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PubMed2000

The spatial and temporal expression of outer segment proteins during development of Macaca monkey cones.

PURPOSE: To characterize the spatial and temporal expression of key structural and phototransduction cascade proteins in the monkey cone outer segment (OS). METHODS: Retinas from Macaca monkeys from ages fetal day (Fd) 89 through adulthood were double labeled using immunofluorescence for short (S) or long/medium (L/M) wavelength-sensitive cone opsin and either a structural protein (peripherin) or a phototransduction cascade protein (alpha-transducin [alpha-T], phosphodiesterase [PDE], or rhodopsin kinase [RK]). The spatial and temporal patterns of expression for each protein at each age were determined and graphed as a percentage of retinal coverage. RESULTS: In both cone types, opsins and phototransduction proteins appear first in the fovea and last at the retinal edge. Peripherin appears concomitantly with opsin in both S and L/M cones, but S cones express peripherin and opsin 1 to 3 weeks before neighboring L/M cones. Alpha-T, PDE, and RK are expressed together in the L/M cone OS shortly after L/M opsin appears. Phototransduction proteins are not expressed in S cones until 1 to 3 weeks after the appearance of S opsin and at the same time that neighboring cones are expressing both L/M opsin and phototransduction proteins. CONCLUSIONS: The concomitant appearance of opsin and peripherin strongly suggests roles in promoting the structural integrity of the developing OS. Phototransduction cascade proteins appear in the developing OS at the same time as one another, but after opsin. The significant lag between their expression and that of S cone opsin indicates that phototransduction proteins are not essential for OS formation, nor does opsin expression trigger their expression. The different temporal but similar spatial expression patterns of phototransduction proteins within S and L/M cones suggests that some local signal(s) coordinates their appearance.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2004

Ancestral loss of short wave-sensitive cone visual pigment in lorisiform prosimians, contrasting with its strict conservation in other prosimians.

Mammals are basically dichromatic in color vision, possessing middle to long wave-sensitive (M/LWS) and the short wave-sensitive (SWS) cone opsins in the retina, whereas some nocturnal mammals lack functional SWS opsins. Prosimians, primitive primates consisting of three extant groups (Lorisiformes, Lemuriformes, and Tarsiiformes), include many nocturnal species. Among nocturnal prosimians, a species of lorisiforms, the greater galago (Otolemur crassicaudatus), is known to lack a functional SWS opsin gene, while lemuriforms and tarsiiforms appear to retain SWS opsins in the retina. It has not been established, however, whether the loss of SWS opsin is a universal phenomenon among lorisiforms and whether the functional SWS opsin genes of lemuriforms and tarsiiforms are under strict or relaxed selective constraint. To gain better insight into an association between nocturnality and loss of SWS function, we isolated and sequenced the SWS opsin genes from two species of lorisiforms, the slow loris (Nycticebus coucang; nocturnal) and the lesser galago (Galago senegalensis; nocturnal), and one species each of lemuriforms and tarsiiforms, the brown lemur (Eulemur fulvus; cathemeral) and the western tarsier ( Tarsius bancanus; nocturnal), respectively. Our sequence analysis revealed that (1) the SWS opsin gene was disrupted in the common ancestor of galagids and lorisids and (2) the rate of nonsynonymous nucleotide substitution has been kept significantly lower than that of synonymous substitution in tarsier and lemur, demonstrating the presence of strict selective constraint on the SWS opsin genes in tarsiiforms and lemuriforms.

Animals↗
PubMed1997

A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin.

Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation.

Animals↗
PubMed2003

Identification of circadian brain photoreceptors mediating photic entrainment of behavioural rhythms in lizards.

We have shown previously that in ruin lizards (Podarcis sicula) the ablation of all known photoreceptive structures (lateral eyes, pineal and parietal eye) in the same individual animal does not prevent entrainment of their circadian locomotor rhythms to light. The present study was aimed at identifying the circadian brain photoreceptors mediating entrainment. For this purpose, we looked for opsin expression in the brain by means of immunocytochemistry. Using anti-cone-opsin antiserum CERN 874 we have localized photoreceptors in the periventricular area of hypothalamus, near the third cerebral ventricle. We also cloned a brain opsin cDNA that, on the basis of the deduced amino acid sequence, appears to belong to the RH2 class of cone-opsins. We named the cloned cone-opsin Ps-RH2. To examine whether brain cone-opsins mediate photic entrainment of circadian locomotor rhythms, we performed post-transcriptional inactivation experiments by injecting an expression eukaryotic vector transcribing the antisense cone-opsin Ps-RH2 mRNA in the third cerebral ventricle of pinealectomized-retinectomized lizards previously entrained to a light-dark (LD) cycle. Injections of the antisense construct abolished photic entrainment of circadian locomotor rhythms of pinealectomized-retinectomized lizards to the LD cycle for 6-9 days. CERN 874 completely failed to label cells within the periventricular area of hypothalamus of brains injected with antisense construct. Thus, abolishment of photic entrainment is due to inactivation of endogenous brain cone-opsins mRNA. The present results demonstrate for the first time in a vertebrate that brain cone-opsins are part of a true circadian brain photoreceptor participating in photic entrainment of behavioural rhythms.

