Movement of retinal along the visual transduction path.

Movement of the ligand/receptor complex in rhodopsin (Rh) has been traced. Bleaching of diazoketo rhodopsin (DK-Rh) containing 11-cis-3-diazo-4-oxo-retinal yields batho-, lumi-, meta-I-, and meta-II-Rh intermediates corresponding to those of native Rh but at lower temperatures. Photoaffinity labeling of DK-Rh and these bleaching intermediates shows that the ionone ring cross-links to tryptophan-265 on helix F in DK-Rh and batho-Rh, and to alanine-169 on helix D in lumi-, meta-I-, and meta-II-Rh intermediates. It is likely that these movements involving a flip-over of the chromophoric ring trigger changes in cytoplasmic membrane loops resulting in heterotrimeric guanine nucleotide-binding protein (G protein) activation.

Formation of hypsorhodopsin at room temperature by picosecond green pulse.

Excitation of squid rhodopsin with a single laser pulse (532 nm, 25 ps) at 18 degrees C yielded photorhodopsin, a precursor of bathorhodopsin. In the linear region, no relation between amount of photorhodopsin and excitation-energy hypsorhodopsin was detected, while in a photon saturation region this was observed. The time constant of hypsorhodopsin to bathorhodopsin decay was about 125 ps. Dependencies of formation of photorhodopsin and hypsorhodopsin on the excitation energy suggest that hypsorhodopsins of squid and octopus are formed by a two-photon reaction. No cattle hypsorhodopsin was detected in our experimental conditions.

Circular dichroism of squid rhodopsin and its intermediates.

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates was measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the alpha- and beta-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at alpha-band and 348 nm at beta-band) as well as at room temperature (CD maxima: 474 nm at alpha-band and 347 nm at beta-band). The rotational strength of the alpha-band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at alpha-band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may de derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, The CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.

Identification of a new intermediate state that binds but not activates transducin in the bleaching process of bovine rhodopsin.

Using time-resolved low-temperature spectroscopy, we have examined whether or not bovine rhodopsin has a unique transducin-binding state, meta Ib, previously detected from chicken rhodopsin. Unlike chicken meta Ib, bovine meta Ib was detected only by detailed kinetics analysis of the bleaching process, but it was stabilized by transducin and visualized in the observed spectral changes. From the effect of GTPgammaS, it was revealed that meta Ib induced no GDP-GTP exchange reaction in transducin. Thus meta Ib is a common intermediate of vertebrate rhodopsin and transducin is activated in two steps by meta Ib and meta II.

Selective activation of G-protein subtypes by vertebrate and invertebrate rhodopsins.

We have quantitatively investigated specificities in activating G-protein subtype by bovine and squid rhodopsins to examine whether or not the phototransduction cascade in each of the photoreceptor cells is determined by the colocalization of a large amount of G-protein subtype (Gt or Gq). In contrast to the efficient activation of respective Gt and Gq, bovine and squid rhodopsins scarcely activated G-protein counterparts. Exchange of alpha- and betagamma-subunits of Gt and Gq indicated the critical role of the alpha-subunit in specific binding to respective rhodopsins. Thus the specific recognition of G-protein subtype by each rhodopsin is a major mechanism in determining the phototransduction cascade.

Thermal recovery of iodopsin from its meta I-intermediate.

The thermal reaction of meta I-intermediate of iodopsin (metaiodopsin I), a chicken red-sensitive cone pigment, was studied by low-temperature spectrophotometry at -20 degrees C. Irradiation of iodopsin at -20 degrees C produced metaiodopsin I, whose absorption maximum was at about 470 nm. An incubation of metaiodopsin I at -20 degrees C resulted in a conversion to metaiodopsin II having absorption maximum at about 380 nm, as well as a concurrent formation of a red-shifted product stable at room temperature. Since the absorption spectrum and photo-reactivity of the red-shifted product were identical with those of iodopsin, the red-shifted product should be iodopsin. Thus a part of metaiodopsin I can revert to iodopsin by the thermal reaction unlike metarhodopsin I.

