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PubMed1990

Bathoiodopsin, a primary intermediate of iodopsin at physiological temperature.

Measurement of the primary photochemical reaction of iodopsin, a chicken red-sensitive cone visual pigment, was carried out at room temperature by using picosecond (ps) laser photolysis. Excitation of iodopsin with a ps green pulse (pulse width, 21 ps) caused the instantaneous formation of a bathochromic product, which was stable on a ps time scale. This product may correspond to "bathoiodopsin," which was detected by low-temperature spectrophotometry. Although bathoiodopsin produced at the temperature of liquid nitrogen or helium reverted to the original pigment (iodopsin) on warming (above -170 degrees C), the bathoiodopsin produced at physiological temperature decayed to all-trans-retinal and R-photopsin (the protein moiety of iodopsin) presumably through several intermediates. The absorption maximum of bathoiodopsin at room temperature was at 625 nm, a wave-length slightly shorter than that measured at low temperature (lambda max, 640 nm). The extinction coefficient of bathoiodopsin at room temperature was lower than that at low temperature and close to that of the original iodopsin at room temperature.

Animals↗
PubMed1991

Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa.

We searched for point mutations in every exon of the rhodopsin gene in 150 patients from separate families with autosomal dominant retinitis pigmentosa. Including the 4 mutations we reported previously, we found a total of 17 different mutations that correlate with the disease. Each of these mutations is a single-base substitution corresponding to a single amino acid substitution. Based on current models for the structure of rhodopsin, 3 of the 17 mutant amino acids are normally located on the cytoplasmic side of the protein, 6 in transmembrane domains, and 8 on the intradiscal side. Forty-three of the 150 patients (29%) carry 1 of these mutations, and no patient has more than 1 mutation. In every family with a mutation so far analyzed, the mutation cosegregates with the disease. We found one instance of a mutation in an affected patient that was absent in both unaffected parents (i.e., a new germ-line mutation), indicating that some "isolate" cases of retinitis pigmentosa carry a mutation of the rhodopsin gene.

Amino Acid Sequence↗
PubMed1992

Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subunit.

The beta subunit of the cGMP phosphodiesterase (PDE) gene has been identified as the candidate gene for retinal degeneration in the rd mouse. To study the molecular mechanisms underlying degeneration and the potential for gene repair, we have expressed a functional bovine cGMP PDE beta subunit in transgenic rd mice. One transgenic mouse line showed complete photoreceptor rescue across the entire span of the retina. A second independently derived line showed partial rescue in which photoreceptors in the superior but not the inferior hemisphere of the retina were rescued. In the latter animals, intermediate stages of degeneration were observed in the transition zone between rescued and diseased photoreceptors. Pathologic changes in the retina ranged from vesiculation of the basalmost outer segment discs in otherwise structurally intact rod cells to photoreceptors with highly disorganized outer segments and intact inner segments. Totally or partially rescued retinas showed a corresponding restoration of cGMP PDE activity, whereas nonrescued retinas had minimal enzyme activity, characteristic of the rd phenotype. These transgenic animals provide models for studying the molecular basis of retinal degenerative disease and conclusively demonstrate that the phenotype of rd mice is produced by a defect in the beta subunit of cGMP PDE.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments.

The chicken retina contains rhodopsin (a rod visual pigment) and four kinds of cone visual pigments. The primary structures of chicken red (iodopsin) and rhodopsin have been determined previously. Here we report isolation of three cDNA clones encoding additional pigments from a chicken retinal cDNA library. Based on the partial amino acid sequences of the purified chicken visual pigments together with their biochemical and spectral properties, we have identified these clones as encoding the chicken green, blue, and violet visual pigments. Chicken violet was very similar to human blue not only in absorption maximum (chicken violet, 415 nm; human blue, 419 nm) but also in amino acid sequence (80.6% identical). Interestingly, chicken green was more similar (71-75.1%) than any other known cone pigment (42.0-53.7%) to vertebrate rhodopsins. The fourth additional cone pigment, chicken blue, had relatively low similarity (39.3-54.6%) in amino acid sequence to those of the other vertebrate visual pigments. A phylogenetic tree of vertebrate visual pigments constructed on the basis of amino acid identity indicated that an ancestral visual pigment evolved first into four groups (groups L, S, M1, and M2), each of which includes one of the chicken cone pigments, and that group Rh including vertebrate rhodopsins diverged from group M2 later. Thus, it is suggested that the gene for scotopic vision (rhodopsin) has evolved out of that for photopic vision (cone pigments). The divergence of rhodopsin from cone pigments was accompanied by an increase in negative net charge of the pigment.