Amino Acid Sequence↗
PubMed2003

Molecular evolution of bat color vision genes.

The two suborders of bats, Megachiroptera (megabats) and Microchiroptera (microbats), use different sensory modalities for perceiving their environment. Megabats are crepuscular and rely on a well-developed eyes and visual pathway, whereas microbats occupy a nocturnal niche and use acoustic orientation or echolocation more than vision as the major means of perceiving their environment. In view of the differences associated with their sensory systems, we decided to investigate the function and evolution of color vision (opsin genes) in these two suborders of bats. The middle/long wavelength (M/L) and short wavelength (S) opsin genes were sequenced from two frugivorous species of megabats, Haplonycteris fischeri and Pteropus dasymallus formosus, and one insectivorous species of microbat, Myotis velifer. Contrary to the situation in primates, where many nocturnal species have lost the functional S opsin gene, both crepuscular and strictly nocturnal species of bats that we examined have functional M/L and S opsin genes. Surprisingly, the S opsin in these bats may be sensitive to UV light, which is relatively more abundant at dawn and at dusk. The M/L opsin in these bats appears to be the L type, which is sensitive to red and may be helpful for identifying fruits among leaves or for other purposes. Most interestingly, H. fischeri has a recent duplication of the M/L opsin gene, representing to date the only known case of opsin gene duplication in non-primate mammals. Some of these observations are unexpected and may provide insights into the effect of nocturnal life on the evolution of opsin genes in mammals and the evolution of the life history traits of bats in general.

Amino Acid Sequence↗
PubMed2003

A novel Xenopus SWS2, P434 visual pigment: structure, cellular location, and spectral analyses.

PURPOSE: The purpose of this study was to clone and characterize the green rod pigment in Xenopus laevis. METHODS: The cDNA for the Xenopus "green rod" pigment was cloned and sequenced from Xenopus retina mRNA by reverse transcription polymerase chain reaction and the 5' end cloned by rapid amplification of the cDNA ends. The cellular localization of the Xenopus opsin was determined by immunolabeling of flat-mounted retinas using a specific antibody against this opsin. Spectral properties of the expressed protein were determined by absorption spectroscopy using recombinant pigment. RESULTS: A novel Xenopus opsin cDNA containing a full-length coding region has been cloned and sequenced. The deduced amino acid sequence predicts a protein of 362 amino acids, forming 7 hydrophobic helices. Sequence analysis indicates that it belongs firmly to the SWS2 class of visual pigments and has 89%, 80%, and 75% amino acid sequence identity with bullfrog, tiger salamander, and newt SWS2 pigments, respectively. Staining of Xenopus retina with a Xenopus SWS2 opsin-specific polyclonal antibody demonstrated that the SWS2 pigment is expressed in green rods. After expression in COS cells, reconstitution with 11-cis retinal, and purification, the SWS2 pigment exhibits an absolute absorption maximum of 434 nm Thus, the name "SWS2, P434" was assigned for this opsin. The pigment decays rapidly in hydroxylamine in the dark, unlike the red rod pigment, rhodopsin. CONCLUSIONS: A novel green rod opsin cDNA has been cloned and sequenced from the retina of adult Xenopus laevis, which encodes a protein belonging to the SWS2 group of opsins. The expressed opsin possesses cone-opsin-like properties although it was identified only in the Xenopus green rod cells.

Amino Acid Sequence↗
PubMed1994

Precocious IRBP gene expression during mouse development.