Reason for the lack of light-dark adaptation in pharaonis phoborhodopsin: reconstitution with 13-cis-retinal.

The reconstitution of pharaonis phoborhodopsin was performed by incubation of its opsin with 13-cis-retinal. Spectrum change was very slow, and two phases of the change were observed: the first and second phases are due to the transient formation of 13-cis pigment and spontaneous isomerization to all-trans-retinal, respectively. Slow binding supports an idea that the retinal binding pocket of ppR is highly restricted. Being bent in the configuration, 13-cis-retinal cannot be accommodated in the pocket due to the steric hindrance. This is a possible reason for the lack of light-dark adaptation.

O-Glycosylation of G-protein-coupled receptor, octopus rhodopsin. Direct analysis by FAB mass spectrometry.

In addition to the N-glycan that is evidently conserved in G-protein-coupled receptors (GPCRs), O-glycans in the N-terminus of GPCRs have been suggested. Using a combination of enzymatic and manual Edman degradation in conjunction with G-protein coupled receptor mass spectrometry, the structure and sites of O-glycans in octopus rhodopsin are determined. Two N-acetylgalactosamine residues are O-linked to Thr4 and Thr5 in the N-terminus of octopus rhodopsin. Further, we found chicken iodopsin, but not bovine rhodopsin, contains N-acetylgalactosamine. This is the first direct evidence to determine the structure and sites of O-glycans in GPCRs.

The primary structure of iodopsin, a chicken red-sensitive cone pigment.

A purified iodopsin was digested by CNBr or several proteolytic enzymes into fragments, the amino acid sequences of which were determined. A partial sequence of the C-terminal fragment was utilized for synthesizing an oligonucleotide probe which identified the iodopsin cDNA (1339 bases). The deduced amino acid sequence (362 residues) had 80%, 42%, or 43% homology to that of human red-sensitive cone pigment, cattle or chicken rhodospin, respectively. Although the hydropathy profile implies that iodopsin, like rhodopsin, has 7 transmembrane alpha-helical segments, iodopsin may have a hydrophilic pocket near the seventh segment on the basis of the unexpected cleavages in the middle of the segment VII by chymotrypsin under nondenaturing conditions.

Demonstration of a rhodopsin-retinochrome system in the stalk eye of a marine gastropod, Onchidium, by immunohistochemistry.

The stalk eye of Onchidium sp. (Gastropoda, Mollusca) is the principal photoreceptor in a multiple photoreceptive system that consists of the stalk and dorsal eyes, dermal photoreceptor cells, and photosensitive neurons. To examine the localization of photopigments, the stalk eyes were immunostained with specific antibodies to rhodopsin, retinochrome, and retinal-binding protein (RALBP), which had been generated against squid retinal proteins. The retina of the stalk eye was divided into villous, pigmented, somatic, and neural layers. It was comprised mainly of two types of visual and pigmented supportive cells. The type 1 visual (VC1) cell was characterized by well-developed microvilli on its apical protrusion and photic vesicles in the cytoplasm. The photic vesicles were specifically blackened by prolonged osmification. The type 2 visual (VC2) cell had less numerous, shorter microvilli on its concave apical surface and lacked photic vesicles. The anti-squid rhodopsin antiserum was localized specifically to the villous layer that corresponded to the VC1 microvilli. With the anti-retinochrome peptide antibody, the somatic layer showed specific but patchy, positive staining that corresponded to the cytoplasm of the VC1 cells. Because the photic vesicles are known to contain retinochrome, these results indicate that this retinochrome is localized in the VC1 cytoplasm. Anti-RALBP antibody stained the supranuclear cytoplasm to the distal cytoplasm of VC1 cells. This is the first demonstration of the localization of RALBP in the Gastropoda Onchidium stalk eye. In squid retina that were immunostained as positive controls, the anti-rhodopsin antibody stained rhabdomeric microvilli, the anti-retinochrome antibody stained the inner segment and the basal region of the outer segment, and the anti-RALBP antibody stained the outer and inner segments, respectively. These results suggest that the rhodopsin-retinochrome system that has been established in cephalopod eyes is present in the Onchidium stalk eye.