Amino Acid Sequence↗
PubMed1993

Zebrafish ultraviolet visual pigment: absorption spectrum, sequence, and localization.

In many vertebrates, UV-sensitive photoreceptors have been identified by microspectrophotometry and UV-visual sensitivity has been identified by behavioral studies, but as yet no vertebrate UV-sensitive pigment gene has been isolated. We have sequenced a cDNA clone that hybridizes to short single cone cells in the zebrafish (Brachydanio rerio). These cells, which make up 25% of the cone population in zebrafish retinae, are UV-sensitive (lambda max approximately 360 nm). The visual pigment encoded by this gene is unusual in that its amino acid sequence is more homologous to the rod pigment rhodopsin (up to 89%) than to other cone pigments (35-83%). Like all other vertebrate visual pigments, it contains a lysine residue at position 296, the presumptive retinal binding site, and a glutamate residue at position 113. However, it is unique in possessing a lysine residue at position 126, which may account for the UV-sensitivity of the pigment.

Amino Acid Sequence↗
PubMed1994

Sequence divergence of the red and green visual pigments in great apes and humans.

We have determined the coding sequences of red and green visual pigment genes of the chimpanzee, gorilla, and orangutan. The deduced amino acid sequences of these pigments are highly homologous to the equivalent human pigments. None of the amino acid differences occurred at sites that were previously shown to influence pigment absorption characteristics. Therefore, we predict the spectra of red and green pigments of the apes to have wavelengths of maximum absorption that differ by < 2 nm from the equivalent human pigments and that color vision in these nonhuman primates will be very similar, if not identical, to that in humans. A total of 14 within-species polymorphisms (6 involving silent substitutions) were observed in the coding sequences of the red and green pigment genes of the great apes. Remarkably, the polymorphisms at 6 of these sites had been observed in human populations, suggesting that they predated the evolution of higher primates. Alleles at polymorphic sites were often shared between the red and green pigment genes. The average synonymous rate of divergence of red from green sequences was approximately 1/10th that estimated for other proteins of higher primates, indicating the involvement of gene conversion in generating these polymorphisms. The high degree of homology and juxtaposition of these two genes on the X chromosome has promoted unequal recombination and/or gene conversion that led to sequence homogenization. However, natural selection operated to maintain the degree of separation in peak absorbance between the red and green pigments that resulted in optimal chromatic discrimination. This represents a unique case of molecular coevolution between two homologous genes that functionally interact at the behavioral level.

Amino Acid Sequence↗
PubMed1994

Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state.

A disulfide bond that is evidently conserved in the guanine nucleotide-binding protein-coupled receptors is present in rhodopsin between Cys-110 and Cys-187. We have replaced these two cysteine residues by alanine residues and now report on the properties of the resulting rhodopsin mutants. The mutant protein C110A/C187A expressed in COS cells resembles wild-type rhodopsin in the ground state. It folds correctly to bind 11-cis-retinal and form the characteristic rhodopsin chromophore. It is inert to hydroxylamine in the dark, and its stability to dark thermal decay is reduced, relative to that of the wild type, by a delta delta G not equal to of only -2.9 kcal/mol. Further, the affinities of the mutant and wild-type rhodopsins to the antirhodopsin antibody rho4D2 are similar, both in the dark and in light. However, the metarhodopsin II (MII) and MIII photointermediates of the mutant are less stable than those formed by the wild-type rhodopsin. Although the initial rates of transducin activation are the same for both mutant and wild-type MII intermediates at 4 degrees C, at 15 degrees C the MII photointermediate in the mutant decays more than 20 times faster than in wild type. We conclude that the disulfide bond between Cys-110 and Cys-187 is a key component in determining the stability of the MII structure and its coupling to transducin activation.