PURPOSE: To determine if the time course for the onset of gene and protein expression for interphotoreceptor binding protein (IRBP) precedes that of opsin in the developing mouse retina. METHODS: Relative mRNA levels of the IRBP and opsin genes were determined in prenatal and postnatal retinal RNA with RNase protection analysis (RPA). To determine if IRBP and opsin protein expressions are differentially regulated, dissociated retinal cells from postnatal (P) days 2 and 3 mice, that were injected with BrdU, were then double-labeled with antibodies against BrdU and either opsin or IRBP. RESULTS: With RPA, IRBP mRNA was detected on embryonic (E) day 11 at the time of cone formation, whereas opsin mRNA was not detected until P0. It took until P3 for opsin expression to reach significant levels, whereas rods already appear during embryonic development. IBRP transcription preceded that of opsin because it rapidly increased from E13 to an early postnatal day. By P20, the expression levels of IRBP and opsin achieved constancy. Double antibody labeling revealed positive staining for both IRBP and BrdU as soon as 2 hours after injection, but it took until 40 hours for double positive staining for opsin and BrdU. CONCLUSION: Because only IRBP protein expression was observed before the last mitosis of the photoreceptor precursor cells, IRBP could be essential for retinal development.

Actins↗
PubMed1999

The nop-1 gene of Neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins.

Opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. Previously, genes encoding opsins had been identified in animals and the Archaea but not in fungi or other eukaryotic microorganisms. Here, we report the identification and mutational analysis of an opsin gene, nop-1, from the eukaryotic filamentous fungus Neurospora crassa. The nop-1 amino acid sequence predicts a protein that shares up to 81.8% amino acid identity with archaeal opsins in the 22 retinal binding pocket residues, including the conserved lysine residue that forms a Schiff base linkage with retinal. Evolutionary analysis revealed relatedness not only between NOP-1 and archaeal opsins but also between NOP-1 and several fungal opsin-related proteins that lack the Schiff base lysine residue. The results provide evidence for a eukaryotic opsin family homologous to the archaeal opsins, providing a plausible link between archaeal and visual opsins. Extensive analysis of Deltanop-1 strains did not reveal obvious defects in light-regulated processes under normal laboratory conditions. However, results from Northern analysis support light and conidiation-based regulation of nop-1 gene expression, and NOP-1 protein heterologously expressed in Pichia pastoris is labeled by using all-trans [3H]retinal, suggesting that NOP-1 functions as a rhodopsin in N. crassa photobiology.

Amino Acid Sequence↗
PubMed2003

Expression of photoreceptor-associated molecules during human fetal eye development.

PURPOSE: A characteristic feature of the human retina is the early differentiation of foveal cells followed by a central to peripheral wave of maturation. This can obscure the true onset of differentiation when regions other that the fovea are sampled, or when methods based on whole retina or whole eye tissue are employed, such as reverse transcription-polymerase chain technique (RT-PCR). In order to assess the suitability of RT-PCR based approaches during human retinal development and to gain insight into the developmental progression of photoreceptor differentiation and maturation in the human, we analyzed the expression of several photoreceptor-associated genes by immunocytochemical labeling (ICC) of the foveal region as well as by RT-PCR of total RNA from whole fetal eyes from different developmental stages. METHODS: Expression of phosphodiesterase beta (PDEB), interphotoreceptor binding protein (IRBP), tubby-like protein (TULP), short wavelength specific (S) opsin, long and medium wavelength specific (L/M) opsin, rod opsin and the transcription factors Crx and Nrl were assessed by RT-PCR from total RNA prepared from snap frozen intact human fetal eyes ranging from fetal week 9 (Fwk 9) to Fwk 18. ICC labeling was performed in a large number of eyes within an age group for IRBP, TULP, Nrl, S opsin, L/M opsin and rod opsin on frozen sections that included the fovea centralis. RESULTS: All ICC markers appeared first in or around the fovea. We detected PDEB and Crx expression as early as Fwk 10, by RT-PCR. TULP and IRBP were first observed with ICC in a small number of foveal cones at Fwk 9, although the first transcripts were not detected until Fwk 12. Nrl-positive nuclei appeared around the fovea by Fwk 11 and S opsin-positive cones by Fwk 12. L/M opsin-positive cones and rod opsin-positive rods were first detected between Fwk 15-16. In general, ICC labeling in the fovea was present for most genes up to 2 weeks before the corresponding transcripts could be successfully amplified by RT-PCR from whole eye tissue. CONCLUSIONS: Our results indicate that in order to pinpoint exactly when and where a molecule appears, ICC labeling of the fovea is a more reliable indicator. RT-PCR was prone to underestimate the exact onset of expression of the molecules tested, yet it faithfully recapitulated the sequence in which they appeared. In addition, our data show that in the human fetal retina, Crx and Nrl are both expressed when the first rod photoreceptors are being generated. This agrees well with previous in vitro results suggesting a synergistic action of both proteins during differentiation of human rod photoreceptors.

