Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin

Isomerization of the 11-cis retinal chromophore in the visual pigment rhodopsin is coupled to motion of transmembrane helix H6 and receptor activation. We present solid-state magic angle spinning NMR measurements of rhodopsin and the metarhodopsin II intermediate that support the proposal that interaction of Trp265(6.48) with the retinal chromophore is responsible for stabilizing an inactive conformation in the dark, and that motion of the beta-ionone ring allows Trp265(6.48) and transmembrane helix H6 to adopt active conformations in the light. Two-dimensional dipolar-assisted rotational resonance NMR measurements are made between the C19 and C20-methyl groups of the retinal and uniformly 13C-labeled Trp265(6.48). The retinal C20-Trp265(6.48) contact present in the dark-state of rhodopsin is lost in metarhodopsin II, and a new contact is formed with the C19 methyl group. We have previously shown that the retinal translates 4-5 A toward H5 in metarhodopsin II. This motion, in conjunction with the Trp-C19 contact, implies that the Trp265(6.48) side-chain moves significantly upon rhodopsin activation. NMR measurements also show that a packing interaction in rhodopsin between Trp265(6.48) and Gly121(3.36) is lost in metarhodopsin II, consistent with H6 motion away from H3. However, a close contact between Gly120(3.35) on H3 and Met86(2.53) on H2 is observed in both rhodopsin and metarhodopsin II, suggesting that H3 does not change orientation significantly upon receptor activation.     

Renewal of visual pigment in photoreceptors of the blowfly

Summary Spectrophotometric measurements of photoreceptors 1–6 in the blowfly demonstrate that rhodopsin undergoes a continuous renewal. This involves, in the dark, the slow degradation of rhodopsin whereas metarhodopsin is degraded at a much faster rate. The effect of light is to reduce the rate at which metarhodopsin is degraded, i.e. the rate is inversely related to the intensity of the light. Rhodopsin synthesis is dependent on the presence of 11-cis retinal which is formed via a photoreaction from all-trans retinal resulting from the breakdown of rhodopsin and/or metarhodopsin: the biosynthesis of rhodopsin is therefore a light dependent process. Light of the blue/violet spectral range was found to mediate the isomerization of all-trans retinal into the 11-cis form. It is proposed that this stereospecificity is the result of all-trans retinal being bound to a protein. On the basis of the results a visual pigment cycle is proposed.     

Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane

Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane.     

Orientation of retinal in bovine rhodopsin determined by cross-linking using a photoactivatable analog of 11-cis-retinal

A photoactivatable analog of 11-cis-retinal has been used to probe the orientation of retinal in bovine rhodopsin. The analog binds to the opsin to regenerate a chromophore with lambda max at 458 nm. The linkage site of the analog to the opsin was confirmed to be Lys-296 as in 11-cis-retinal rhodopsin. The analog-reconstituted rhodopsin activated transducin and was phosphorylated by rhodopsin kinase on illumination. On photolysis of rhodopsin containing the radioactively labeled analog at 365 nm at -15 degrees C, 20-25% of the analog was covalently linked to the protein. Proteolysis of the labeled protein and characterization of the appropriate peptides showed that cross-linking of the analog was predominantly to helices C or F. When analog reconstituted rhodopsin in rod outer segments was photolyzed, cross-linking was predominantly to helix C. However, when analog-reconstituted rhodopsin, purified in lauryl maltoside, was photolyzed, labeling occurred mainly in helix F. Sequence analysis showed major sites of cross-linking to be Phe-115, Ala-117, Glu-122, Trp-126, and Ser-127 in helix C while Trp-265 was the major site in helix F. The results suggest that the beta-ionone ring of retinal orients toward helices C and F.     

Reduced rhodopsin phosphorylation during retinal dystrophy

During inherited retinal dystrophy in Irish Setter dogs, decreased activity of cGMP phosphodiesterase (PDE) results in high cGMP levels and retinal degeneration (1-3). This defect could be in PDE itself, or in its interactions with other proteins of the rod outer segment. We report herein that when retinas from 8-week-old dogs were phosphorylated with gamma-32P-ATP, and separated on SDS-PAGE, phosphorylation of rd dog rhodopsin was reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Since rd-mediated inhibition was prevented by 1 mM NaF, the results suggest that the cause of reduced rd phosphorylation is increased phosphatase activity. Together, these results demonstrate that decreased phosphorylation of rhodopsin due to increased phosphatase activity is a fundamental biochemical change which may partially account for the degenerative process and loss of visual acuity during inherited retinal dystrophy.     

