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.

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.

Interaction of GTP-binding protein Gq with photoactivated rhodopsin in the photoreceptor membranes of crayfish.

Interaction of G-protein with photoactivated rhodopsin (Rh*) in crayfish photoreceptor membranes was investigated by immunoprecipitation using an antibody against rhodopsin. Two kinds of protein were co-precipitated with rhodopsin. One is an alpha subunit of class-q G-protein (42 kDa, CGq alpha) which showed light-induced, dose-dependent binding to rhodopsin, and the other is an actin-like protein (44 kDa) with light-independent binding. Most of the CGq alpha was available for binding to Rh* but was dissociated from Rh* in the presence of GTP gamma S. These findings demonstrate that, in the crayfish photoreceptor, a Gq class of G-protein is activated by Rh*.

Squid photoreceptor phospholipase C is stimulated by membrane Gq alpha but not by soluble Gq alpha.

Phospholipase C (PLC) was purified from squid retina. Soluble Gq alpha, membrane Gq alpha and G beta gamma were isolated from GTP gamma S-treated and light-illuminated photoreceptor membranes. The membrane Gq alpha stimulated phosphatidyl inositol-phospholipase C (PI-PLC) activity in a dose-dependent manner. Soluble Gq alpha and membrane G beta gamma showed no stimulating effects on PLC. GTP gamma S-binding was found exclusively in membrane fraction, with very little present in the KCl-soluble fraction which contained soluble Gq alpha. These results indicate that light-activated rhodopsin activates PLC through membrane-bound Gq alpha and suggest that the rhodopsin/Gq/PLC cascade might be the pathway of phototransduction in squid photoreceptors.

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.

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.

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).

Retinal-binding protein as a shuttle for retinal in the rhodopsin-retinochrome system of the squid visual cells.

The molluscan visual cell is characterized by having two photopigment systems, rhodopsin and retinochrome. In connection with these systems, located separately in the rhabdomal microvilli and in the nucleated cell bodies, the physiological role of retinal-binding protein (RALBP) was investigated in the squid (Todarodes pacificus) by using 3-dehydroretinal (retinal 2) as a tracer for retinal chromophore. In dark-adapted eyes, squid RALBP is combined abundantly with 11-cis-retinal. However, upon incubation with an excess of all-trans-retinal or retinol, RALBP took up great amounts of each of them, releasing its native retinoid ligands. When an all-trans-retinal-rich RALBP thus produced was incubated in the dark with metaretinochrome 2-carrying membranes, the RALBP released all-trans-retinal to the membranes to regenerate retinochrome, taking up 11-cis-retinal 2 from metaretinochrome 2. Upon further incubation of this 11-cis-retinal 2-rich RALBP with metarhodopsin-carrying membranes, the RALBP released the 11-cis-retinal 2 to the membranes to form rhodopsin 2, receiving all-trans-retinal from metarhodopsin. These findings show that squid RALBP is capable of serving as a shuttle during the recycling of retinal in the rhodopsin-retinochrome conjugate system to maintain the photoreceptive function of the visual cells.

Rhodopsin and retinochrome in the retina of a marine gastropod, Conomulex luhuanus.

Photopigments in the conch retina were examined with special attention given to the photic vesicles characteristic of gastropod photoreceptors. Three different fractions of visual cell fragments were prepared from the retina: the MV-fraction containing the rhabdomal microvilli, and the PVH- and PVL-fractions containing the photic vesicles located in the visual cell body. Rhodopsin was found in the MV-fraction (lambda max = 474 nm), and yielded a photoequilibrium mixture with metarhodopsin (lambda max = 512 nm) on irradiation with blue light. Retinochrome was found in both of the PVH- and PVL-fractions (lambda max = approximately 510 nm), and was bleached into metaretinochrome by exposure to orange light, showing no marked shift of the absorption peak. Unlike the PVH-fraction, the PVL-fraction contains much aporetinochrome in addition to retinochrome, suggesting that the large mass of photic vesicles around the nucleus may serve as storage for retinal in retinochrome and for newly synthesized aporetinochrome. The total amount of retinochrome in the retina was several times higher than that of rhodopsin, distinguishing the gastropod eye from the cephalopod eye.

