (an inhibitory protein) is located in the rod inner segment (RIS), and transducin (an activating protein) is located in the rod outer segment (ROS). In light-adapted retinas, the proteins have reciprocal localizations. In this study, our data demonstrate that the temporal and spatial changes in the subcellular localization of arrestin and beta-transducin are correlated with the amount of light to which the animals are exposed. By using the frog Xenopus laevis and immunofluorescence confocal microscopy, our results also show that in the dark-adapted retina some arrestin remains in the ROS. The data most dramatically demonstrate that this residual arrestin is highly concentrated in the connecting cilium, the axoneme, and the microtubules associated with the disc incisures. These data suggest a structure-function relationship between the light-dependent positional status of arrestin and the elements of the rod photoreceptor cytoskeleton. The massive, rapid, light-induced reciprocal changes in the subcellular concentrations of these proteins must directly affect phototransduction and appear to be a general phenomenon by which photoreceptor cells rapidly and transiently regulate the trafficking and subcellular concentration of a variety of signal transduction proteins within the RIS and ROS. Hereditary mutations in the components of the movement mechanism should lead to defects in vision and possibly blindness.

Animals↗
PubMed1988

Light-dependent subcellular movement of photoreceptor proteins.

The intracellular localization of photoreceptor-specific proteins 33 kd, beta-transducin, and 48 kd, as determined by immunocytochemistry, is transient and dependent on the lighting environment to which the retina is exposed. Western analysis of the proteins in isolated rod outer segments from mouse retina demonstrates that beta-transducin actually moves from the outer segment to the inner segment in response to light and that 48 kd moves simultaneously in the opposite direction. The light-induced movements appear to be initiated by the absorption of light by rhodopsin because red light, which does not bleach rhodopsin, does not produce this redistribution of photoreceptor proteins. Time course analysis of these movements suggests that the light-induced shift is detectable at the earliest time examined (30 seconds). The bidirectional movement suggests that the photoreceptor cells have at least two distinct dynein-like or kinesin-like translocator molecules that act as microtubule-based motors. This movement appears to be a basic mechanism by which photoreceptor cells rapidly and radically alter the subcellular concentrations of photoreceptor-specific proteins, which in turn may affect the rapid changes in membrane potential that occur during phototransduction.

Animals↗
PubMed2005

Top-down analysis of protein isoprenylation by electrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry; the mouse Tgamma protein.

Protein isoprenylation, an important post-translational modification with a lipid, involves the selective attachment of two types of isoprenoids, farnesyl (C15) and geranylgeranyl (C20). The isoprenoid is linked via a thioether bond to the C-terminal cysteine residue of a variety of cellular proteins, including the heterotrimeric G protein gamma-subunits. One member of the G protein family, transducin (Talpha/Tbetagamma), plays a central role in visual transduction, and the structure-function relationship has been extensively studied with purified proteins, predominantly with bovine transducin that was shown to be farnesylated at the C-terminal cysteine residue of the gamma-subunit (Tgamma). We report here the structure of the C-terminal modification of mouse Tgamma, which has not yet been elucidated owing to the low amount of protein that can be isolated from the mouse retina. Electrospray ionization mass spectrometry (ESI-MS) of the high-performance liquid chromatography (HPLC)-purified Tgamma was in good agreement with the calculated mass of the farnesylated and methylated form of mouse Tgamma (Pro1-Cys70). A 'top-down' analysis of intact Tgamma using an ESI hybrid quadrupole time-of-flight (TOF) tandem mass spectrometer provided isoprenyl-specific ions that were observed to produce ions separated by 204 Da from the conventional (unmodified) precursor ion or the C-terminal sequence ions. Such characteristic fragmentation on an isoprenoid observed in top-down analysis could be useful in general for determining the type of isoprenylation as well as probing the site of modification in the protein sequence.

Animals↗
PubMed1997

Differential expression of photoreceptor-specific proteins during disease and degeneration in the progressive rod-cone degeneration (prcd) retina.