Biomarkers↗
PubMed1994

Photoreceptor outer segment development in Xenopus laevis: influence of the pigment epithelium.

Opsin gene expression, synthesis, and photoreceptor outer segment morphology were evaluated during retinal development in Xenopus laevis. Retinal rudiments were harvested during in vivo development from embryonic stages 31 through 46 or were allowed to develop in vitro after removal from stage 33/34 embryos for 1, 2, or 3 days either with or without an investing pigment epithelium. Opsin mRNA was detected at stage 33/34 and the transcript level increased until stage 40 and remained at this level through stage 46. Opsin was first detected at stage 37/38 and progressively increased through stage 46. Rudimentary photoreceptor outer segment membranes occasionally appeared as early as stage 33/34 and they gradually increased in length, forming well-defined stacks of collapsed membranous saccules (discs) during in vivo development. The maturation of eye rudiments in culture was followed to determine how closely in vivo and in vitro development compare and to examine the ability of photoreceptors to differentiate when maintained in the absence of an overlying pigment epithelium (PE) layer. With the PE present, opsin mRNA as well as opsin content steadily increased over the entire culture period. After 1 day of culture, short cilia with minimal amounts of outer segment membranous material were present. By Day 3, the degree of outer segment differentiation corresponded morphologically to approximately stage 43 of in vivo development. When cultured in the absence of an investing PE, the opsin mRNA level increased minimally during the 3 days in culture. Opsin content increased, yet the relative amount was approximately 50% less than that present in retinas developing in the presence of the PE. Membranous material was elaborated; however, the outer segments appeared to be highly disorganized and formed whorl-like structures rather than the normal stacked disc morphology. These results suggest that the PE may be involved in regulating opsin at the transcriptional and/or translational levels and also participates in the organization of rod outer segment membranes.

Animals↗
PubMed1986

The acylation of rat rhodopsin in vitro and in vivo.

Rat retinas were incubated with [3H] palmitate and [14C] leucine and subsequently detergent-extracted. Glycoproteins were isolated on Con A-Sepharose columns and separated by gel electrophoresis. Leucine labeled the newly synthesized opsin but palmitate was esterified to both mature rhodopsin and newly synthesized opsin which migrated more slowly because of its untrimmed oligosaccharide chains. Crude rod outer segments were found to contain most of the palmitate-labeled mature rhodopsin, while the retinal debris contained most of the doubly labeled newly synthesized opsin. Homogenization of double-labeled retinas followed by centrifugation in a linear sucrose gradient gave rise to several bands of particles including purified rod outer segments, a Golgi-enriched fraction and a pellet enriched in endoplasmic reticulum. Newly synthesized opsin was first found in the pellet at the earliest incubation times and subsequently appeared in the Golgi fraction and finally in the rod outer segments. Palmitate-labeled mature rhodopsin was found only in the rod outer segments. It appeared at the earliest time points and increased with time. Thus the acylation of opsin occurs in the endoplasmic reticulum, shortly after polypeptide synthesis, and in the rod outer segments, the latter possibly as an exchange reaction. Most of the newly synthesized opsin remained in the pellet and did not pass through the Golgi to the rod outer segments. Intravitreal injection of [3H] palmitate and [14C] leucine gave rise to doubly labeled opsin that appeared to remain untrimmed for at least 6 hr in vivo. After 17 hr, both labels were found only in mature rhodopsin, thus accumulation of new molecules in the endoplasmic reticulum may occur in vivo. In addition, leucine maximally labeled the opsin-rhodopsin pool early in the first day whereas palmitate did not maximally label rhodopsin until 2- or 3 days post injection. Moreover, while leucine label was lost at day 9 because of rod outer-segment renewal and shedding, the palmitate label in rhodopsin remained unchanged. Thus, palmitate labeling in vivo reflects the pattern seen in vitro with a prolonged equilibration of rod outer-segment rhodopsin with the fatty-acid pool, probably mediated by a fatty acyl exchange reaction.

Acylation↗
PubMed1996

Opsin/all-trans-retinal complex activates transducin by different mechanisms than photolyzed rhodopsin.