Localization of iodopsin in the chick retina during in vivo and in vitro cone differentiation.

Using highly specific antibodies against a chick red-sensitive cone pigment, iodopsin, we investigated the localization of iodopsin in the developing and mature chick retina. The chick retina contains several different photoreceptor types, including a rod, a double cone with a principal and accessory cone, and four different types of single cones. Immunocytochemical observations revealed that outer segments (OS) of one of the single cones (type 1) and both cells of the double cone were strongly immunoreactive to anti-iodopsin antibodies. The Golgi regions and small vesicular structures in the inner segments (IS) of these cells also were intensely stained, indicating a continuous synthesis of iodopsin and its addition to the newly formed cone OS. In the differentiating cones of the developing but immature chick retina, iodopsin immunoreactivity was found at the plasma membranes of both the IS and the terminals (pedicles). This suggests that unidirectional transport of iodopsin to the outer segment may be established during cone differentiation. Immunostaining in the outer plexiform layer (OPL) produced two bands, suggesting that the pedicles of the double cones and type 1 single cones terminate at different positions in this layer. Application of the antibodies to a cell culture system of the chick retina revealed that cells immunoreactive to anti-iodopsin differ slightly in morphology from those reactive with anti-rhodopsin. Since antibodies to iodopsin and rhodopsin stained different types of photoreceptors in the intact chick retina, it will be possible to analyze cell lineage of rods and cones in vitro by use of these antibodies.

Amino acid residues controlling the properties and functions of rod and cone visual pigments.

The visual transduction processes in rod and cone photoreceptor cells are initiated by photon absorption by the different types of visual pigments. In relation to the functional difference between these cells, cone visual pigments in chicken retinas exhibit faster regeneration from 11-cis-retinal and opsin and faster decay of physiologically active intermediate (Meta II) than rod visual pigment, rhodopsin. Replacement of the amino acid residue at position 122 of chicken rhodopsin by the residues present in the respective cone pigments dramatically changes both the decay rate of Meta II and the rate of regeneration into those of the cone pigment-type, indicating that the residue at this position is a major determinant controlling these properties. Thus, the single replacement of amino acid residue at this position would be one of the key steps of the divergence into twilight and daylight vision.

The second cytoplasmic loop of metabotropic glutamate receptor functions at the third loop position of rhodopsin.

G protein-coupled receptors identified so far are classified into at least three major families based on their amino acid sequences. For the family of receptors homologous to rhodopsin (family 1), the G protein activation mechanism has been investigated in detail, but much less for the receptors of other families. To functionally compare the G protein activation mechanism between rhodopsin and metabotropic glutamate receptor (mGluR), which belong to distinct families, we prepared a set of bovine rhodopsin mutants whose second or third cytoplasmic loop was replaced with either the second or third loop of Gi/Go- or Gq-coupled mGluR (mGluR6 or mGluR1). Among these mutants, the mutants in which the second or third loop was replaced with the corresponding loop of mGluR exhibited no G protein activation ability. In contrast, the mutant whose third loop was replaced with the second loop of Gi/Go-coupled mGluR6 efficiently activated Gi but not Gt: this activation profile is almost identical with those of the mutant rhodopsins whose third loop was replaced with those of the Gi/Go-coupled receptors in family 1 [Yamashita et al. (2000) J. Biol. Chem. 275, 34272-34279]. The mutant whose third loop was replaced with the second loop of Gq-coupled mGluR1 partially retained the Gi coupling ability of rhodopsin, which is in contrast to the fact that all the rhodopsin mutants having the third loops of Gq-coupled receptors in family 1 exhibit no detectable Gi activation. These results strongly suggest that the molecular architectures of rhodopsin and mGluR are different, although the G protein activation mechanism involving the cytoplasmic loops is common.

Regulatory mechanism for the stability of the meta II intermediate of pinopsin.