Targeting of Drosophila Rhodopsin Requires Helix 8 but Not the Distal C-Terminus

Background

The fundamental role of the light receptor rhodopsin in visual function and photoreceptor cell development has been widely studied. Proper trafficking of rhodopsin to the photoreceptor membrane is of great importance. In human, mutations in rhodopsin involving its intracellular mislocalization, are the most frequent cause of autosomal dominant Retinitis Pigmentosa, a degenerative retinal pathology characterized by progressive blindness. Drosophila is widely used as an animal model in visual and retinal degeneration research. So far, little is known about the requirements for proper rhodopsin targeting in Drosophila.

Methodology/Principal Findings

Different truncated fly-rhodopsin Rh1 variants were expressed in the eyes of Drosophila and their localization was analyzed in vivo or by immunofluorescence. A mutant lacking the last 23 amino acids was found to properly localize in the rhabdomeres, the light-sensing organelle of the photoreceptor cells. This constitutes a major difference to trafficking in vertebrates, which involves a conserved QVxPA motif at the very C-terminus. Further truncations of Rh1 indicated that proper localization requires the last amino acid residues of a region called helix 8 following directly the last transmembrane domain. Interestingly, the very C-terminus of invertebrate visual rhodopsins is extremely variable but helix 8 shows conserved amino acid residues that are not conserved in vertebrate homologs.

Conclusions/Significance

Despite impressive similarities in the folding and photoactivation of vertebrate and invertebrate visual rhodopsins, a striking difference exists between mammalian and fly rhodopsins in their requirements for proper targeting. Most importantly, the distal part of helix 8 plays a central role in invertebrates. Since the last amino acid residues of helix 8 are dispensable for rhodopsin folding and function, we propose that this domain participates in the recognition of targeting factors involved in transport to the rhabdomeres.     

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.     

Visual pigment and photoreceptor sensitivity in the isolated skate retina

Photoreceptor potentials were recorded extracellularly from the aspartate-treated, isolated retina of the skate (Raja oscellata and R. erinacea), and the effects of externally applied retinal were studied both electrophysiologically and spectrophotometrically. In the absence of applied retinal, strong light adaptation leads to an irreversible depletion of rhodopsin and a sustained elevation of receptor threshold. For example, after the bleaching of 60% of the rhodopsin initially present in dark-adapted receptors, the threshold of the receptor response stabilizes at a level about 3 log units above the dark-adapted value. The application of 11-cis retinal to strongly light-adapted photoreceptors induces both a rapid, substantial lowering of receptor threshold and a shift of the entire intensity-response curve toward greater sensitivity. Exogenous 11-cis retinal also promotes the formation of rhodopsin in bleached photoreceptors with a time-course similar to that of the sensitization measured electrophysiologically. All-trans and 13-cis retinal, when applied to strongly light-adapted receptors, fail to promote either an increase in receptor sensitivity or the formation of significant amounts of light-sensitive pigment within the receptors. However, 9-cis retinal isin. These findings provide strong evidence that the regeneration of visual pigment in the photoreceptors directly regulates the process of photochemical dark adaptation.     

Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin

Spin labeling EPR spectroscopy has been used to characterize light-induced conformational changes of bacteriorhodopsin (bR). Pairs of nitroxide spin labels were attached to engineered cysteine residues at strategic positions near the cytoplasmic ends of transmembrane alpha-helices B, F, and G in order to monitor distance changes upon light activation. The EPR analysis of six doubly labeled bR mutants indicates that the cytoplasmic end of helix F not only tilts outwards, but also rotates counter-clockwise during the photocycle. The direction of the rotation of helix F is the opposite of the clockwise rotation previously reported for bovine rhodopsin. The opposite chirality of the F helix rotation in the two systems is perhaps related to the differences in the cis-trans photoisomerization of the retinal in the two proteins. Using time-resolved EPR, we monitored the rotation of helix F also in real time, and found that the signal from the rotation arises concurrently with the reprotonation of the retinal Schiff base.     

Structural insights into the early steps of receptor-transducer signal transfer in archaeal phototaxis

Electron paramagnetic resonance-based inter-residue distance measurements between site-directed spin-labelled sites of sensory rhodopsin II (NpSRII) and its transducer NpHtrII from Natronobacterium pharaonis revealed a 2:2 complex with 2-fold symmetry. The core of the complex is formed by the four transmembrane helices of a transducer dimer. Upon light excitation, the previously reported flap-like movement of helix F of NpSRII induces a conformational change in the transmembrane domain of the transducer. The inter-residue distance changes determined provide strong evidence for a rotary motion of the second transmembrane helix of the transducer. This helix rotation becomes uncoupled from changes in the receptor during the last step of the photocycle.     