In rhodopsin, the 11-cis-retinal chromophore forms a complex with Lys296 of opsin via a protonated Schiff base. Absorption of light initiates the activation of rhodopsin by cis/trans photoisomerization of retinal. Thermal relaxation through different intermediates leads into the metarhodopsin states which bind and activate transducin (Gt) and rhodopsin kinase (RK). all-trans-Retinal also recombines with opsin independent of light, forming activating species of the receptor. In this study, we examined the mechanism by which all-trans-retinal activates opsin. To exclude other amines except active site Lys296 from formation of Schiff bases, we reductively methylated rhodopsin (PM-rhodopsin), which we then bleached to generate PM-opsin. Using spectroscopic methods and a Gt activation assay, we found that all-trans-retinal interacted with PM-opsin, producing a noncovalent complex that activated Gt. The residual nucleotide exchange in Gt catalyzed by opsin was approximately 1/250 lower relative to that of photoactivated rhodopsin (pH 8.0, 23 degrees C). Addition of equimolar all-trans-retinal led to an occupancy of one-tenth of the putative retinal binding site(s) of opsin and enhanced the Gt activation rate 2-fold. When the concentration of all-trans-retinal was increased to saturation, the Gt activation rate of the opsin/all-trans-retinal complex was approximately 1/33 lower compared to that of photoactivated rhodopsin. We conclude that all-trans-retinal can form a noncovalent complex with opsin that activates Gt by different mechanisms than photolyzed rhodopsin.

Animals↗
PubMed2006

Exploring the molecular mechanism for color distinction in humans.

We examine here the role of the red, green, and blue human opsin structures in modulating the absorption properties of 11-cis-retinal bonded to the protein via a protonated Schiff base (PSB). We built the three-dimensional structures of the human red, green, and blue opsins using homology modeling techniques with the crystal structure of bovine rhodopsin as the template. We then used quantum mechanics (QM) combined with molecular mechanics (MM) (denoted as QM/MM) techniques in conjunction with molecular dynamics to determine how the room temperature molecular structures of the three human color opsin proteins modulate the absorption frequency of the same bound 11-cis-retinal chromophore to account for the differences in the observed absorption spectra. We find that the conformational twisting of the 11-cis-retinal PSB plays an important role in the green to blue opsin shift, whereas the dipolar side chains in the binding pocket play a surprising role of red-shifting the blue opsin with respect to the green opsin, as a fine adjustment to the opsin shift. The dipolar side chains play a large role in the opsin shift from red to green.

Amino Acid Sequence↗
PubMed1958

The rhodopsin system of the squid.

Squid rhodopsin (lambda(max) 493 mmicro)-like vertebrate rhodopsins-contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (lambda(max) 500 mmicro) near neutrality, yellow (lambda(max) 380 mmicro) in alkaline solution. The two forms-acid and alkaline metarhodopsin-are interconverted according to the equation, Alkaline metarhodopsin + H(+) right harpoon over left harpoonacid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.

Animals↗
PubMed2003

Gene duplication and spectral diversification of cone visual pigments of zebrafish.

Zebrafish is becoming a powerful animal model for the study of vision but the genomic organization and variation of its visual opsins have not been fully characterized. We show here that zebrafish has two red (LWS-1 and LWS-2), four green (RH2-1, RH2-2, RH2-3, and RH2-4), and single blue (SWS2) and ultraviolet (SWS1) opsin genes in the genome, among which LWS-2, RH2-2, and RH2-3 are novel. SWS2, LWS-1, and LWS-2 are located in tandem and RH2-1, RH2-2, RH2-3, and RH2-4 form another tandem gene cluster. The peak absorption spectra (lambdamax) of the reconstituted photopigments from the opsin cDNAs differed markedly among them: 558 nm (LWS-1), 548 nm (LWS-2), 467 nm (RH2-1), 476 nm (RH2-2), 488 nm (RH2-3), 505 nm (RH2-4), 355 nm (SWS1), 416 nm (SWS2), and 501 nm (RH1, rod opsin). The quantitative RT-PCR revealed a considerable difference among the opsin genes in the expression level in the retina. The expression of the two red opsin genes and of three green opsin genes, RH2-1, RH2-3, and RH2-4, is significantly lower than that of RH2-2, SWS1, and SWS2. These findings must contribute to our comprehensive understanding of visual capabilities of zebrafish and the evolution of the fish visual system and should become a basis of further studies on expression and developmental regulation of the opsin genes.

Amino Acid Sequence↗
PubMed2001

Topography of different photoreceptor cell types in the larval retina of Atlantic halibut (Hippoglossus hippoglossus).