Pinopsin is a chicken pineal photoreceptive molecule with a possible role in photoentrainment of the circadian clock. Sequence comparison among members of the rhodopsin family has suggested that pinopsin might have properties more similar to cone visual pigments than to rhodopsin, but the lifetime of the physiologically active intermediate (meta II) of pinopsin is rather similar to that of metarhodopsin II, which is far more stable than meta II intermediates of cone visual pigments [Nakamura, A. et al., (1999) Biochemistry 38, 14738-14745]. In the present study, we investigated the amino acid residue(s) contributing to this unique property of pinopsin by using site-directed mutagenesis to pinopsin-specific structural features, (i) Ser171, (ii) Asn184, and (iii) the second extracellular loop two-amino acids shorter than that of cone visual pigments. The meta II stability of the 171/184 double mutant of pinopsin (S171R/N184D) is almost the same as that of wild-type pinopsin. In contrast, the meta II lifetime is markedly shortened (one third) by introduction of the third mutation (replacement of a six-amino acid stretch, 188-193, by the corresponding eight residues of chicken green-sensitive cone pigment) to the 171/184 double mutant of pinopsin. Consistently, meta II of the green-sensitive pigment mutant, in which the eight-amino acid stretch is inversely replaced by the corresponding six residues of pinopsin, is more stable than meta II of the wild-type pigment. These results strongly suggest that the specific sequence and/or the number of residues at amino acids 188-193 in pinopsin play an important role in the stabilization of the meta II intermediate.

Differentiation of both rod and cone types of photoreceptors in the in vivo and in vitro developing pineal glands of the quail.

The avian pineal is a photo-endocrinal organ and is considered to synthesize and secrete melatonin in an intrapineal rhythm which can be modified by direct light stimulation of the pineal photoreceptors. Since the avian retina contains numerous different types of photoreceptors, at least 6 types in the quail retina, it is interesting to ask how many types of photoreceptors are present in the avian pineal. In the present study, we have identified two types of photoreceptors in the quail pineal organ, one appears rod-like and the other cone-like, using an immunohistochemical method with highly specific anti-chicken rhodopsin and anti-iodopsin monoclonal antibodies. Rhodopsin-immunoreactive (Rho-I) cells were much larger in number than iodopsin-immunoreactive (Iodo-I) cells. During pineal development, Rho-I cells were first observed at embryonic day 13 (E13: 13 days of incubation), whereas Iodo-I cells were found at day E15. Rho-I cells showed numerous neurite-like processes, but Iodo-I cells had few, if any, processes. We developed a new culture system for avian pineal cell differentiation by seeding cells on nitrocellulose membrane filters. By this method both types of pineal photoreceptors differentiated in vitro: Rho-I cells were much larger in number and had much more fine processes than Iodo-I cells, similar to those seen in the intact developing pineal. With the new culture system the relation between pineal photoreceptor differentiation and sympathetic innervation was examined in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Dependency of photon density on primary process of cattle rhodopsin.

The primary photochemical reactions of cattle rhodopsin suspended in H2O or D2O were compared between excitation with both a weak and an intense picosecond laser pulse (wavelength, 532 nm; duration, 25 ps) at room temperature. The time-dependent change of absorbance at about 575 nm demonstrated that photohodopsin, a precursor of bathorhodopsin, was produced immediately after the excitation with a weak picosecond laser pulse. It decayed to bathorhodopsin with a time constant of 45 ps which is close to the value reported previously [Shichida et al., (1984) Photobiochem. Photobiophys., 7, 221-228]. No deuterium effect was observed in this process. Excitation with an intense laser pulse induced instantaneous increase of the absorbance at about 575 nm and remained at almost constant level on the picosecond time scale, which was in good agreement with the pioneering work [Busch et al., (1972) Proc. Natl. Acad. Sci., USA, 69, 2802-2806]. No deuterium effect was observed in this photochemical process.

Photosensitivities of iodopsin and rhodopsins.

