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PubMed1994

Relief of opsin desensitization and prolonged excitation of rod photoreceptors by 9-desmethylretinal.

The 9-methyl group of 11-cis-retinal plays a crucial role in photoexcitation of the visual pigment rhodopsin. A hydrogen-substituted analogue, 11-cis-9-desmethylretinal, combines with opsin to form a pigment that produces abnormal photoproducts and diminished activation of the GTP-binding protein transducin in vitro. We have measured the formation of this analogue pigment in bleached salamander rods and determined the size and shape of its quantal response. In addition, we have characterized the influence of opsin and newly formed analogue pigment on the quantal response to native porphyropsin. We find that, as 11-cis-9-desmethylretinal combines with opsin in bleached rods, the amplitude of the quantal response from residual native pigment is elevated by approximately 7.5-fold to 0.15 +/- 0.09 pA, a value close to the amplitude of the quantal response before bleach (0.31 +/- 0.10 pA). When activated by light, the new analogue pigment produces a quantal response that is approximately 30-fold smaller and decays approximately 5 times more slowly than that of native pigment in unbleached cells. We conclude that the 9-methyl group of retinal is not critical for conversion of opsin to its nondesensitizing state but that it is critical for the normal processes of activation and deactivation of metarhodopsin that give rise to the quantal response.

Action Potentials↗
PubMed1996

Structure and function in rhodopsin: expression of functional mammalian opsin in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been investigated for expression of mammalian opsin as an alternative to the currently used expression in COS-1 mammalian cells. The synthetic opsin gene was placed under the control of the inducible promoter GAL1 in the multicopy yeast/ Escherichia coli shuttle vector YEpRF1. Transformation of a GAL+ S. cerevisiae strain with the vector and growth of galactose-induced cultures to saturation showed the production of 2.0 +/- 0.5 mg of opsin from about 10(10) cells by ELISA. The addition of 11-cis-retinal to either cell spheroplasts or lysed cells showed that a fraction (2-4%) of the total expressed opsin reconstituted to rhodopsin. This fraction was purified to homogeneity and was shown to be fully functional and indistinguishable from bovine rhodopsin by the following criteria: (i) UV-visible absorption spectra, (ii) the formation of metarhodopsin II and its rate of decay, and (iii) initial rate of transducin activation as measured by the formation of a complex between transducin (alpha subunit) and guanosine 5'-[gamma-[35S]thio]triphosphate. The purified fraction was homogeneously glycosylated. However, glycosylation was distinct from that of bovine rhodopsin as judged by mobility on SDS/PAGE and endoglycosidase H sensitivity.

Animals↗
PubMed1999

The endogenous chromophore of retinal G protein-coupled receptor opsin from the pigment epithelium.

The recent identification of nonvisual opsins has revealed an expanding family of vertebrate opsin genes. The retinal pigment epithelium (RPE) and Müller cells contain a blue and UV light-absorbing opsin, the RPE retinal G protein-coupled receptor (RGR, or RGR opsin). The spectral properties of RGR purified from bovine RPE suggest that RGR is conjugated in vivo to a retinal chromophore through a covalent Schiff base bond. In this study, the isomeric structure of the endogenous chromophore of RGR was identified by the hydroxylamine derivatization method. The retinaloximes derived from RGR in the dark consisted predominantly of the all-trans isomer. Irradiation of RGR with 470-nm monochromatic or near-UV light resulted in stereospecific isomerization of the bound all-trans-retinal to an 11-cis configuration. The stereospecificity of photoisomerization of the all-trans-retinal chromophore of RGR was lost by denaturation of the protein in SDS. Under the in vitro conditions, the photosensitivity of RGR is at least 34% that of bovine rhodopsin. These results provide evidence that RGR is bound in vivo primarily to all-trans-retinal and is capable of operating as a stereospecific photoisomerase that generates 11-cis-retinal in the pigment epithelium.

Animals↗
PubMed2000

Diffusible ligand all-trans-retinal activates opsin via a palmitoylation-dependent mechanism.