Alanine↗
PubMed1995

Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration.

Retinitis pigmentosa (RP) is a group of hereditary human diseases that cause retinal degeneration and lead to eventual blindness. More than 25% of all RP cases in humans appear to be caused by dominant mutations in the gene encoding the visual pigment rhodopsin. The mechanism by which the mutant rhodopsin proteins cause dominant retinal degeneration is still unclear. Interestingly, the great majority of these mutants appear to produce misfolded rhodopsin. We now report the isolation and characterization of 13 rhodopsin mutations that act dominantly to cause retinal degeneration in Drosophila; four of these correspond to identical substitutions in human autosomal dominant RP patients. We demonstrate that retinal degeneration results from interference in the maturation of wild-type rhodopsin by the mutant proteins.

Alleles↗
PubMed1996

Retinoic acid alters photoreceptor development in vivo.

Application of exogenous retinoic acid (RA) to zebrafish during the initial stages of photoreceptor differentiation results in a precocious development of rod photoreceptors and an inhibition of cone photoreceptor maturation. The acceleration of rod differentiation is observed initially within the ventral retina 3 days after fertilization, following 24 hr of RA application, and within the dorsal retina 4 days after fertilization, following 48 hr of RA application. The differentiation of rods was impeded significantly when the synthesis of endogenous retinoic acid was inhibited by citral prior to the initial stage of rod differentiation. RA-treated embryos labeled for bromodeoxyuridine (BrdU) uptake revealed that RA exerts its effect on a postmitotic cell population within the developing retina. During normal development in zebrafish, rod differentiation is most robust within the ventral retina, a region previously shown to be rich in RA. Our data suggest that the RA signaling pathway is involved in the differentiation and maturation of both the rod and cone photoreceptors within the developing zebrafish retina.

Animals↗
PubMed1999

Adaptive evolution of color vision of the Comoran coelacanth (Latimeria chalumnae).

The coelacanth, a "living fossil," lives near the coast of the Comoros archipelago in the Indian Ocean. Living at a depth of about 200 m, the Comoran coelacanth receives only a narrow range of light, at about 480 nm. To detect the entire range of "color" at this depth, the coelacanth appears to use only two closely related paralogous RH1 and RH2 visual pigments with the optimum light sensitivities (lambdamax) at 478 nm and 485 nm, respectively. The lambdamax values are shifted about 20 nm toward blue compared with those of the corresponding orthologous pigments. Mutagenesis experiments show that each of these coadapted changes is fully explained by two amino acid replacements.

Adaptation, Physiological↗
PubMed1995

Efficient interaction with a receptor requires a specific type of prenyl group on the G protein gamma subunit.

Post-translational prenylation of the carboxyl-terminal cysteine is a characteristic feature of the guanine nucleotide-binding protein (G protein) gamma subunits. Recent findings show that the farnesylated COOH-terminal tail of the gamma 1 subunit is a specific determinant of rhodopsin-transducin coupling. We show here that when synthetic peptides specific to the COOH-terminal tail of gamma 1 are chemically modified with geranyl, farnesyl, or geranylgeranyl groups and tested for their ability to interact with light activated rhodopsin, the farnesylated peptide is significantly more effective. These results show that an appropriate isoprenoid on the G protein gamma subunit serves not only a membrane anchoring function but in combination with the COOH-terminal domain specifies receptor-G protein coupling.

Acyclic Monoterpenes↗
PubMed1996

Characterization of the Xenopus rhodopsin gene.