Isolation and characterization of a retinal-binding protein from the squid retina.

A retinal-binding protein (RALBP) was isolated from the squid retina, and purified by anion-exchange and size-exclusion chromatography. The molecular weight was determined to be 51,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration. The purified sample showed absorption maxima at about 330 and 400 nm in addition to a protein band, indicating the occurrence of retinol and retinal, respectively. The relative heights of these two peaks varied from preparation to preparation, depending on retinoid ligands. Irradiation of RALBP caused no marked change in absorption, but the amount of 11-cis-retinal decreased to form a photosteady state mixture with all-trans- and 13-cis-retinals. RALBP was fairly stable even in the presence of hydroxylamine (100 mM), but was affected by sodium borohydride (30 mM) or borane dimethylamine (400 mM), with the retinal reduced to retinol. When incubated with metaretinochrome-carrying membranes in the dark, RALBP specifically took up 11-cis-retinal and lost all-trans-retinol. Upon further incubation of this RALBP with opsin-containing membranes, rhodopsin was progressively formed in the dark. Squid RALBP may act as a shuttle in transferring the 11-cis-retinal from metaretinochrome to opsin in the visual cells.

Light-induced binding of proteins to rhabdomeric membranes in the retina of crayfish (Procambarus clarkii).

Light-induced protein interaction as part of the process of visual transduction in arthropods with rhabdomeric photoreceptors was investigated biochemically by using crayfish retina. Two kinds of retinal buffer soluble proteins (one of 40 kDa and the other of 46 kDa) were found to bind to the irradiated rhabdomeric membranes both in vitro and in vivo. The proteins bound to the membranes in the presence of metarhodopsin. An antibody against mouse arrestin (S-antigen) cross-reacted with the 40 kDa protein. These results suggest that the binding of the proteins to the membranes is caused by the formation of metarhodopsin, and that the 40 kDa protein has a similar structure to arrestin.

High-performance frontal analysis-high-performance liquid chromatographic system for stereoselective determination of unbound ketoprofen enantiomers in plasma after direct sample injection.

An on-line, high-performance frontal analysis (HPFA)-high-performance liquid chromatographic system was developed for the enantioselective determination of a low level of unbound ketoprofen (KP) that is in equilibrium with KP that is bound to protein. The system consists of three subsystems (HPFA system, preconcentration system, and chiral separation system) connected in series in the stated order via column-switching valves. When either a 300-microL portion of buffer solution containing 300 or 550 microM human serum albumin and 100 or 300 microM racemic KP or a 300-microL portion of human plasma containing 12.5-100 microM racemic KP was directly injected onto the HPFA column with the mobile phase at a low flow rate, KP was separated from proteins and eluted as a zonal peak with a plateau. The KP concentration in the eluant of the plateau region was the same as the unbound-KP concentration that was in equilibrium with protein-bound KP in the initial sample solution. A 1-mL portion of the eluant of the plateau region was switched to the preconcentration system, where KP was adsorbed and condensed on an octadecylsilyl silica (ODS) column. The adsorbed KP was eluted out of the ODS column and transferred to the chiral separation system, via another switching valve, where the enantiomers of unbound KP were separated and determined. The results agree well with those obtained by the conventional ultrafiltration method.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the unbound concentration of hydrophobic drugs in albumin solutions by high-performance frontal analysis using a diol-silica column.

High-performance frontal analysis (HPFA) using diol-silica column allows the determination of the unbound concentration of hydrophobic drugs under protein binding equilibrium, which is often difficult by the conventional methods. After the direct injection of a drug-protein mixed solution onto a diol-silica column, the drug is eluted as a zonal peak containing a plateau region under a mild mobile phase condition (phosphate buffer, pH 7.4, ionic strength = 0.17). The unbound drug concentration was determined as the drug concentration in the plateau region. The reliability of this method was confirmed by comparison with the results obtained by the conventional ultrafiltration-HPLC method. The versatility of the HPFA method using a diol-silica column was demonstrated by developing a novel on-line HPFA-chiral HPLC system for a simple and easy determination of the unbound concentration of nilvadipine (NV) enantiomers. The direct injection of 1.33 mL of a sample solution containing 20 nM-1 microM NV and 550 microM HSA gave the plateau region due to unbound NV, which was heart-cut and transferred on-line into a preconcentration and chiral HPLC separation system. As low as a few hundred picomolar level of unbound NV enantiomers was determined by use of UV detection (at 244 nm). The binding between NV and HSA is enantioselective; (R)-NV binds more strongly than (S)-NV.

