Fumio Tokunaga

A novel subtype of G-protein-coupled receptor kinase, GRK7, in teleost cone photoreceptors

Two kinds of retinal cDNA fragments (OlGRK‐R and ‐C) encoding the putative G‐protein‐coupled receptor kinases (GRKs) were isolated from medaka, Oryzias latipes. OlGRK‐R appears to be closely related to the rhodopsin kinase (RK) found in the outer segments of mammalian photoreceptors, but the deduced amino acid sequence of OlGRK‐C shows less than 50% identity to those of GRKs known to date, suggesting that OlGRK‐C is a novel GRK subtype (GRK7). The mRNA of OlGRK‐R is detectable in rods, and that of OlGRK‐C is found in all four types of cone photoreceptor. The C‐terminal of OlGRK‐R has a consensus sequence for farnesylation, whereas, surprisingly, OlGRK‐C has a consensus sequence for geranylgeranylation. Our result are consistent with the concept that lower vertebrates have rod‐ and cone‐specific opsin kinases.

The primary structure and distribution of killifish visual pigments

Five cDNA fragments (KFH-R, -V, -G, -B and -Rh) encoding the putative visual pigments of killifish were isolated and sequenced. Judging from the deduced amino acid sequences, each cDNA falls into a different group of the five major families of vertebrate visual pigment genes. In situ hybridization localized the mRNA of KFH-R and -G to the principle and accessory members, respectively, of double cones. Visual pigment genes KFH-Rh, -B and -V were expressed in the rods, and the long and short single cones, respectively. It is suggested that the relationships between the cell types and their respective visual pigment gene groups may be a common pattern among teleost fishes.

Evidence for Proton Transfer from Glu-46 to the Chromophore during the Photocycle of Photoactive Yellow Protein

Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at −40u2009°C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995)Biochemistry 34, 12669–12672) were observed at 1482, 1437, and 1163 cm−1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm−1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm−1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 → Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM.

Primary structure of a visual pigment in bullfrog green rods.

In frog retina there are special rod photoreceptor cells (‘green rods’) with physiological properties similar to those of typical vertebrate rods (‘red rods’). A cDNA fragment encoding the putative green rod visual pigment was isolated from a retinal cDNA library of the bullfrog, Rana catesbeiana. Its deduced amino acid sequence has more than 65% identity with those of blue‐sensitive cone pigments such as chicken blue and goldfish blue. Antisera raised against its C‐terminal amino acid sequence recognized green rods. It is concluded that bullfrog green rods contain a visual pigment which is closely related to the blue‐sensitive cone pigments of other non‐mammalian vertebrates.

Primary structure and characterization of a bullfrog visual pigment contained in small single cones

A cDNA fragment encoding a putative visual pigment (FCV pigment) was isolated from the bullfrog, Rana catesbeiana. Its deduced amino acid sequence shows high similarities to those of short wavelength-sensitive pigments such as human blue-, chicken violet- and goldfish ultraviolet-sensitive pigments. An antiserum against its C-terminal amino acid sequence recognized the outer segments of small cone photoreceptor cells without oil droplets. It is suggested that the FCV pigment is a short wavelength-sensitive pigment contained in small single cones which have not been characterized previously.

Evolution of visual pigments in geckos

Most geckos are nocturnal forms and possess rod retinas, but some diurnal genera have pure‐cone retinas. We isolated cDNAs encoding the diurnal gecko opsins, dg1 and dg2, similar to nocturnal gecko P521 and P467, respectively. Despite the large morphological differences between the diurnal and nocturnal gecko photoreceptor types, they express phylogenetically closely related opsins. These results provide molecular evidence for the reverse transmutation, that is, rods of an ancestral nocturnal gecko have backed into cones of diurnal geckos. The amino acid substitution rates of dg1 and dg2 are higher than those of P521 and P467, respectively. Changes of behavior regarding photic environment may have contributed to acceleration of amino acid substitutions in the diurnal gecko opsins.