The abundant Xenopus rhodopsin gene and cDNA have been cloned and characterized. The gene is composed of five exons spanning 3.5 kilobase pairs of genomic DNA and codes for a protein 82% identical to the bovine rhodopsin. The cDNA was expressed in COS1 cells and regenerated with 11-cis-retinal, forming a light-sensitive pigment with maximal absorbance at 500 nm. Both Southern blots and polymerase chain reaction amplification of intron 1 revealed multiple products, indicating more than one allele for the rhodopsin gene. Comparisons with other vertebrate rhodopsin 5 upstream sequences showed significant nucleotide homologies in the 200 nucleotides proximal to the transcription initiation site. This homology included the TATA box region, Ret 1/PCE1 core sequence (CCAATTA), and surrounding nucleotides. To functionally characterize the rhodopsin promoter, transient embryo transfections were used to assay transcriptional control elements in the 5 upstream region using a luciferase reporter. DNA sequences encompassing -5500 to +41 were able to direct luciferase expression in embryo heads. Reporter gene expression was also observed in embryos microinjected with reporter plasmids during early blastomere stages. These results locate transcriptional control elements upstream of the Xenopus rhodopsin gene and show the feasibility of embryo transfections for promoter analysis of rod-specific genes.

Amino Acid Sequence↗
PubMed1997

Transducin-alpha C-terminal peptide binding site consists of C-D and E-F loops of rhodopsin.

The binding of heterotrimeric GTP-binding proteins (G-proteins) to serpentine receptors involves several independent contacts. We have deduced the points of interaction between mutant bovine rhodopsins and alphat-(340-350), a peptide corresponding to the C terminus of the alpha subunit (alphat) of bovine retinal G-protein, transducin. Direct binding of alphat-(340-350) to rhodopsin stabilizes the activated metarhodopsin II state (M II), consequently uncoupling the rhodopsin-transducin interaction. This peptide action requires two segments on the cytoplasmic domain of rhodopsin: the Tyr136-Val137-Val138-Val139 sequence on the C-D loop and the Glu247-Lys248-Glu249-Val250-Thr251 sequence on the E-F loop. We propose that a tertiary interaction of these two loop regions forms a pocket for binding the alphat C terminus of the transducin during light transduction in vivo. In most G-proteins, the C termini of alpha subunits are important for interaction with receptors, and, in several serpentine receptors, regions similar to those in rhodopsin are essential for G-protein activation, indicating that the interaction described here may be a generally applicable mode of G-protein binding in signal transduction.

Amino Acid Sequence↗
PubMed1999

Similar structures and shared switch mechanisms of the beta2-adrenoceptor and the parathyroid hormone receptor. Zn(II) bridges between helices III and VI block activation.

The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, Gt (Sheikh, S. P., Zvyaga, T. A. , Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the beta2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works.

Animals↗
PubMed2004

Proximal and distal sequences control UV cone pigment gene expression in transgenic zebrafish.

The molecular basis of cone photoreceptor-specific gene expression is largely unknown. In this study, we define cis-acting DNA sequences that control the cell type-specific expression of the zebrafish UV cone pigment gene by transient expression of green fluorescent protein transgenes following their injection into zebrafish embryos. These experiments show that 4.8 kb of 5'-flanking sequences from the zebrafish UV pigment gene direct expression specifically to UV cones and that this activity requires both distal and proximal sequences. In addition, we demonstrate that a proximal region located between -215 and -110 bp (with respect to the initiator methionine codon) can function in the context of a zebrafish rhodopsin promotor to convert its specificity from rod-only expression to rod and UV cone expression. These experiments demonstrate the power of transient transgenesis in zebrafish to efficiently define cis-acting regulatory sequences in an intact vertebrate.

5' Untranslated Regions↗
PubMed2004

Conserved transcriptional activators of the Xenopus rhodopsin gene.