Progressive rod-cone degeneration (prcd) is a late-onset hereditary retinal degeneration characterized by normal development of photoreceptors prior to degeneration and death of visual cells. We reported previously that expression of opsin mRNA and protein decreases prior to visual cell degeneration. To examine the specificity of this reduction, we have used immunocytochemistry to correlate photoreceptor-specific protein expression with visual cell disease progression. Eyes from light-adapted age-matched control and prcd-affected dogs were fixed in paraformaldehyde, embedded in diethylene glycol distearate (DGD) wax, and reacted with antibodies specific to interphotoreceptor retinoid-binding protein (IRBP), S-antigen, opsin, phosducin, gamma-phosphodiesterase (gamma-PDE), and beta 1-transducin. While IRBP expression did not change with disease progression, immunoreactivity to other proteins varied. For S-antigen and opsin, immunoreactivity decreased dramatically with the transition from photoreceptor disease to degeneration; gamma-PDE immunolabeling in rods also decreased, but the reduction was less abrupt. However, for two other proteins (phosducin and beta 1-transducin), immunoreactivity increased initially and was redistributed (particularly to the rod outer segment) in early disease (stage 1). Our results show that there is a differential expression of photoreceptor-specific proteins with disease and degeneration that is not uniform for all the gene products examined; expression can be decreased, altered in distribution or remain unchanged. It is clear that the decrease of opsin expression described previously is not an isolated phenomenon in the progression of prcd, but is part of a more generalized degenerative process which eventually culminates in cell death.

Animals↗
PubMed1998

Enhancement of opsin activity by all-trans-retinal.

The rod cell photoreceptor apoprotein, opsin, activates the G-protein, transducin, although at a much reduced level than light-activated rhodopsin. The ability of all-trans-retinal to enhance opsin apoprotein activity was investigated using a guanyl nucleotide exchange assay on transducin. All-trans-retinal enhanced opsin activity in a concentration-dependent manner. At high concentrations of all-trans-retinal, the activity of the all-trans-retinal-opsin complex was comparable to that from an equimolar amount of metarhodopsin(II). However, in contrast to metarhodopsin(II), the active all-trans-retinalopsin complex did not require a stable Schiff base linkage between opsin and all-trans-retinal. The lack of a stable Schiff base and differences in activity at high pH imply that opsin and all-trans-retinal form a complex that is distinct from metarhodopsin(II). The ability of all-trans-retinal to stimulate the transduction cascade may be a source of post-bleach noise in photoreceptors.

Animals↗
PubMed1987

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail.

The retinal proteins opsin, alpha-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocytochemistry we have employed antibodies directed against these "photoreceptor proteins" in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and alpha-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina, in the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.

Animals↗
PubMed1989

Visual transduction in vertebrate photoreceptors. Light activation of guanylate cyclase.

Light activation of guanylate cyclase at different calcium concentrations was studied in the rod outer segments of the toad retina. The enzyme becomes sensitive to calcium ions after a flash of light, showing an enhancement of its activity when Ca2+ concentration is lowered from 10(-4) M to 10(-8) M. A possible pathway of guanylate cyclase activation by light was also investigated by means of the antibody 4A to transducin. When added in excess to transducin, the antibody inhibits light activation of phosphodiesterase as well as of cyclase, suggesting a possible coupling of the two enzymes.

Animals↗
PubMed1986

A 48 kDa protein arrests cGMP phosphodiesterase activation in retinal rod disk membranes.

Photolyzed rhodopsin (R) catalyzes GTP-binding to alpha-transducins (T alpha); T alpha X GTPs then activate cGMP phosphodiesterase (PDE). PDE activation is arrested by ATP in two ways: (i) initial velocity is suppressed, and (ii) PDE velocity rapidly returns to preactivation levels (turnoff). Arrestin (a 48 kDa protein) markedly enhances turnoff while not affecting initial velocity. Arrestin in the presence of ATP achieves rapid turnoff by directly inhibiting activated PDE, as indicated by its ability to inhibit the direct activation of PDE by T alpha X GMP--PNP (guanylyl-imidodiphosphate). Double reciprocal plots reveal a competition between arrestins and activated transducins for sites on PDE. Blocking R phosphorylation blocks initial velocity suppression but does not disturb rapid turnoff. Our data suggest a 2-fold mechanism for PDE deactivation: (i) formation of T alpha X GTPs is suppressed by R phosphorylation, while (ii) activation of PDE by T alpha X GTPs is competitively inhibited by arrestins when ATP is present.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1986

Primary structure of the alpha-subunit of bovine adenylate cyclase-inhibiting G-protein deduced from the cDNA sequence.

