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PubMed1988

A monoclonal antibody that binds to photoreceptors in the turtle retina.

We have raised monoclonal antibodies to photoreceptor cells in the retina of the turtle (Pseudemys scripta elegans). One of these antibodies, 15-18 (an IgG1), was studied by immunoelectron microscopy using colloidal gold, and found to bind to the outer segments of all rods and some single cones, but did not stain turtle double cones. Immunoblotting and immunoprecipitation show that antibody 15-18 binds to an antigen of apparent Mr approximately 34,5000 which is probably turtle opsin. Antibody 15-18 binds visual pigments from several species, including bovine opsin. In order to determine the antigenic site bound by 15-18 in bovine opsin, synthetic peptides were used as competitors in an enzyme-linked immunoassay (ELISA). The antigenic site is located in the surface loop connecting rhodopsin helices IV-V, in the sequence 190-197. Antibody 15-18 binds to the external surface of rod cell outer segments, thus providing direct evidence for the predicted orientation of rhodopsin in the plasma membrane.

Animals↗
PubMed1984

Autoradiographic and biochemical analysis of photoreceptor membrane renewal in Octopus retina.

Using autoradiographic and biochemical methods, we have demonstrated the renewal of light-sensitive membranes and photopigments in Octopus visual cells. After the injection of Octopus with [3H]leucine, electron microscope autoradiography revealed an intracellular pathway similar to that in vertebrates for the synthesis and transport of nascent protein from the inner segments to the rhabdomes. However, migration of labelled protein from synthetic sites to the light-sensitive rhabdomes took longer in Octopus than the equivalent process in vertebrates. Biochemical analysis of [3H]leucine-labelled retinas identified some of the labelled protein observed in autoradiographs of the rhabdomes as the visual pigment, rhodopsin. We have shown that retinochrome, a second photopigment in cephalopod retinas, is also renewed. Biochemical analysis 8 h after injection of [3H]leucine revealed heavy labelling of this photoprotein. Light microscope autoradiography of Octopus retina 8 h after injection of [3H]retinol showed labelling of both the rhabdomes and the myeloid bodies of the inner segments. Biochemical data gathered 8 h after injection of [3H]retinol indicated chromophore addition to both rhodopsin and retinochrome with retinochrome being more heavily labelled than rhodopsin. Thus, silver grains observed over the rhabdomes and inner segments could arise from one or both photopigments. These data suggest that retinal is stored in the myeloid bodies of the photoreceptor inner segments. Retinal could then be transferred, perhaps via retinochrome, to newly synthesized opsin before the visual pigment is assembled into new rhabdomeric membranes. Alternatively, retinochrome may serve to transport retinal from the myeloid bodies to the rhabdomes to regenerate rhodopsin as previously proposed.

Animals↗
PubMed1986

Tunicamycin-induced dysgenesis of retinal rod outer segment membranes. II. Quantitative freeze-fracture analysis.

Tunicamycin (TM), a selective inhibitor of dolichylphosphate-dependent oligosaccharide biosynthesis, effectively blocks glycosylation, but not synthesis, of opsin, the rod visual pigment apoglycoprotein. In parallel with this inhibition, vesicular membrane material accumulates in the compartment between rod inner and outer segments (the intersegmental space) in TM-treated retinas (Fliesler et al, J Cell Biol 100:574-587, 1985). Freeze-fracture analysis was applied to isolated Xenopus laevis retinas which were incubated in the presence or absence of TM in order to clarify the relationship of those intersegmental membranous structures to others in the rod outer segment (ROS) assembly pathway. The membranes in the intersegmental space display characteristics similar to those of the ROS, but distinct from those of the inner segment. They exhibit densely particulate convex protoplasmic face (PF)-leaflets and relatively particle-free concave exoplasmic face (EF)-leaflets, similar to comparable leaflets of the ROS plasmalemma and nascent (immature) disc membranes. Quantitative analysis further demonstrates that the size distribution and densities of intramembrane particles (IMPs) in PF-leaflets of the intersegmental membranes are indistinguishable from those of ROS membranes, suggesting that both membrane systems contain similar integral proteins (i.e., opsin). Finally, the topology of the intersegmental membranes is most closely related to that of the ROS plasmalemma or nascent disc membranes, suggesting that they arise as a result of aberrant disc morphogenesis, rather than by breakdown of mature discs. Overall, the data support the conclusion that the tubulo-vesicular membranes in the intersegmental space represent newly assembled, opsin-containing material which has been efficiently compartmentalized in preparation for disc morphogenesis, but is incapable of forming normal, topologically closed discs.

