Immunoreactivity of rhodopsin and opsin

An examination by a radioimmunoassay of the relative affinity of opsin and rhodopsin for rabbit antibody raised against bovine rhodopsin revealed that opsin was the preferred antigen. About 10-fold greater amounts of rhodopsin than opsin were required to achieve 50% inhibition of binding of 125I-labeled ligand in the RIA. Opsin was more reactive when examined in the light or dark, compared to rhodopsin incubated in the dark. Mixtures of opsin and rhodopsin (prepared by partial bleaching of rhodopsin or synthetic mixtures) exhibited increased reactivity with increasing mole fraction of opsin. This response was nonlinear, with small increases in opsin producing relatively large increases in reactivity. A partial fractionation of the antibody into two groups showing differential reactivities toward opsin and rhodopsin was achieved by affinity chromatography on opsin-Sepharose. However, with both groups, opsin was still the preferred antigen. Scatchard analysis of 125I-labeled rhodopsin and opsin produced nonlinear plots, indicating the presence of multiple species of antibody. The affinities and binding capacities were similar for both labeled antigens. In competitive binding studies, the antibody showed a strong preference for either labeled ligand (rhodopsin or opsin) as compared to the unlabeled material. These latter observations indicate that altering rhodopsin either by bleaching or iodination produced changes in the relative immunoreactivity of the molecule.     

Mechanism and specificity of rhodopsin phosphorylation.

Partial separation of protein kinase activity from rhodopsin in isolated bovine retinal photoreceptor outer segments was accomplished by mild ultrasonic treatment followed by ultracentrifugation. Residual kinase activity in the rhodopsin-rich sediment was destroyed by chemical denaturation which did not affect the spectral properties of the rhodopsin. The retinal outer segment kinase was found to be specific for rhodopsin, since in these preparations it alone of several bovine protein kinases was capable of phosphorylating rhodopsin in the light. The phosphorylation reaction apparently requires a specific conformation of the rhodopsin molecule since it is abolished by heat denaturation of rhodopsin, and it is greatly reduced or abolished by treatment of the visual pigment protein with potassium alum after the rhodopsin has been "bleached" by light. When kinase and rhodopsin or opsin fractions were prepared from dark-adapted and bleached outer segments and the resultant fractions were mixed in various combinations of bleached and unbleached preparations, the observed pattern of light-activated phosphorylation was consistent only with the interpretation that a conformational change in the rhodopsin molecule in the light exposes a site on the visual pigment protein to the kinase and ATP. These results rule out the possibility of a direct or indirect (rhodopsin-mediated) light activation of the kinase. Finally, phosphorylation of retinal outer segment protein in monochromatic lights of various wavelengths followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both rhodopsin and the higher molecular weight visual pigment protein reported by several laboratories have the same action spectrum for phosphorylation. This result is consistent with the suggestion that the higher molecular weight species is a rhodopsin dimer.     

M opsin phosphorylation in intact mammalian retinas

The deactivation of visual pigments involved in phototransduction is critical for recovering sensitivity after exposure to light in rods and cones of the vertebrate retina. In rods, phosphorylation of rhodopsin by rhodopsin kinase (GRK1) and the subsequent binding of visual arrestin completely terminates phototransduction. Although signal termination in cones is predicted to occur via a similar mechanism as in rods, there may be differences due to the expression of related but distinct gene products. While rods only express GRK1, cones in some species express only GRK1 or GRK7 and others express both GRKs. In the mouse, cone opsin is phosphorylated by GRK1, but this has not been demonstrated in mammals that express GRK7 in cones. We compared cone opsin phosphorylation in intact retinas from the 13-lined ground squirrel (GS) and pig, cone- and rod-dominant mammals, respectively, which both express GRK7. M opsin phosphorylation increased during continuous exposure to light, then declined between 3 and 6 min. In contrast, rhodopsin phosphorylation continued to increase during this time period. In GS retina homogenates, anti-GS GRK7 antibody blocked M opsin phosphorylation by 73%. In pig retina homogenates, only 20% inhibition was observed, possibly due to phosphorylation by GRK1 released from rods during homogenization. Our results suggest that GRK7 phosphorylates M opsin in both of these mammals. Using an in vitro GTPgammaS binding assay, we also found that the ability of recombinant M opsin to activate G(t) was greatly reduced by phosphorylation. Therefore, phosphorylation may participate directly in the termination of phototransduction in cones by decreasing the activity of M opsin.     