In rhodopsin's function as a photoreceptor, 11-cis-retinal is covalently bound to Lys(296) via a protonated Schiff base. 11-cis/all-trans photoisomerization and relaxation through intermediates lead to the metarhodopsin II photoproduct, which couples to transducin (G(t)). Here we have analyzed a different signaling state that arises from noncovalent binding of all-trans-retinal (atr) to the aporeceptor opsin and enhances the very low opsin activity by several orders of magnitude. Like with metarhodopsin II, coupling of G(t) to opsin-atr is sensitive to competition by synthetic peptides from the COOH termini of both G(t)alpha and G(t)gamma. However, atr does not compete with 11-cis-retinal incorporation into the Lys(296) binding site and formation of the light-sensitive pigment. Blue light illumination fails to photorevert opsin-atr to the ground state. Thus noncovalently bound atr has no access to the light-dependent binding site and reaction pathway. Moreover, in contrast to light-dependent signaling, removal of the palmitoyl anchors at Cys(322) and Cys(323) in the rhodopsin COOH terminus impairs the atr-stimulated activity. Repalmitoylation by autoacylation with palmitoyl-coenzyme A restores most of the original activity. We hypothesize that the palmitoyl moieties are part of a second binding pocket for the chromophore, mediating hydrophobic interactions that can activate a large part of the catalytic receptor/G-protein interface.

Acylation↗
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↗
PubMed2006

Opsin oligomerization in a heterologous cell system.

Using bioluminescence resonance energy transfer (BRET) we studied opsin oligomerization in heterologous expression systems and quantitatively assessed its oligomerization state. BRET2 saturation and competition experiments were performed with live COS-7 cells expressing Rluc-and GFP2-tagged receptor constructs. BRET2 saturation curves obtained were hyperbolic, and the calculated oligomerization state (N = 1 for dimers) suggested that opsin (N = 1.34 +/- 0.25) forms higher oligomers. Very high BRET2 values obtained for the opsin homo-dimer pair indicated a large energy transfer efficiency (E) and for cases where E >> 0.1 a modified saturation curve was proposed. The existence of homo-dimer complexes was additionally supported by competition assay results and was also observed in HEK-293 cells. Furthermore, evidence was provided for homo-and hetero-dimerization of family A (beta2-adrenergic) and B (gastric inhibitory polypeptide, GIP) receptors. In summary, these experiments demonstrate homo-and hetero-dimerization for opsin, beta 2-adrenergic, and GIP receptors.

Animals↗
PubMed1997

Site-directed mutagenesis of highly conserved amino acids in the first cytoplasmic loop of Drosophila Rh1 opsin blocks rhodopsin synthesis in the nascent state.

The cytoplasmic surface of Drosophila melanogaster Rh1 rhodopsin (ninaE) harbours amino acids which are highly conserved among G-protein-coupled receptors. Site-directed mutations which cause Leu81Gln or Asn86Ile amino acid substitutions in the first cytoplasmic loop of the Rh1 opsin protein, are shown to block rhodopsin synthesis in the nascent, glycosylated state from which the mutant opsin is degraded rapidly. In mutants Leu81Gln and Asn86Ile, only 20-30% and <2% respectively, of functional rhodopsins are synthesized and transported to the photoreceptive membrane. Thus, conserved amino acids in opsin's cytoplasmic surface are a critical factor in the interaction of opsin with proteins of the rhodopsin processing machinery. Photoreceptor cells expressing mutant rhodopsins undergo age-dependent degeneration in a recessive manner.

Amino Acid Sequence↗
PubMedDate not supplied

Strategies for identification of mutations causing hereditary retinal diseases in dogs: evaluation of opsin as a candidate gene.