Animals↗
PubMed1986

Tunicamycin-induced dysgenesis of retinal rod outer segment membranes. I. A scanning electron microscopy study.

Incubation of Xenopus retinas with tunicamycin has been shown to block the glycosylation of opsin, the rod visual pigment apoglycoprotein, with concomitant accumulation of vesicular membrane material in the compartment between the rod inner and outer segments (i.e., the intersegmental space) (Fliesler et al, J Cell Biol 100:574-587, 1985). To further assess the morphology, topology, and cellular origin of this membranous material, Xenopus retinas were incubated in the presence or absence of tunicamycin and the photoreceptor cells were examined by scanning electron microscopy. The material which accumulated in the intersegmental space appeared to be a complex of membranous structures consisting of cisternae with numerous tubular projections, as well as closely associated individual vesicles of various sizes. This tubulo-vesicular material was exclusively associated with the basal surface of the rod outer segment. The connecting cilium, periciliary ridge complex, and the apical surface of the rod inner segment were devoid of such membrane material. Nascent (open) discs (i.e., evaginations of the plasma membrane at the base of the outer segment) often observed in control retinas were not present in tunicamycin-treated tissue. These results support the hypothesis that the membranous material which accumulates in the intersegmental space of rods in tunicamycin-treated retinas represents incompletely and aberrantly formed nascent disc membranes. The formation of this material is apparently a consequence of a deficiency in newly synthesized, asparagine-linked membrane glycoconjugates (e.g., the oligosaccharide chains of opsin) at the site of disc assembly.

Animals↗
PubMed1987

Isolation and characterization of a retinal-binding protein from the squid retina.

A retinal-binding protein (RALBP) was isolated from the squid retina, and purified by anion-exchange and size-exclusion chromatography. The molecular weight was determined to be 51,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration. The purified sample showed absorption maxima at about 330 and 400 nm in addition to a protein band, indicating the occurrence of retinol and retinal, respectively. The relative heights of these two peaks varied from preparation to preparation, depending on retinoid ligands. Irradiation of RALBP caused no marked change in absorption, but the amount of 11-cis-retinal decreased to form a photosteady state mixture with all-trans- and 13-cis-retinals. RALBP was fairly stable even in the presence of hydroxylamine (100 mM), but was affected by sodium borohydride (30 mM) or borane dimethylamine (400 mM), with the retinal reduced to retinol. When incubated with metaretinochrome-carrying membranes in the dark, RALBP specifically took up 11-cis-retinal and lost all-trans-retinol. Upon further incubation of this RALBP with opsin-containing membranes, rhodopsin was progressively formed in the dark. Squid RALBP may act as a shuttle in transferring the 11-cis-retinal from metaretinochrome to opsin in the visual cells.

Animals↗
PubMed1986

Enzyme-linked immunosorbent assay for quantitative determination of the visual pigment rhodopsin in total-eye extracts.

A versatile, multispecies enzyme-linked immunosorbent assay for the rod visual pigment (rhod)opsin has been developed. For this quantitative inhibition assay a monospecific polyclonal antiserum is used which is elicited in rabbits against bovine rod outer-segment membranes. Detergent concentrations as high as 1.0% can be used in the assay with only a slight loss in sensitivity. The assay allows quantitative determination of the apoprotein opsin with a detection level of about 0.04 pmol per sample by using standards prepared by illumination of spectrophotometrically determined amounts of rhodopsin. The antiserum shows considerable cross-reactivity with opsin from several species (mouse, rat, quail, monkey and man). The high degree of monospecificity and cross-reactivity of the antiserum already allowed quantitation of opsin content in crude eye extracts of mouse, rat and quail with a sensitivity comparable to that of bovine opsin. Similar types of multispecies immunoassays for quantitation of highly conserved membrane proteins can be developed using the described approach, requiring only a monospecific antiserum elicited against an easily accessible species and crude tissue extracts both for coating and as a source of the inhibitory antigen.

Animals↗
PubMed1987

cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor.

We have isolated and sequenced a cDNA encoding the human beta 2-adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster beta 2-adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. We have localized the gene for the beta 2-adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor.

Amino Acid Sequence↗
PubMed2005

Expression of UV-, blue-, long-wavelength-sensitive opsins and melatonin in extraretinal photoreceptors of the optic lobes of hawk moths.