Engineering of an artificial light-modulated potassium channel

Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1(F137S) (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools.     

Affinity labeling of bovine opsin by trans-retinoyl chloromethane

All-trans-retinoyl chloromethane is a potent irreversible inactivator of bovine opsin in retinal rod outer segments. The inactivation appears to be due to specific modification of the apoprotein at the 11-cis-retinaldehyde binding site. The reaction follows pseudo first order kinetics at 37 degrees C. A moderate dissociation constant for the initial reversible complex of 6.1 mM could be derived with a first order rate constant of 1.8x10(-1) min-1 for the conversion to the irreversible inactivated form. Native rhodopsin or rhodopsin regenerated from opsin by addition of 11-cis-retinaldehyde is completely protected against inactivation by trans-retinoyl chloromethane. All-trans-retinaldehyde does not provide this protection for the irreversible inactivation.     

The thermal stability of rhodopsin and opsin

Rhodopsin, the red photosensitive pigment of rod vision, is composed of a specific cis isomer of retinene, neo-b (11-cis), joined as chromophore to a colorless protein, opsin. We have investigated the thermal denaturation of cattle rhodopsin and opsin in aqueous digitonin solution, and in isolated rod outer limbs. Both rhodopsin and opsin are more stable in rods than in solution. In solution as well as in rods, moreover, rhodopsin is considerably more stable than opsin. The chromophore therefore protects opsin against denaturation. This is true whether rhodopsin is extracted from dark-adapted retinas, or synthesized in vitro from neo-b retinene and opsin. Excess neo-b retinene does not protect rhodopsin against denaturation. The protection involves the specific relationship between the chromophore and opsin. Similar, though somewhat less, protection is afforded opsin by the stereoisomeric iso-a (9-cis) chromophore in isorhodopsin. The Arrhenius activation energies (E_a) and entropies of activation (ΔS‡) are much greater for thermal denaturation of rhodopsin and isorhodopsin than of opsin. Furthermore, these values differ considerably for rhodopsins from different species —frog, squid, cattle—presumably due to species differences in the opsins. Heat or light bleaches rhodopsin by different mechanisms, yielding different products. Light stereoisomerizes the retinene chromophore; heat denatures the opsin. Photochemical bleaching therefore yields all-trans retinene and native opsin; thermal bleaching, neo-b retinene and denatured opsin.     

The molar extinction of rhodopsin

The molar extinction of rhodopsin is 40,600 cm.² per mole equivalent of retinene; i.e., this is the extinction of a solution of rhodopsin which is produced by, or yields on bleaching, a molar solution of retinene. The molar extinctions of all-trans retinene and all-trans retinene oxime have also been determined in ethyl alcohol and aqueous digitonin solutions. On the assumption that each chromophoric group of rhodopsin is made from a single molecule of retinene, it is concluded that the primary photochemical conversion of rhodopsin to lumi-rhodopsin has a quantum efficiency of 1; though the over-all bleaching of rhodopsin in solution to retinene and opsin may have a quantum efficiency as low as one-half. On bleaching cattle rhodopsin, about two sulfhydryl groups appear for each molecule of retinene liberated. In frog rhodopsin the —SH:retinene ratio appears to be higher, 5:2 or perhaps even 3:1. Some of this sulfhydryl appears to have been engaged in binding retinene to opsin; some may have been exposed as the result of changes in opsin which accompany bleaching, comparable with protein denaturation.     