Progressive retinal atrophy (PRA), like retinitis pigmentosa (RP) in man, represents a clinical classification grouping together a variety of hereditary diseases of the visual cells which have broadly similar clinical characteristics. At least six distinct autosomal recessive and one X-linked retinal disease locus have been identified. As one of the strategies to look for the gene defect causing the different forms of PRA, we are examining first the most promising candidate genes. These include those coding for photoreceptor-specific structural proteins and enzymes of the phototransduction pathway, especially those reported to cause RP. Preeminent among these candidates is the gene for rod opsin, in which multiple causative mutations have been identified in both dominant and recessive forms of RP. In addition, mutations in this gene are also causally associated with congenital stationary night blindness (CSNB) in man. We have used two strategies to examine the rod opsin gene for association with inherited retinal disease in dogs: (1) linkage to determine cosegregation of the disease locus with an intragenic polymorphic marker in the opsin gene in those breeds where suitable informative pedigrees were available; and (2) scanning the coding sequence of the gene in cases where only a limited number of affected or obligate heterozygous samples were available for a breed. We conclude that mutations in the rod opsin gene are not associated with PRA or CSNB in the 11 different dog breeds tested.

Animals↗
PubMed1995

Investigation of the role of opsin gene polymorphism in generalized progressive retinal atrophies in dogs.

The generalized progressive retinal atrophies (gPRAs) form a group of retinal degenerations of pedigree dogs and cats, which have a variety of genetic origins (mostly unknown). We have examined the opsin gene for polymorphisms in several breeds of pedigree dog suffering from distinct forms of gPRA, by methods including single-strand conformation polymorphism analysis, microsatellite analysis and direct sequencing. The breeds examined included the Tibetan terrier, the miniature schnauzer, the Irish setter, the miniature poodle, the Labrador retriever and the English cocker spaniel, as well as individuals from breeds in which PRA has not been described and of mixed breed. Individuals from each of the named breeds suffering from PRA were compared with clinically normal dogs. Two polymorphisms were found. One, segregating within the Tibetan terrier population, but not seen in other breeds, was a synonymous transition at nucleotide position 780 in exon 3. Inheritance of this polymorphism suggests that opsin is unlikely to contain mutations causative of gPRA in this breed. The other polymorphism occurred between all miniature schnauzers examined and dogs of other breeds. It consisted of a single base insertion in intron 2. No polymorphisms in the opsin sequence were detected in any other breed. DNA sequencing allowed rigorous exclusion of mutations in opsin as a cause of gPRA in miniature poodles, English cocker spaniels or Labrador retrievers.

Animals↗
PubMed1991

Quantum yield of CHAPSO-solubilized rhodopsin and 3-hydroxy retinal containing bovine opsin.

The quantum yields of bleaching for two artificial pigments, bovine opsin combined with (3R)-3-hydroxy retinal or (3R,S)-3-methoxy retinal, were determined in comparison to the value for regenerated bovine rhodopsin. Regeneration of the visual pigments was performed by incubation of 3-[(3-Cholamidopropyl)-dimethylammonio]-2-hydroxy-1- propanesulfonate (CHAPSO)-solubilized opsin with the 11-cis isomers of retinal and the respective retinal derivatives. The extinction coefficients of the pigments in CHAPSO were determined to 35,000 M-1 cm-1 (native rhodopsin), 35,300 M-1 cm-1 (regenerated rhodopsin) and 34,500 M-1 cm-1 (3-OH retinal opsin). With respect to rhodopsin (lambda max: 500 nm), the pigments carrying the substituted chromophores exhibit blue shifted absorbance maxima (3-hydroxy and 3-methoxy retinal opsin: 488 nm). In parallel experiments under absolutely identical conditions we find related to the value of CHAPSO solubilized rhodopsin (identical to 1) a quantum efficiency of bleaching for the 3-hydroxy pigment of 1.2.

Animals↗
PubMed1990

Time course of opsin expression in developing rod photoreceptors.

We have investigated the time course of rod photoreceptor determination in the goldfish retina. Rod precursor cells located in the outer nuclear layer of the mature retina continuously generate rod photoreceptors. In this study, we asked when rod precursor cells begin to express opsin, which would signal their commitment to the rod pathway of differentiation. There are three possibilities: a rod precursor could express opsin while still mitotic, at or shortly after the terminal mitosis but before differentiation, or during differentiation. We used immunocytochemistry with antibodies against bromodeoxyuridine, BrdU (a thymidine analogue) and against opsin to determine when during the mitotic history of a cell the expression of opsin first occurred, taking a double labelled cell to be evidence of commitment to the rod cell fate. We found that the first double labelled cells appeared at 4 days after BrdU injection. The number of double labelled cells increased to peak at 10 days, and then fell. These results support the hypothesis that dividing rod precursor cells are probably multipotent stem cells not committed to the rod cell fate.