Lepidopterans display biological rhythms associated with egg laying, eclosion and flight activity but the photoreceptors that mediate these behavioural patterns are largely unknown. To further our progress in identifying candidate light-input channels for the lepidopteran circadian system, we have developed polyclonal antibodies against ultraviolet (UV)-, blue- and extraretinal long-wavelength (LW)-sensitive opsins and examined opsin immunoreactivity in the adult optic lobes of four hawk moths, Manduca sexta, Acherontia atropos, Agrius convolvuli and Hippotion celerio. Outside the retina, UV and blue opsin protein expression is restricted to the adult stemmata, with no apparent expression elsewhere in the brain. Melatonin, which is known to have a seasonal influence on reproduction and behaviour, is expressed with opsins in adult stemmata together with visual arrestin and chaoptin. By contrast, the LW opsin protein is not expressed in the retina or stemmata but rather exhibits a distinct and widespread distribution in dorsal and ventral neurons of the optic lobes. The lamina, medulla, lobula and lobula plate, accessory medulla and adjacent neurons innervating this structure also exhibit strong LW opsin immunoreactivity. Together with the adult stemmata, these neurons appear to be functional photoreceptors, as visual arrestin, chaoptin and melatonin are also co-expressed with LW opsin. These findings are the first to suggest a role for three spectrally distinct classes of opsin in the extraretinal detection of changes in ambient light and to show melatonin-mediated neuroendocrine output in the entrainment of sphingid moth circadian and/or photoperiodic rhythms.

Animals↗
PubMed1997

Mutation of a conserved proline disrupts the retinal-binding pocket of the X-linked cone opsins.

PURPOSE: To test the effects of disruption of a conserved proline in the green cone opsin molecule on light-activated isomerization, transducin activation, protein accumulation, glycosylation, and transport. METHODS: Stable cell lines were established by transfecting EBNA-293 cells with a plasmid containing wild-type or mutant (P307L) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of CdCl2 and analyzed by spectra, transducin activation, Western blotting, and immunocytochemistry. RESULTS: The P307L mutation diminished ability of the visual pigment to absorb light at the appropriate wavelength and to activate transducin. Protein glycosylation and transport to the cell membrane were unaffected. Although there was some diminution in the accumulation of the opsin, this was insufficient to account for the observed effect. CONCLUSIONS: Like rhodopsin, the formation of the cone opsins visual pigments is dependent on the binding of retinal into a hydrophobic pocket that is formed by the second and fourth transmembranous loops. Disruption of a conserved proline near the retinal binding site represents a cause of color vision deficiency that is unrelated to spectral shifts of the photopigment.

Biological Transport↗
PubMed1986

Photosensitivity of 10-substituted visual pigment analogues: detection of a specific secondary opsin-retinal interaction.

The photosensitivities of the bovine rhodopsin and gecko pigment 521 analogues regenerated from C-10-substituted analogues of 11-cis- and 9-cis-retinals were determined by two different methods. A similar reactivity trend was noted for both pigment systems as revealed in the photosensitivity of the gecko pigments and relative quantum yields of the bovine analogues. The 10-fluoro-11-cis photopigments had a photosensitivity less than, but approaching, that of the native (11-cis) visual pigment while the 10-fluoro-9-cis photopigments had a much lower photosensitivity than the parent 9-cis regenerated pigment. The results are interpreted in terms of recently described models of rhodopsin architecture and of the primary molecular reaction of visual pigments to light. The unusually low photoreactivity of the 10-fluoro-9-cis pigment molecule is viewed as the result of a regiospecific hydrogen-bonding interaction of the electronegative fluorine atom to the opsin.

Animals↗
PubMed1995

The molecular basis for the green-blue sensitivity shift in the rod visual pigments of the European eel.

When the European eel matures sexually and migrates back to deep sea breeding grounds the visual pigments in its rod photoreceptors change from being maximally sensitive to green light to being maximally sensitive to blue light. In part, this change in sensitivity is due to a change in the opsin component of the visual pigment molecule. We used hormone injection to induce these developmental changes in a group of eels and from these animals an opsin coding region was cloned and sequenced using cDNA made from retinal mRNA. From the retinae of hormone-injected eels and those not injected with hormones, distinct opsin mRNAs were isolated. These mRNAs encode two rod opsin proteins that are very similar but have significant amino acid substitutions in key positions that are likely to be involved in spectral tuning of the eel green and blue sensitive rod visual pigment molecules.