The retinal G protein-coupled receptor (RGR) enhances isomerohydrolase activity independent of light

Rod and cone visual pigments use 11-cis-retinal, a vitamin A derivative, as their chromophore. Light isomerizes 11-cis- into all-trans-retinal, triggering a conformational transition of the opsin molecule that initiates phototransduction. After bleaching all-trans-retinal leaves the opsin, and light sensitivity must be restored by regeneration of 11-cis-retinal. Under bright light conditions the retinal G protein-coupled receptor (RGR) was reported to support this regeneration by acting as a photoisomerase in a proposed photic visual cycle. We analyzed the contribution of RGR to rhodopsin regeneration under different light regimes and show that regeneration, during light exposure and in darkness, is slowed about 3-fold in Rgr(-/-) mice. These findings are not in line with the proposed function of RGR as a photoisomerase. Instead, RGR, independent of light, accelerates the conversion of retinyl esters to 11-cis-retinal by positively modulating isomerohydrolase activity, a key step in the "classical" visual cycle. Furthermore, we find that light accelerates rhodopsin regeneration, independent of RGR.     

The opsins

The photosensitive molecule rhodopsin and its relatives consist of a protein moiety - an opsin - and a non-protein moiety - the chromophore retinal. Opsins, which are G-protein-coupled receptors (GPCRs), are found in animals, and more than a thousand have been identified so far. Detailed molecular phylogenetic analyses show that the opsin family is divided into seven subfamilies, which correspond well to functional classifications within the family: the vertebrate visual (transducin-coupled) and non-visual opsin subfamily, the encephalopsin/tmt-opsin subfamily, the G_q-coupled opsin/melanopsin subfamily, the G_o-coupled opsin subfamily, the neuropsin subfamily, the peropsin subfamily and the retinal photoisomerase subfamily. The subfamilies diversified before the deuterostomes (including vertebrates) split from the protostomes (most invertebrates), suggesting that a common animal ancestor had multiple opsin genes. Opsins have a seven-transmembrane structure similar to that of other GPCRs, but are distinguished by a lysine residue that is a retinal-binding site in the seventh helix. Accumulated evidence suggests that most opsins act as pigments that activate G proteins in a light-dependent manner in both visual and non-visual systems, whereas a few serve as retinal photoisomerases, generating the chromophore used by other opsins, and some opsins have unknown functions.     