The identities of single cone cells in the retina of Atlantic halibut (Hippoglossus hippoglossus) larvae were studied by in situ hybridisation using RNA probes for the five different halibut opsins. Four different cone opsins (ultraviolet-, blue-, green- and red-sensitive) are expressed in Atlantic halibut at the end of the yolk-sac period, whereas rod opsin is expressed later in development. Photoreceptor cells expressing ultraviolet-sensitive opsin are found only in the ventral retina, presumably to optimise detection of the downwelling ultraviolet light. The majority of the photoreceptors (approximately 90%) in the retina express green-sensitive opsin and its distribution shows no regional differences. In contrast, blue- and red-sensitive opsins are expressed much less frequently (in approximately 10% of photoreceptors), although these two opsins are also found over the entire retina. The expression patterns of the different visual pigments indicate some form of mosaic expression in the single-coned larval retina, and this is reminiscent of the square mosaic expression found in post-metamorphic Atlantic halibut. These findings suggest plasticity in green-opsin-expressing cells during development, resulting in a square mosaic expression pattern.

Animals↗
PubMed1996

Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa.

The rhodopsin mutants P23H and G188R, identified in autosomal dominant retinitis pigmentosa (ADRP), and the site-specific mutants D190A and DeltaY191-Y192 were expressed in COS cells from synthetic mutant opsin genes containing these mutations. The proteins expressed from P23H and D190A partially regenerated the rhodopsin chromophore with 11-cis-retinal and were mixtures of the correctly folded (retinal-binding) and misfolded (non-retinal-binding) opsins. The mixtures were separated into pure, correctly folded mutant rhodopsins and misfolded opsins. The proteins expressed from the ADRP mutant G188R and the mutant DeltaY191-Y192 were composed of totally misfolded non-retinal-binding opsins. Far-UV CD spectra showed that the correctly folded mutant rhodopsins had helical content similar to that of the wild-type rhodopsin, whereas the misfolded opsins had helical content 50-70% of the wild type. The near-UV CD spectra of the misfolded mutant proteins lack the characteristic band pattern seen in the wild-type opsin, indicative of a different tertiary structure. Further, whereas the folded mutant rhodopsins were essentially resistant to trypsin digestion, the misfolded opsins were degraded to small fragments under the same conditions. Therefore, the misfolded opsins appear to be less compact in their structures than the correctly folded forms. We suggest that most, if not all, of the point mutations in the intradiscal domain identified in ADRP cause partial or complete misfolding of rhodopsin.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of solubilised dark-adapted rhodopsin. Insights into the activation of rhodopsin kinase.

A protocol for the separation of phosphorhodopsin from phospho-opsin has been developed. The method takes advantage of the finding that, while 0.5% N,N-dimethyldodecylamine-N-oxide completely solubilises membrane-embedded phosphorhodopsin, at this concentration of detergent, phospho-opsin is only sparingly soluble. Phosphorhodopsin solubilised in this manner may be freed from contaminant phospho-opsin by chromatography on hydroxyapatite. Using this method, the rhodopsin-kinase-catalysed phosphorylation of photoexcited rhodopsin and native rhodopsin was studied in rod outer-segment membranes at different levels of bleaching. Prior to analysis of the phosphorylation mixture, the phosphorylated form of photoexcited rhodopsin was converted into phospho-opsin by treatment with NH2OH. It was found that, while at a 5% bleach level the amount of phosphorhodopsin produced was 15% that of phospho-opsin, at 60% bleaching the phosphorhodopsin was less than 1% of phospho-opsin. The phosphorylation reaction under different bleaching conditions was also studied in a completely soluble system (using 2% dodecyl maltoside) and the pattern of phosphate incorporation into rhodopsin versus opsin was identical to that in the membrane system. We have previously proposed that rhodopsin kinase normally exists in an inactive form and is only activated following interaction with photoexcited rhodopsin. The present work strengthens this conclusion and also shows that, following activation, the kinase preferentially phosphorylates photoexcited rhodopsin but can also act upon unbleached rhodopsin. Two possible mechanisms for the activation of the kinase are considered. From the distribution of phosphorhodopsin and phospho-opsin at different bleaching levels, the relative rates of the phosphorylation of photoexcited rhodopsin (kR*) and rhodopsin (kR) were calculated. kR*/kR values for the membrane system of 71 +/- 20 and, for the solubilised system, of 80 +/- 19 were obtained. The algebraic equation used to obtain these values highlights the fact that the ratio of the concentrations of the two substrates, photoexcited rhodopsin and rhodopsin, in a sample, determines the final distribution of phosphate between bleached and unbleached rhodopsin. This conclusion may contribute to the understanding of 'high-gain' phosphorylation observed previously.

Animals↗