Vertebrate rhodopsin promoters exhibit striking sequence identities proximal to the initiation site, suggesting that conserved transcription factors regulate rhodopsin expression in these animals. We identify and characterize two transcriptional activators of the Xenopus rhodopsin gene: homologs of the mammalian Crx and Nrl transcription factors, XOtx5 and XL-Nrl (originally named XL-maf), respectively. XOtx5 stimulated transcription approximately 10-fold in human 293 cells co-transfected with a plasmid containing the rhodopsin promoter (-508 to +41) upstream of luciferase, similar to the approximately 6-fold stimulation with human Crx. XL-Nrl stimulated transcription approximately 27-fold in mammalian 293 cells co-transfected with the rhodopsin luciferase reporter, slightly more than the approximately 17-fold stimulation with Nrl. Together, the Xenopus transcription factors synergistically activated the rhodopsin promoter (approximately 140-fold), as well as in combination with mammalian homologs. Deletion of the Nrl-response element, TGCTGA, eliminated the synergistic activation by both mammalian and Xenopus transcription factors. Deletion of the conserved ATTA sequences (Ret-1 or BAT-1), binding sites for Crx, did not significantly decrease activation by Crx/XOtx5. However, there was increased activation by Nrl/XL-Nrl and an increased synergy when the Ret-1 site was disrupted. These results illustrate conservation of mechanisms of retinal gene expression among vertebrates. In transgenic tadpoles, XOtx5 and XL-Nrl directed premature and ectopic expression from the Xenopus rhodopsin promoter-GFP transgene. Furthermore, activation of the endogenous rhodopsin gene was also observed in some animals, showing that XOtx5 and XL-Nrl can activate the promoter in native chromatin environment.

Animals↗
PubMed2005

Partial agonism in a G Protein-coupled receptor: role of the retinal ring structure in rhodopsin activation.

The visual process in rod cells is initiated by absorption of a photon in the rhodopsin retinal chromophore and consequent retinal cis/trans-isomerization. The ring structure of retinal is thought to be needed to transmit the photonic energy into conformational changes culminating in the active metarhodopsin II (Meta II) intermediate. Here, we demonstrate that cis-acyclic retinals, lacking four carbon atoms of the ring, can activate rhodopsin. Detailed analysis of the activation pathway showed that, although the photoproduct pathway is more complex, Meta II formed with almost normal kinetics. However, lack of the ring structure resulted in a low amount of Meta II and a fast decay of activity. We conclude that the main role of the ring structure is to maintain the active state, thus specifying a mechanism of activation by a partial agonist of the G protein-coupled receptor rhodopsin.

Animals↗
PubMed2006

Conformational changes associated with receptor-stimulated guanine nucleotide exchange in a heterotrimeric G-protein alpha-subunit: NMR analysis of GTPgammaS-bound states.

Solution NMR studies of a (15)N-labeled G-protein alpha-subunit (G(alpha)) chimera ((15)N-ChiT)-reconstituted heterotrimer have shown previously that G-protein betagamma-subunit (G(betagamma)) association induces a "pre-activated" conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the (15)N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5'-O-3-thiotriphosphate (GTPgammaS)/Mg(2+)-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPgammaS/Mg(2+)-bound ChiT revealed (1)HN, (15)N chemical shift changes relative to GDP/Mg(2+)-bound ChiT that were similar, but not identical, to those observed for the GDP.AlF(4)(-)/Mg(2+)-bound state. Line widths observed for R*-generated GTPgammaS/Mg(2+)-bound (15)N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg(2+)- and GDP.AlF(4)(-)/Mg(2+)-bound states. The increased dynamics appeared to be correlated with G(betagamma) and R* interactions because they are not observed for GTPgammaS/Mg(2+)-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPgammaS/Mg(2+)-exchanged heterotrimer. The HSQC spectrum of (15)N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPgammaS/Mg(2+)-bound ChiT. Overall, these results demonstrated that R*-induced changes in G(alpha) can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation.

Amino Acid Sequence↗