The relative photosensitivity and the molar extinction coefficient of a highly purified iodopsin (chicken red sensitive cone visual pigment) solubilized in a mixture of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and phosphatidylcholine (CHAPS-PC) were measured using bovine rhodopsin solubilized in 2% digitonin as a standard and compared with those of chicken and bovine rhodopsins. The photosensitivity obtained (1.08) was close to those of rhodopsins (chicken, 1.04; bovine, 0.99) in CHAPS-PC. The molar extinction coefficient of iodopsin (47,200) was 1.15-1.17 times higher than those of rhodopsins (chicken, 40,500; bovine, 41,200). The oscillator strength of iodopsin (0.60) calculated from the extinction coefficient was nearly identical to that of chicken rhodopsin (0.61), suggesting that the chromophore of iodopsin is similar in configuration to rhodopsin. In contrast, the difference in quantum yield between iodopsin (0.62) and chicken rhodopsin (0.70) suggests that the chromophore-opsin interaction after absorption of a photon by the chromophore may be different.

Photoreceptor cell types in the retina of various vertebrate species: immunocytochemistry with antibodies against rhodopsin and iodopsin.

Types of photoreceptor cells in the retinas of 36 species of vertebrates (5 classes, 14 orders) were investigated immunocytochemically with monoclonal antibodies against chicken iodopsin (Io-mAb) and antiserum against bovine rhodopsin (Rh-As). In mammals, Rh-As labeled the outer segments of some photoreceptor cells in striped squirrels (a diurnal mammal) and those of most photoreceptor cells in mice (a nocturnal mammal), while Io-mAb labeled any photoreceptor cells in either of them. In all species of birds studied, Io-mAb labeled the principal and accessory members of double cones and single cones with a red oil droplet. Rh-As labeled single cones with a yellow or clear oil droplet in addition to rods. In turtles, both Rh-As and Io-mAb labeled single cones with a red or clear oil droplet and the principal (with a yellow oil droplet) and accessory members of double cones. This suggests that the visual pigments in these cones of turtles have common epitopes with bovine rhodopsin and chicken iodopsin. In Japanese grass lizards, single cones with a yellow oil droplet and double cones were immunoreactive to both Rh-As and Io-mAb. In snakes, rods and cones could not be distinguished but both positively and negatively stained cells were observed by the use of each antibody. In geckos, however, all photoreceptor cells were immunonegative to Io-mAb. In all species studied in amphibians, Rh-As labeled rods but not cones. Neither rods nor cones reacted with Io-mAb. In fishes, almost all species studied had well developed cones, and some of these cones were labeled by Rh-As. However, Io-mAb labeled the outer segments of some cones only in loaches. Rh-As labeled photoreceptor cells in all species of fishes studied. Thus, Rh-As recognized the outer segments of rods in all species studied from fishes to mammals, whereas the epitope recognized by Io-mAb is conserved in some species of fishes, most species of reptiles and all species of birds studied.

Shape of the chromophore binding site in pharaonis phoborhodopsin from a study using retinal analogs.

To investigate the shape of the chromophore binding site of pharaonis phoborhodopsin (ppR), ppR-opsin was incubated with five ring-modified retinal analogs: an acyclic retinal, phenylretinal, alpha-retinal, cyclohexylretinal and 5-isopropyl-alpha-retinal. The experimental results were compared with those obtained from bacteriorhodopsin-opsin (bR-opsin) and the same retinal analogs. It was suggested that ring chain conformation is important in affecting the spectral shoulder unique for the absorption spectrum of ppR. The rate of pigment formation depended greatly on the analogs used with the planar analogs showing rapid formation. Thus, we concluded that the space of the retinal binding site of ppR is restricted to the plane of the cyclohexenyl ring of the chromophore, whereas that of bR is less restricted.

Probing for the threshold energy for visual transduction: red-shifted visual pigment analogs from 3-methoxy-3-dehydroretinal and related compounds.

While azulenic retinal analogs failed to yield a red-shifted visual pigment analog, the 9-cis isomers of the push-pull polyenals 3-methoxy-3-dehydroretinal and 14F-3-methoxy-3-dehydroretinal yielded iodopsin pigment analogs with absorption maxima at, respectively, 663 and 720 nm. The former gave a relatively stable batho product (700 nm) and was able to activate transducin. A lower activity was observed for the latter. One possible explanation for the combined results is that the excitation energies of these red-shifted pigments are approaching the threshold energy for visual transduction (although at this time we cannot rigorously exclude a role of the added F-atom in reducing the transducin activity).