Amino Acid Sequence↗
PubMed2005

Characterization of the long-wavelength opsin from Mecoptera and Siphonaptera: does a flea see?

Mecoptera and Siphonaptera represent two insect orders that have largely been overlooked in the study of insect vision. Recent phylogenetic evidence demonstrates that Mecoptera (scorpionflies) is paraphyletic, with the order Siphonaptera (fleas) nesting as sister to the family Boreidae (snow fleas), showing an evolutionary trend towards reduction in gross eye morphology within fleas. We provide the first molecular characterization of long-wavelength opsins from these three lineages (opsin gene from fleas [FL-Opsin], the Boreidae [B-Opsin], and a mecopteran family [M-Opsin]) and assess the effects of loss of visual acuity on the structure and function of the opsin gene. Phylogenetic analysis implies a physiological sensitivity in the red-green spectrum for these opsins. Analysis of intron splice sites reveals a high degree of similarity between FL-Opsin and B-Opsin as well as conserved splice sites across insect blue-green and long-wavelength opsins. Calculated rates of evolution and tests for destabilizing selection indicate that FL-Opsin, B-Opsin, and M-Opsin are evolving at similar rates with no radical selective pressures, implying conservative evolution and functional constraint across all three lineages.

Animals↗
PubMed2001

Interaction of 11-cis-retinol dehydrogenase with the chromophore of retinal g protein-coupled receptor opsin.

Vertebrate opsins in both photoreceptors and the retinal pigment epithelium (RPE) have fundamental roles in the visual process. The visual pigments in photoreceptors are bound to 11-cis-retinal and are responsible for the initiation of visual excitation. Retinochrome-like opsins in the RPE are bound to all-trans-retinal and play an important role in chromophore metabolism. The retinal G protein-coupled receptor (RGR) of the RPE and Müller cells is an abundant opsin that generates 11-cis-retinal by stereospecific photoisomerization of its bound all-trans-retinal chromophore. We have analyzed a 32-kDa protein (p32) that co-purifies with bovine RGR from RPE microsomes. The co-purified p32 was identified by mass spectrometric analysis as 11-cis-retinol dehydrogenase (cRDH), and enzymatic assays have confirmed the isolation of an active cRDH. The co-purified cRDH showed marked substrate preference to 11-cis-retinal and preferred NADH rather than NADPH as the cofactor in reduction reactions. cRDH did not react with endogenous all-trans-retinal bound to RGR but reacted specifically with 11-cis-retinal that was generated by photoisomerization after irradiation of RGR. The reduction of 11-cis-retinal to 11-cis-retinol by cRDH enhanced the net photoisomerization of all-trans-retinal bound to RGR. These results indicate that cRDH is involved in the processing of 11-cis-retinal after irradiation of RGR opsin and suggest that cRDH has a novel role in the visual cycle.

Alcohol Oxidoreductases↗
PubMed1999

Genetic analyses of visual pigments of the pigeon (Columba livia).

We isolated five classes of retinal opsin genes rh1(Cl), rh2(Cl), sws1(Cl), sws2(Cl), and lws(Cl) from the pigeon; these encode RH1(Cl), RH2(Cl), SWS1(Cl), SWS2(Cl), and LWS(Cl) opsins, respectively. Upon binding to 11-cis-retinal, these opsins regenerate the corresponding photosensitive molecules, visual pigments. The absorbance spectra of visual pigments have a broad bell shape with the peak, being called lambdamax. Previously, the SWS1(Cl) opsin cDNA was isolated from the pigeon retinal RNA, expressed in cultured COS1 cells, reconstituted with 11-cis-retinal, and the lambdamax of the resulting SWS1(Cl) pigment was shown to be 393 nm. In this article, using the same methods, the lambdamax values of RH1(Cl), RH2(Cl), SWS2(Cl), and LWS(Cl) pigments were determined to be 502, 503, 448, and 559 nm, respectively. The pigeon is also known for its UV vision, detecting light at 320-380 nm. Being the only pigments that absorb light below 400 nm, the SWS1(Cl) pigments must mediate its UV vision. We also determined that a nonretinal P(Cl) pigment in the pineal gland of the pigeon has a lambdamax value at 481 nm.

Amino Acid Sequence↗
PubMedDate not supplied

Identification and distribution of photoreceptor subtypes in the neotenic tiger salamander retina.