In vitro identification of rhodopsin in the green alga Chlamydomonas

The unicellular alga Chlamydomonas can detect both intensity and direction of the ambient light and adjust its swimming speed and direction accordingly. On the basis of physiological experiments, the functional photoreceptor for this visual process has recently shown to be a rhodopsin. We here report the in vitro identification of endogenous retinal and a rhodopsin in Chlamydomonas cell extracts and purified membrane preparations. The rhodopsin absorption spectrum has fine structure with the maximum at 495 nm and matches the action spectra for the behavioral light responses. The rhodopsin can be bleached and subsequently reconstituted with exogenous retinal. Labeling with [3H]retinal occurs in the final preparation only with a single protein with a molecular weight of 32,000. We conclude that this protein is the visual photoreceptor in Chlamydomonas.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.     

Structural and functional role of helices I and II in rhodopsin. A novel interplay evidenced by mutations at Gly-51 and Gly-89 in the transmembrane domain

The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced lysine. Increased formation of non-active forms of rhodopsin, like metarhodopsin III, may have some influence in the molecular defect underlying retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of rhodopsin.     

Rhodopsin, 11-cis vitamin A, and interstitial retinol-binding protein (IRBP) during retinal development in normal and rd mutant mice

Biochemical and immunological techniques were used to determine the emergence of interstitial retinol binding protein (IRBP), rhodopsin, and stored retinyl esters (all-trans and 11-cis) during retinal development in normal and rd mice. IRBP could be demonstrated at embryonic Day 17 (E17), corresponding to an early stage of inner segment development. Although all-trans retinyl esters were present earlier, 11-cis retinyl esters did not appear until postnatal Days 6-7 (P6-P7), corresponding to rod outer segment (ROS) disc formation. Rhodopsin was detected at the same developmental stage. The proportion of 11-cis retinyl esters reached a maximum of 40-50% at P15-P20. Thereafter, the proportion dropped, due to more rapid accumulation of the all-trans isomer. Rhodopsin and IRBP increased in parallel with ROS elongation up to P25, when the ROS had reached their mature lengths. The increases then continued up to P40-P50. In rd (retinal degeneration) mice, IRBP and rhodopsin were identical with the controls until P12, but then dropped as the photoreceptors degenerated. Synthesis and secretion of IRBP in vitro was less than 10% of the controls in rd retinas at P26, when only 4-5% of the photoreceptors survived. The quantities of retinyl esters (mainly stearate and palmitate in the ratio of 6:1, respectively) stored in dark-adapted mouse eyes progressively increased as the animals aged, representing 0.5 mole eq. of the rhodopsin at 8 months. Although retinyl esters (11-cis and all-trans) also accumulated in rd mouse eyes up to P12, little further increase occurred. At P93, the retinyl esters (0.01 nmole X eye-1) were only 4% of the controls at P91. A peak in the proportion of 11-cis isomer occurred at P10-P20, but it averaged only 15% of the total ester and declined to 5% at P93. These findings support the hypothesis that IRBP is synthesized by the rods and cones, and suggest that its synthesis and secretion are initiated when the photoreceptor inner segments start to differentiate. 11-cis Retinoids and rhodopsin do not appear until the outer segments start to form. It is suggested that in the rd mouse the absence of photoreceptors, perhaps coupled with lack of normal interphotoreceptor matrix, leads to a loss in the ability of the pigment epithelium to store retinyl esters.     

Demonstration of a sensory rhodopsin in eubacteria

We report the first sensory rhodopsin observed in the eubacterial domain, a green light-activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all-trans retinal to form a pink pigment (lambda max 543 nm) with a photochemical reaction cycle of 110 ms half-life (pH 6.8, 18 degrees C). Co-expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane-embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS-PAGE and autofluorography of 3H-labelled all-trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix-helix interactions with membrane-embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.     