Animals↗
PubMed1997

Defective phototransductive disk membrane morphogenesis in transgenic mice expressing opsin with a mutated N-terminal domain.

Retinitis pigmentosa is a heterogeneous group of inherited retinal disorders in which the photoreceptor cells degenerate. A line of transgenic mice expresses a mutant opsin gene that encodes three missense mutations near the amino terminus, including P23H, which is the basis for a common form of dominant retinitis pigmentosa. By studying the photoreceptor cells of these mice and their normal littermates, we found that: (1) opsin was routed correctly, (2) the concentration of opsin in the disk membranes appeared normal by freeze fracture analysis, (3) the amount of disk membrane shedding was normal, but (4) the basal disks of the outer segments were disorganized, indicating defective disk membrane morphogenesis. Defective disk membrane morphogenesis appears to result in the formation of fewer mature disks, thus accounting for observed gradual shortening of the photoreceptor outer segments with age. We suggest that abnormal disk membrane morphogenesis is the primary cellular defect that leads to blindness, and that it arises from the inability of nascent disk membranes, containing normal and mutant opsin, to interact normally with each other.

Animals↗
PubMed2006

Color discrimination in the red range with only one long-wavelength sensitive opsin.

The basic precondition for color vision is the presence of at least two receptor types with different spectral sensitivities. The sensitivity of a receptor is mostly defined by the opsin-based visual pigment expressed in it. We show here, through behavioral experiments, that the nymphalid butterfly Heliconius erato, although it expresses short and medium wavelength opsins and only one long wavelength opsin, discriminates colors in the long-wavelength range (590 nm, 620 nm and 640 nm), whereas another nymphalid, Vanessa atalanta, despite having color vision, is unable to do so. In the eyes of H. erato we identified filtering pigments very close to the rhabdom which differ between ommatidia and produce the yellow and red ommatidial reflection seen under orthodromic illumination. The eyes of V. atalanta lack the filtering pigments, and reflect a homogeneous orange. We hypothesize that the filtering pigments found in the eyes of H. erato may shift the spectral sensitivity peak of the long wavelength receptors in some ommatidia towards longer wavelengths. The comparison of the signals between the two new receptor types makes color discrimination in the red range possible. To our knowledge, this is the first behavioral proof of color vision based on receptors expressing the same opsin.

Animals↗
PubMed1997

Three opsin-encoding cDNAS from the compound eye of Manduca sexta.

Three distinct opsin-encoding cDNAs, designated MANOP1, MANOP2 and MANOP3, were isolated from the retina of the sphingid moth Manduca sexta. MANOP1 codes for a protein with 377 amino acid residues. It is similar in sequence to members of a phylogenetic group of long-wavelength-sensitive arthropod photopigments, most closely resembling the opsins of ants, a praying mantis, a locust and the honeybee. MANOP2 and MANOP3 opsins have 377 and 384 residues respectively. They belong to a related group of insect visual pigments that include the ultraviolet-sensitive rhodopsins of flies as well as other insect rhodopsins that are also thought to absorb at short wavelengths. The retina of Manduca sexta contains three rhodopsins, P520, P450 and P357, with absorbance peaks, respectively, at green, blue and ultraviolet wavelengths. There is evidence that MANOP1 encodes the opsin of P520. We suggest that MANOP2 encodes P357 and that MANOP3, representing a class of blue-sensitive insect photopigments, encodes P450.

Amino Acid Sequence↗
PubMed1989

A longitudinal study on the expression of the opsin gene in the degenerating retina of C3H/He mice.

By a combination of immunohistochemistry and in situ hybridization using a 35S-labeled opsin cDNA probe, it was revealed in rd mutant C3H/He mice that opsin mRNA is not detectable in photoreceptor cells which still exhibit opsin-immunoreactivity at later stages of retinal degeneration. This indicates that the photoreceptor cells of rd mutant mice cease to express the opsin gene at earlier stages of retinal degeneration.