The neotenic tiger salamander retina is a major model system for the study of retinal physiology and circuitry, yet there are unresolved issues regarding the organization of the photoreceptors and the photoreceptor mosaic. The rod and cone subtypes in the salamander retina were identified using a combination of morphological and immunocytochemical markers for specific rod and cone opsin epitopes. Because the visual pigment mechanisms present in the tiger salamander retina are well characterized and the antibodies employed in these studies are specific for particular rod and cone opsin epitopes, we also were able to identify the spectral class of the various rod and cone subtypes. Two classes of rods corresponding to the "red" and "green" rods previously reported in amphibian retinas were identified. In serial semithin section analyses, rods and cones comprised 62.4+/-1.4% and 37.6+/-1.4% of all photoreceptors, respectively. One rod type comprising 98.0+/-0.7% of all rods showed the immunological and morphological characteristics of "red" rods, which are maximally sensitive to middle wavelengths. The second rod subtype comprised 2.0+/-0.7% of all rods and possessed the immunological and morphological characteristics of "green" rods, which are maximally sensitive to short wavelengths. By morphology four cone types were identified, showing three distinct immunological signatures. Most cones (84.8+/-1.5% of all cones), including most large single cones, the accessory and principal members of the double cone, and some small single cones, showed immunolabeling by antisera that recognize long wavelength-sensitive cone opsins. A subpopulation of small single cones (8.4+/-1.7% of all cones) showed immunolabeling for short wavelength-sensitive cone opsin. A separate subpopulation of single cones which included both large and small types (6.8+/-1.4% of all cones) was identified as the UV-Cone population and showed immunolabeling by antibodies that recognize rod opsin epitopes. Analysis of flatmounted retinas yielded similar results. All photoreceptor types appeared to be distributed in all retinal regions. There was no obvious crystalline organization of the various photoreceptor subtypes in the photoreceptor mosaic.

Animals↗
PubMedDate not supplied

Cloning and characterization of six zebrafish photoreceptor opsin cDNAs and immunolocalization of their corresponding proteins.

Zebrafish (Danio rerio) represents an excellent genetic model for vertebrate visual system studies. Because the opsin proteins are ideal markers of specific photoreceptor cell types, we cloned six different zebrafish opsin cDNAs. Based on pairwise alignments and phylogenetic comparisons between the predicted zebrafish opsin amino acid sequences and other vertebrate opsins, the cDNAs encode rhodopsin, two different green opsins (zfgr1 and zfgr2), a red, a blue, and an ultraviolet opsin. Phylogenetic analysis indicates the zfgr1 protein occupies a well-resolved dendrogram branch separate from the other green opsins examined, while zebrafish ultraviolet opsin is closely related to the human blue- and chicken violet-sensitive proteins. Polyclonal antisera were generated against individual bacterial fusion proteins containing either the red, blue, or ultraviolet amino termini or the rod or green opsin carboxyl termini. Immunolocalization on adult zebrafish frozen sections demonstrates the green and red opsins are each expressed in different members of the double cone cell pair, the blue opsin is detected in long single cones, and the ultraviolet opsin protein is expressed in the short single cones. In 120-h postfertilization wholemounts, green, red, blue, and ultraviolet opsin-positive cells are detected in an orderly arrangement throughout the entire retina. The antibodies' photoreceptor-type specificity indicates they will be useful for characterizing both wild-type and mutant zebrafish retinas.

Amino Acid Sequence↗
PubMed1998

Mechanisms of spectral tuning in blue cone visual pigments. Visible and raman spectroscopy of blue-shifted rhodopsin mutants.

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket. We identified 12 amino acid residues in the human blue cone pigment that might induce the required green-to-blue opsin shift. The simultaneous substitution of nine of these sites in rhodopsin (M86L, G90S, A117G, E122L, A124T, W265Y, A292S, A295S, and A299C) shifted the absorption maximum from 500 to 438 nm, accounting for 2,830 cm-1, or 80%, of the opsin shift between rhodopsin and the blue cone pigment. Raman spectroscopy of mutant pigments shows that the dielectric character and architecture of the chromophore-binding pocket are specifically altered. An increase in the number of dipolar side chains near the protonated Schiff base of retinal increases the ground-excited state energy gap via long range dipole-dipole Coulomb interaction. In addition, the W265Y substitution causes a decrease in solvent polarizability near the chromophore ring structure. Finally, two substitutions on transmembrane helix 3 (A117G and E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the protonated Schiff base group with the surrounding dipolar groups and the counterion. Taken together, these results identify the amino acid side chains and the underlying physical mechanisms responsible for a majority of the opsin shift in blue visual pigments.

Amino Acid Sequence↗