Cis-trans isomers of vitamin A and retinene in the rhodopsin system

Vitamin A and retinene, the carotenoid precursors of rhodopsin, occur in a variety of molecular shapes, cis-trans isomers of one another. For the synthesis of rhodopsin a specific cis isomer of vitamin A is needed. Ordinary crystalline vitamin A, as also the commercial synthetic product, both primarily all-trans, are ineffective. The main site of isomer specificity is the coupling of retinene with opsin. It is this reaction that requires a specific cis isomer of retinene. The oxidation of vitamin A to retinene by the alcohol dehydrogenase-cozymase system displays only a low degree of isomer specificity. Five isomers of retinene have been isolated in crystalline condition: all-trans; three apparently mono-cis forms, neoretinenes a and b and isoretinene a; and one apparently di-cis isomer, isoretinene b. Neoretinenes a and b were first isolated in our laboratory, and isoretinenes a and b in the Organic Research Laboratory of Distillation Products Industries. Each of these substances is converted to an equilibrium mixture of stereoisomers on simple exposure to light. For this reaction, light is required which retinene can absorb; i.e., blue, violet, or ultraviolet light. Yellow, orange, or red light has little effect. The single geometrical isomers of retinene must therefore be protected from low wave length radiation if their isomerization is to be avoided. By incubation with opsin in the dark, the capacity of each of the retinene isomers to synthesize rhodopsin was examined. All-trans retinene and neoretinene a are inactive. Neoretinene b yields rhodopsin indistinguishable from that extracted from the dark-adapted retina (λ_max· 500 mµ). Isoretinene a yields a similar light-sensitive pigment, isorhodopsin, the absorption spectrum of which is displaced toward shorter wave lengths (λ_max· 487 mµ). Isoretinene b appears to be inactive, but isomerizes preferentially to isoretinene a, which in the presence of opsin is removed to form isorhodopsin before the isomerization can go further. The synthesis of rhodopsin in solution follows the course of a bimolecular reaction, as though one molecule of neoretinene b combines with one of opsin. The synthesis of isorhodopsin displays similar kinetics. The bleaching of rhodopsin, whether by chemical means or by exposure to yellow or orange (i.e., non-isomerizing) light, yields primarily or exclusively all-trans retinene. The same appears to be true of isorhodopsin. The process of bleaching is therefore intrinsically irreversible. The all-trans retinene which results must be isomerized to active configurations before rhodopsin or isorhodopsin can be regenerated. A cycle of isomerization is therefore an integral part of the rhodopsin system. The all-trans retinene which emerges from the bleaching of rhodopsin must be isomerized to neoretinene b before it can go back; or if first reduced to all-trans vitamin A, this must be isomerized to neovitamin Ab before it can regenerate rhodopsin. The retina obtains new supplies of the neo-b isomer: (a) by the isomerization of all-trans retinene in the eye by blue or violet light; (b) by exchanging all-trans vitamin A for new neovitamin Ab from the blood circulation; and (c) the eye tissues may contain enzymes which catalyze the isomerization of retinene and vitamin A in situ. When the all-trans retinene which results from bleaching rhodopsin in orange or yellow light is exposed to blue or violet light, its isomerization is accompanied by a fall in extinction and a shift of absorption spectrum about 5 mµ toward shorter wave lengths. This is a second photochemical step in the bleaching of rhodopsin. It converts the inactive, all-trans isomer of retinene into a mixture of isomers, from which mixtures of rhodopsin and isorhodopsin can be regenerated. Isorhodopsin, however, is an artefact. There is no evidence that it occurs in the retina; nor has isovitamin Aa or b yet been identified in vivo. In rhodopsin and isorhodopsin, the prosthetic groups appear to retain the cis configurations characteristic of their retinene precursors. In accord with this view, the β-bands in the absorption spectra of both pigments appear to be cis peaks. The conversion to the all-trans configuration occurs during the process of bleaching. The possibility is discussed that rhodopsin may represent a halochromic complex of a retinyl ion with opsin. The increased resonance associated with the ionic state of retinene might then be responsible both for the color of rhodopsin and for the tendency of retinene to assume the all-trans configuration on its release from the complex. A distinction must be made between the immediate precursor of rhodopsin, neovitamin Ab, and the vitamin A which must be fed in order that rhodopsin be synthesized in vivo. Since vitamin A isomerizes in the body, it is probable that any geometrical isomer can fulfill all the nutritional needs for this vitamin.     

Rhodopsin activation causes retinal degeneration in Drosophila rdgC mutant

Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgC-induced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the G protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation.     

Kinetic study of photoregeneration process of digitonin-solubilized squid rhodopsin

In the photoregeneration process of squid rhodopsin, an intermediate has been found at neutral pH values (phosphate buffer) with a flash light (λ > 540 nm). An intermediate R430, with the 11-cis retinal as chromophore, is produced from metarhodopsin in light and is converted to rhodopsin through the processes R430 → P380 and P380 → rhodopsin. The pH dependence of the velocity of the conversions suggests that processes R430 → P380 and P380 → rhodopsin involve a protolytic reaction and that the ionized group is a histidine residue of opsin. Kinetic parameters show that the largest conformational change in opsin occurs in the conversion of R430 → P380.     