Arrestins expressed in killifish photoreceptor cells

Two kinds of cDNA fragments (KfhArr‐R and KfhArr‐C) encoding the putative arrestins of killifish, Oryzias latipes, were isolated. The distributions of these transcripts were investigated by in situ hybridization, and it was demonstrated that KfhArr‐R and KfhArr‐C are expressed in, respectively, rod and all four types of cone cells. The deduced amino acid sequences of KfhArr‐R and KfhArr‐C are closely related to human S‐antigen (rod arrestin) and X‐arrestin (cone arrestin), respectively. Phylogenetic analysis of arrestin sequences suggests that vertebrate visual arrestins form a single cluster distinct from other arrestins and diverged to form rod and cone subtypes before the divergence between teleosts and tetrapods. It is speculated that the divergence pattern of vertebrate visual arrestins may prove to be reflected in the divergence of the proteins participating in the respective phototransduction cascades.

The Role of Calcium-binding Sites in S-modulin Function

S-modulin controls rhodopsin phosphorylation in a calcium-dependent manner, and it has been suggested that it modulates the light sensitivity of the photoreceptor cell. S-modulin binds to the ROS membrane at high Ca2+concentration, and N-terminal myristoylation is necessary for this property (the calcium-myristoyl switch). S-modulin has four EF-hand motifs, of which two (EF-2 and -3) are functional. Here, we report on the roles of EF-2 and -3 in S-modulin function (calcium binding, membrane association, and inhibition of rhodopsin phosphorylation) by site-directed mutants (E85M and E121M). Surprisingly, E121M, which has a mutation in EF-3, neither binds Ca2+ nor inhibits phosphorylation. In contrast, E85M binds one Ca2+ and has the same membrane affinity as wild-type S-modulin, but has lost the ability to inhibit rhodopsin phosphorylation. It is suggested that the binding of Ca2+to EF-3 is probably required for EF-2 to be a functional Ca2+-binding site and to induce exposure of the myristoyl group; and that the binding of Ca2+ to EF-2 is important for the interaction with rhodopsin kinase.

Characterization of lamprey rhodopsin by isolation from lamprey retina and expression in mammalian cells.

Abstract— A visual pigment was extracted from lamprey retina and was expressed in cultured mammalian cells (293S) using a cDNA fragment isolated from lamprey retina. The extracted pigment, a putative lamprey rhodopsin, had an absorption maximum at 503 nm. The recombinant lamprey rhodopsin, reconstituted with 11‐cis‐retinal, showed an absorption maximum at about 500 nm. Both pigments reacted with an anti‐bovine rhodopsin antibody (Rh29), which recognizes the short photoreceptor cells in lamprey retina. Unlike rhodopsins of higher vertebrates, the lamprey rhodopsin bleached gradually in the presence of 100 mM hydroxylamine even in the dark. Our results suggest that, despite its high similarities with other vertebrate rhodopsins, lamprey rhodopsin has a character different from those of higher vertebrates.

X-Ray Diffraction Studies of Bacteriorhodopsin. Determination of the Positions of Mercury Label at Several Engineered Cysteine Residues

Abstract— The single cysteine‐containing bacteriorhodopsin mutants F27C, L100C, T170C, F171C and I222C were labeled with p‐chloromercuribenzoic acid, which specifically reacts with sulfhydryl groups. These cysteines should be located at the cytoplasmic ends of the transmembrane helices A, C, F or G. We determined the positions of the bound mercury atoms by X‐ray diffraction of purple membrane films, with better than 1 Å accuracy. The determined mercury positions were compared with the structural model from cryoelectron microscopy (N. Grigorieff, T. A. Ceska, K. H. Downing, J. M. Baldwin and R. Henderson, J. Mol. Biol 259, 393‐421, 1996). Given that the distance between the mercury and the Cα atom of the cysteine in the xy plane must be shorter than 4.5 Å and that the mercury atom is located at the δ position, the positions obtained for the mercury labels agree with their expected positions from the structural model. The present results give a rationale for detecting structural changes upon illumination as shifts occur in the mercury label position.