Distinct roles of the second and third cytoplasmic loops of bovine rhodopsin in G protein activation.

In contrast to the extensive studies of light-induced conformational changes in rhodopsin, the cytoplasmic architecture of rhodopsin related to the G protein activation and the selective recognition of G protein subtype is still unclear. Here, we prepared a set of bovine rhodopsin mutants whose cytoplasmic loops were replaced by those of other ligand-binding receptors, and we compared their ability for G protein activation in order to obtain a clue to the roles of the second and third cytoplasmic loops of rhodopsin. The mutants bearing the third loop of four other G(o)-coupled receptors belonging to the rhodopsin superfamily showed significant G(o) activation, indicating that the third loop of rhodopsin possibly has a putative site(s) related to the interaction of G protein and that it is simply exchangeable with those of other G(o)-coupled receptors. The mutants bearing the second loop of other receptors, however, had little ability for G protein activation, suggesting that the second loop of rhodopsin contains a specific region essential for rhodopsin to be a G protein-activating form. Systematic chimeric and point mutational studies indicate that three amino acids (Glu(134), Val(138), and Cys(140)) in the N-terminal region of the second loop of rhodopsin are crucial for efficient G protein activation. These results suggest that the second and third cytoplasmic loops of bovine rhodopsin have distinct roles in G protein activation and subtype specificity.

Highly conserved glutamic acid in the extracellular IV-V loop in rhodopsins acts as the counterion in retinochrome, a member of the rhodopsin family.

Retinochrome is a member of the rhodopsin family having a chromophore retinal and functioning as a retinal photoisomerase in squid photoreceptor cells. Unlike vertebrate rhodopsins, but like many invertebrate rhodopsins, retinochrome does not have a glutamic acid at position 113 to serve as a counterion for the protonated retinylidene Schiff base. Here we investigated possible counterions in retinochrome by site-specific mutagenesis. Our results showed that the counterion is the glutamic acid at position 181, at which almost all the pigments in the rhodopsin family, including vertebrate and invertebrate rhodopsins, have a glutamic or aspartic acid. The remarkable exceptions are the long-wavelength visual pigments that have a histidine that, together with a nearby lysine, serves as a chloride-binding site. Replacement of Glu-181 of bovine rhodopsin with Gln caused a 10-nm red-shift of absorption maximum. Because the position at 181 is in the extracellular loop connecting the transmembrane helices VI and V, these results demonstrate the importance of this loop to function for spectral tuning in the rhodopsin family.

Water and peptide backbone structure in the active center of bovine rhodopsin.

Difference FTIR spectra in the conversion of rhodopsin or isorhodopsin to bathorhodopsin were recorded for recombinant wild-type and E113Q bovine rhodopsins. Differences in various vibrational modes between E113Q and the wild-type proteins whose Schiff bases interact with chloride and Glu113, respectively, were analyzed. Water molecules in rhodopsin that change upon formation of bathorhodopsin are detected by a change in frequency of the O-H stretching vibration from 3538 to 3525 cm(-1). This change in the wild-type protein is absent in E113Q. One or a few water molecules are therefore suggested to be located in the proximity of Glu113, the counterion of the Schiff base. Another water vibration at 3564 cm(-1), which is shifted to 3542 cm(-1) in bathorhodopsin in the wild type, persists in E113Q but with approximately 5-cm(-1) shift toward higher frequency. This is due to water molecules that may be located at a site somewhat more remote from Glu113. Structural changes of some peptide carbonyls and amides are also absent in E113Q. On the other hand, the E113Q protein shows shifts of the N-H+ stretching vibrational band, that is probably due to the protonated Schiff base, upon conversion of rhodopsin to bathorhodopsin. No corresponding changes were observed in the wild type. We propose a model in which a water molecule interacts with Glu113, the protonated Schiff base, and peptide carbonyls, and amides. These residues undergo structural changes upon formation of bathorhodopsin.