Development and Regulation of Rhodopsin Kinase in Rat Pineal and Retina

Rhodopsin kinase, once thought to be a retinal enzyme, was recently found at high levels in the pineal gland. In the present study the developmental pattern and the regulation by environmental lighting of this enzyme in both tissues was studied in the rat. Enzyme activity was present in the neonatal pineal gland several days earlier than in the retina, and increased gradually up to 20 days of age and remained at that level thereafter; the retinal enzyme appeared to increase until day 60. Pineal and retinal rhodopsin kinase activities showed a 25% increase in in the middle of the dark and the beginning of the light period, respectively. Exposure to constant light caused a 50% decrease in rhodopsin kinase levels in both tissues. However, only pineal rhodopsin kinase activity declined followed bilateral superior cervical ganglionectomy. This indicates pineal rhodopsin kinase activity is similar to other pineal enzymes in that it is controlled by light acting through the sympathetic nervous system. In contrast, the light-induced decrease in retinal rhodopsin kinase may be due to the direct destructive effect of light on the retina. The finding of neural control of pineal rhodopsin kinase in the pineal gland of adult rats is consistent with a function of the enzyme in the neural regulation of pineal function.     

Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist

The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.     

Tunable laser resonance Raman spectroscopic investigations of the transduction process in vertebrate rod cells.

Tunable laser resonance Raman spectroscopy has been applied to probe (in vivo) the role of rhodopsin in transducing light energy into the chemical necessary to generate a neural response. These in vivo experiments have suggested that the Schiff base linkage through which retinal is attached to opsin in rhodopsin is protonated. Furthermore, it appears that light eventually stimulates the deprotonation of the Schiff base linkage between the Meta I and Meta II steps in the intermediate sequence which is the result of light interacting with rhodopsin. Our data suggest that this deprotonation of the Schiff base occurs on the same time scale as overall proton release and uptake by the rhodopsin molecule. It is interesting to note that this series of protonations and deprotonations also occurs within the same time scale as the neural response generation in vertebrates and the generation of a proton gradient by bacteriorhodopsin, which is used by the bacterium, Halobacterium halobium, for ATP synthesis. If these data are analyzed within the context of the in vivo resonance Raman experiments (which seem to indicate that proton release is stimulated in the disc membrane during transduction) then there is a strong suggestion that the proton will assume an important role in any working hypothesis of visual transduction. In essence it appears that protons along with ATP and calcium ions must all be essential elements in the transduction process.     

Critical role of transmembrane segment zinc binding in the structure and function of rhodopsin

Zinc deficiency and retinitis pigmentosa are both important factors resulting in retinal dysfunction and night blindness. In this study, we address the critical biochemical and structural relevance of zinc ions in rhodopsin and examine whether zinc deficiency can lead to rhodopsin dysfunction. We report the identification of a high-affinity zinc coordination site within the transmembrane domain of rhodopsin, coordinated by the side chains of two highly conserved residues, Glu(122) in transmembrane helix III and His(211) in transmembrane helix V. We also demonstrate that this zinc coordination is critical for rhodopsin folding, 11-cis-retinal binding, and the stability of the chromophore-receptor interaction, defects of which are observed in retinitis pigmentosa. Furthermore, a cluster of retinitis pigmentosa mutations is localized within and around this zinc binding site. Based on these studies, we believe that improvement in zinc binding to rhodopsin at this site within the transmembrane domain may be a pharmacological approach for the treatment of select retinitis pigmentosa mutations. Transmembrane coordination of zinc may also be an important common principle across G-protein-coupled receptors.     

The role of retinal photoisomerase in the visual cycle of the honeybee

The compound eye of the honeybee has previously been shown to contain a soluble retinal photoisomerase which, in vitro, is able to catalyze stereospecifically the photoconversion of all-trans retinal to 11-cis retinal. In this study we combine in vivo and in vitro techniques to demonstrate how the retinal photoisomerase is involved in the visual cycle, creating 11-cis retinal for the generation of visual pigment. Honeybees have approximately 2.5 pmol/eye of retinal associated with visual pigments, but larger amounts (4-12 pmol/eye) of both retinal and retinol bound to soluble proteins. When bees are dark adapted for 24 h or longer, greater than 80% of the endogenous retinal, mostly in the all-trans configuration, is associated with the retinal photoisomerase. On exposure to blue light the retinal is isomerized to 11-cis, which makes it available to an alcohol dehydrogenase. Most of it is then reduced to 11-cis retinol. The retinol is not esterified and remains associated with a soluble protein, serving as a reservoir of 11-cis retinoid available for renewal of visual pigment. Alternatively, 11-cis retinal can be transferred directly to opsin to regenerate rhodopsin, as shown by synthesis of rhodopsin in bleached frog rod outer segments. This retinaldehyde cycle from the honeybee is the third to be described. It appears very similar to the system in another group of arthropods, flies, and differs from the isomerization processes in vertebrates and cephalopod mollusks.