The role of sulfhydryl groups in the bleaching and synthesis of rhodopsin

The condensation of retinene₁ with opsin to form rhodopsin is optimal at pH about 6, a pH which favors the condensation of retinene₁ with sulfhydryl rather than with amino groups. The synthesis of rhodopsin, though unaffected by the less powerful sulfhydryl reagents, monoiodoacetic acid and its amide, is inhibited completely by p-chloromercuribenzoate (PCMB). This inhibition is reversed in part by the addition of glutathione. PCMB does not attack rhodopsin itself, nor does it react with retinene₁. Its action in this system is confined to the —SH groups of opsin. Under some conditions the synthesis of rhodopsin is aided by the presence of such a sulfhydryl compound as glutathione, which helps to keep the —SH groups of opsin free and reduced. By means of the amperometric silver titration of Kolthoff and Harris, it is shown that sulfhydryl groups are liberated in the bleaching of rhodopsin, two such groups for each retinene₁ molecule that appears. This is true equally of rhodopsin from the retinas of cattle, frogs) and squid. The exposure of new sulfhydryl groups adds an important element to the growing evidence that relates the bleaching of rhodopsin to protein denaturation. The place of sulfhydryl groups in the structure of rhodopsin is still uncertain. They may be concerned directly in binding the chromophore to opsin; or alternatively they may furnish hydrogen atoms for some reductive change by which the chromophore is formed from retinene₁. In the amperometric silver titration, the bleaching of rhodopsin yields directly an electrical variation. This phenomenon may have some fundamental connection with the role of rhodopsin in visual excitation, and may provide a model of the excitation process in general.     

Mutation that selectively affects rhodopsin concentration in the peripheral photoreceptors of Drosophila melanogaster

A Drosophila mutant (ninaAP228) that is low in rhodopsin concentration but identical to the wild-type fly in photoreceptor morphology has been isolated. R1-6 photoreceptors of the mutant differ from those of wild type in that (a) the prolonged depolarizing afterpotential (PDA) is absent, (b) concentrations of rhodopsin and opsin are substantially reduced, and (c) intramembrane particle density in the membranes of the rhabdomeres is low. Each of these traits is mimicked by depriving wild- type flies of vitamin A. The ninaAP228 mutation differs from vitamin A deprivation in that in the mutant (a) the rhabdomeric membrane particle density is reduced only in the R1-6 photoreceptors and not in R7 or R8, (b) the PDA can be elicited from the R7 photoreceptors, and (c) photoconversion of R1-6 rhodopsin to metarhodopsin by ultraviolet (UV) light is considerably more efficient than in vitamin A-deprived flies. The absorption properties of the mutant rhodopsin in the R1-6 photoreceptors appear to be identical to those of wild type as judged from rhodopsin difference spectra. The results suggest that the mutation affects the opsin, rather than the chromophore, component of rhodopsin molecules in the R1-6 photoreceptors. The interaction between the chromophore and R1-6 opsin, however, appears to be normal.     

Arrestin-rhodopsin binding stoichiometry in isolated rod outer segment membranes depends on the percentage of activated receptors

In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the "photoactivation density," i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestin-Rho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.     

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.     

Mouse cones require an arrestin for normal inactivation of phototransduction

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.     

Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296.

In previous studies, mutation of either Lys296 or Glu113 in bovine rhodopsin has been shown to result in constitutive activation of the apoprotein form, opsin [Robinson et al. (1992) Neuron 9, 719-725]. In this report, pH-rate profiles for the rhodopsin-catalyzed exchange of GTPgS for GDP on transducin are established for the constitutively active opsin mutants. All of the mutants, including the double-mutant E113Q,K296G, show a bell-shaped pH-rate profile. Therefore, it is evident that at least two ionizable groups in addition to Lys296 and Glu113 control the formation of the active opsin state. The sole effect of mutation at position 113 or 296 is to alter the ionization constant of the group with the higher pKa, called pka2. pKa2 decreases in the following order: rhodopsin/light (9.0) > K296E = K296G = E113Q,K296G (8.0) > E113Q (6.8) > K296H (6.6) > wild-type opsin (< 5.0). These results are consistent with a model where activation of opsin involves (i) breaking of the salt bridge between Lys296 and Glu113, (ii) deprotonation of Lys296, and (iii) the net uptake of a proton from the solvent. Furthermore, exogenous addition of the chromophore all-trans-retinal shifts the wild-type and E113Q opsin equilibrium to favor the active state. In all these respects, the light-independent activation of the opsin mutants appears to proceed by a mechanism similar to that of light-activated rhodopsin.