Animals↗
PubMed1999

Encephalopsin: a novel mammalian extraretinal opsin discretely localized in the brain.

We have identified a mammalian opsin, encephalopsin, that shows strong and specific expression in the brain. Encephalopsin defines a new family of opsins and shows highest homology to vertebrate retinal and pineal opsins. Encephalopsin is highly expressed in the preoptic area and paraventricular nucleus of the hypothalamus, both regions implicated in encephalic photoreception in nonmammalian vertebrates. In addition, encephalopsin shows highly patterned expression in other regions of the brain, being enriched in selected regions of the cerebral cortex, cerebellar Purkinje cells, a subset of striatal neurons, selected thalamic nuclei, and a subset of interneurons in the ventral horn of the spinal cord. Rostrocaudal gradients of encephalopsin expression are present in the cortex, cerebellum, and striatum. Radial stripes of encephalopsin expression are seen in the cerebellum. In the cortex and cerebellum, encephalopsin expression is considerably higher and more highly patterned in the adult than in the neonate. Encephalopsin is the first putative extraocular opsin identified in mammals and may play a role in encephalic photoreception.

Amino Acid Sequence↗
PubMed1999

Photoreceptor autophagy: effects of light history on number and opsin content of degradative vacuoles.

PURPOSE: To investigate whether regulation of rhodopsin levels as a response to changed lighting environment is performed by autophagic degradation of opsin in rod inner segments (RISs). METHODS: Groups of albino rats were kept in 3 lux or 200 lux. At 10 weeks of age, one group was transferred from 3 lux to 200 lux, another group was switched from 200 lux to 3 lux, and two groups remained in their native lighting (baselines). Rats were killed at days 1, 2, and 3 after switching. Another group was switched from 3 lux to 200 lux, and rats were killed at short intervals after the switch. Numbers of autophagic vacuoles (AVs) in RISs were counted, and immunogold labeling was performed for opsin and ubiquitin in electron microscopic sections. RESULTS: The number of AVs increased significantly after switching from 3 lux to 200 lux at days 1 and 2 and declined at day 3, whereas the reverse intensity change did not cause any increase. Early time points after change from 3 lux to 200 lux showed a significant increase of AVs 2 and 3 hours after switching. Distinct opsin label was observed in AVs of rats switched to 200 lux. Ubiquitin label was present in all investigated specimens and was also seen in AVs especially in 200-lux immigrants. CONCLUSIONS: Earlier studies had shown that an adjustment to new lighting environment is performed by changes in rhodopsin levels in ROSs. Autophagic degradation of opsin or rhodopsin may subserve, at least in part, the adaptation to abruptly increased habitat illuminance by removing surplus visual pigment.

Adaptation, Ocular↗
PubMed1990

Mouse opsin. Gene structure and molecular basis of multiple transcripts.

The single copy mouse opsin gene produces five major transcripts, varying in size from 1.7 to 5.1 kilobases. The mRNAs are present at levels that vary over 2 orders of magnitude and can be detected as early as postnatal day 1. Each of the transcripts is polyadenylated and can be identified in polysome-bound RNA, suggesting that each is translated in vivo. To elucidate the molecular basis of this complex transcription pattern, we have characterized genomic fragments covering the entire mouse opsin gene, including several kilobases of 5'- and 3'-flanking regions. Transcription initiates at a single site 97 base pairs upstream of the translation start codon. Northern hybridization with exon- and intron-specific probes demonstrated that the various transcripts are not generated by partial or alternative splicing. Sequence analysis of the 3' end of the gene showed the presence of multiple polyadenylation signals. Analysis by polymerase chain reaction of the 3' end of opsin cDNA demonstrated that the complex transcription pattern originated from the selective use of these polyadenylation sites, generating transcripts that differ only in the length of the 3'-untranslated region. Transcript heterogeneity similar to that observed in mouse was also found in rat and, to a lesser degree, in human and frog opsin mRNAs.

Amino Acid Sequence↗