The primary structure of the alpha-subunit of the adenylate cyclase-inhibiting G-protein (Gi) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. A much higher degree of amino acid sequence homology is observed between the alpha-subunits of Gi and transducin (68%) than between those of Gi and the adenylate cyclase-stimulating G-protein (Gs) (43%) or between those of transducin and Gs (42%).

Adenylyl Cyclase Inhibitors↗
PubMed1993

Activation of phosphatidylinositol lipid-specific phospholipase C-beta 3 by G-protein beta gamma subunits.

A novel member of the inositol lipid-specific phospholipase C family, PtdIns-PLC beta 3, is shown to be activated by beta gamma subunits of the heterotrimeric GTP-binding protein, transducin. The activation is a direct effect since it is observed with the purified proteins. Furthermore, the activation is blocked by the GDP-liganded alpha subunit of transducin, confirming that the effect is due to free beta gamma subunits. The implications with respect to receptor-PtdIns-PLC coupling are discussed.

Animals↗
PubMed1990

Watching G proteins at work.

It has been known for over a century that rod photoreceptors in the living retina contract and swell in response to light. Although it is still not known whether this structural light-response is of any functional significance, it has recently been possible to correlate the underlying molecular processes with the activation and deactivation of the photoreceptor G protein, transducin. The technique of light-scattering allows the monitoring of minute changes in cell dimensions, and using this non-invasive experimental approach it can be shown that certain properties of the coupling between transducin and rhodopsin are different in a structurally well-preserved system as compared with rod material used for conventional biochemical studies. Thus, not unlike a psychiatrist, who often learns more about a patient's 'interiors' by observing the body language than by direct interrogation, a biochemist, studying the 'body language' of a cell, may extract information about delicate 'cell interior processes' that would be perturbed by more direct experimental approaches.

Animals↗
PubMed1994

Critical processes which characterize the photocurrent of retinal rod outer segments to flash stimuli.

The chemical reactions of retinal rod outer segments (ROS) were modeled aimed at finding the critical process for the reconstruction of the photocurrent to flash stimuli. The differential equations, which were derived from the chemical reactions, were numerically integrated. According to the present model, it was found that the most critical process for the recovery of the photocurrent was the synthesis of cGMP by guanylate cyclase in the [Ca2+]i-dependent manner. The other recovery processes, such as rhodopsin phosphorylation, transducin and phosphodiesterase inactivation seemed not to be involved in the recovery of the photocurrent to flash stimuli. Finally, a recently proposed scheme in which transducin remained bound to phosphodiesterase after its activation was examined. The simulation for this scheme showed that the ROS sensitivity was greatly reduced because of the limited amplification in the transduction cascades.

Calcium↗
PubMed1995

A gene responsible for vegetative incompatibility in the fungus Podospora anserina encodes a protein with a GTP-binding motif and G beta homologous domain.

The het-e-1 gene of the fungus Podospora anserina is responsible for vegetative incompatibility through specific interactions with different alleles of the unlinked gene, het-c. Coexpression of two incompatible genes triggers a cell death reaction that prevents heterokaryon formation. The het-e1 allele has been cloned to get information on the function of the locus. It encodes a putative 1356-amino-acid polypeptide that displays two sequence motifs that have not yet been reported to be present on a single polypeptide. They are a GTP-binding domain and a repeated region that shares similarity with that of the beta-transducin. Contrary to other members of the beta-transducin family, sequence conservation between the repeated units is very strong and the number of repeats is different in wild-type het-e alleles.

Alleles↗