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Y Fukada

Publications and source records attributed to Y Fukada.

At least 91 records · Page 5Linked to original sources

Calcium-bound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation.

In rod photoreceptor cells, Ca2+-bound recoverin associates with disk membranes and inhibits light-dependent phosphorylation of rhodopsin. However, the functional significance of Ca2+-induced membrane association of recoverin has not been fully evaluated. We found that Ca2+-bound recoverin forms a complex with rhodopsin kinase preferentially at the membrane surface. Addition of increasing amounts of membranes promoted the membrane association of recoverin, and remarkably suppressed rhodopsin kinase activity. It was concluded that the Ca2+-recoverin-rhodopsin kinase complex is stabilized by membrane association, leading to effective suppression of the kinase activity.

Animals↗

DSP-4 treatment influences olfactory preferences of developing rats.

Control cagemates of rats treated with the norepinephrine (NE) neurotoxin DSP-4, showed normal olfactory learning as infants, but abnormal aversion to home-cage odors as juveniles. Neither age nor social housing conditions influenced the odor preferences of DSP-4-treated rats: they showed tolerance or attraction to familiar odors at both developmental stages. Controls, but not DSP-4-treated juveniles, housed in mixed treatment groups, showed elevated concentrations of a serotonin metabolite and reduced NE concentrations in the hippocampus, suggesting that this social situation was particularly stressful for the controls. DSP-4-treated juveniles, but not infants, produced odors that were discriminable from controls'. Thus, conflicting olfactory signals in the home-cages of mixed juvenile groups may have led to the development of stress in controls. NE depletion appeared to lessen social stress effects in their DSP-4-treated cagemates. These findings support other data suggesting that NE modulates the biobehavioral effects of the social environment.

Adrenergic Agents↗

Molecular properties of chimerical mutants of gecko blue and bovine rhodopsin.

In spite of the high similarity in amino acid sequence between rod visual pigment rhodopsin and gecko blue-sensitive pigment (gecko blue), not only the spectral sensitivities but also the thermal decay rates of the meta II- and III-intermediates are noticeably different from one another [Kojima et al. (1995) Biochemistry 34, 1096-1106]. In order to identify the protein region(s) that contain(s) key residue being responsible for the functional difference, we constructed six chimerical mutants derived from gecko blue and bovine rhodopsin, with the aid of protein production in a human embryonic kidney cell line (293S). While the absorption maximum of every mutant was located in between gecko blue (466 nm) and bovine rhodopsin (500 nm), a large blue-shift (18 nm) was observed when the helices I-III of rhodopsin were replaced with those of gecko blue. A time resolved spectroscopic study demonstrated that this replacement also accelerated the decay rate of the meta II-intermediate. The decay of the meta III-intermediate of the mutants became faster as the compartment of gecko blue was increased. Thus, the faster decay of the meta II-intermediate of gecko blue is largely attributed to residues within helices I-III, while the decay of the meta III-intermediate apparently depends on the overall structure of the protein.

Animals↗

Preparation and characterization of monoclonal antibodies specific for lauroylated isoform of bovine transducin alpha-subunit: immunohistochemical analysis of bovine retinas.

The photoreceptor G protein transducin [alpha- and beta gamma-subunits (T alpha/T beta gamma)] plays a central role in the visual transduction process. The amino-terminus of bovine T alpha is modified by one of four distinct fatty acids-laurate (C12:0), myristate (C14:0), C14:1 (5-cis), and C14:2 (5-cis, 8-cis)-but the biological significance and the localization of the four isoforms of T alpha are poorly understood. To investigate the cellular distribution of each isoform, we prepared monoclonal antibodies against a synthetic C12:0-, C14:0-, C14:1-, or C14:2-nonapeptide corresponding to the N-terminal region of T alpha. Among several types of antibodies isolated, only one type, represented by LA4, reacted specifically with the C12:0-peptide as well as purified T alpha but not with the other proteins in bovine retinal homogenate, including recoverin, indicating that the epitope comprises both C12:0 and the N-terminal amino acids of T alpha. Immunohistochemical analyses of bovine retinal sections by LA4 showed the uniform distribution of C12:0-T alpha in almost all the rod outer segments. Hence, it seemed unlikely that each isoform of T alpha was localized in specific cells. This observation, together with evidence for a possible functional diversity among the isoforms, suggests that the four isoforms of T alpha in a single rod cell may contribute simultaneously to a fine tuning of the photon-signal transduction process.

Acylation↗

The differences in the expressions of visual pigments and transducin in photoreceptor cell differentiation.

The distribution and accumulation of visual pigments, i.e., rod pigment, rhodopsin and red sensitive cone pigment, iodopsin, and transducin in the retina of chicken and chicken embryo were investigated immunohistochemically using their specific antibodies. The immunoreactivities of these proteins appeared at the early stage of photoreceptor differentiation (embryonal day 15) and increased in the photoreceptor cells appeared to reach maximum at the end of the embryonal period (embryonal day 20). On the other hand, although the immunoreactivity of beta gamma subunit of transducin (T beta gamma) was detected at embryonal day 15, the expression level of T beta gamma still remained in low level during the embryonal period. These observations suggest that both T beta gamma and visual pigments are expressed during the embryonic period in chicken photoreceptor cells, but their accumulations in the cells are different.

Animals↗

Primary structure of a gamma subunit of G protein, gamma 12, and its phosphorylation by protein kinase C.

We have determined the primary structure of a novel gamma subunit (gamma 12, previously designated gamma S1) of G protein purified from bovine spleen. The mature gamma 12 protein composed of 68 amino acids had acetylated serine at the N terminus and geranylgeranylated/carboxylmethylated cysteine at the C terminus. This was consistent with the C-terminal prenylation signal in the amino acid sequence, which was predicted from gamma 12 cDNA isolated from a bovine spleen cDNA library. Western blots with the specific antibody against gamma 12 showed that gamma 12 is present in all tissues examined. Among various gamma subunits (gamma 1, gamma 2, gamma 3, gamma 7, and gamma 12), gamma 12 has a unique property to be phosphorylated by protein kinase C. The phosphorylated amino acid residue was Ser1 (or Ser2). The phosphorylated beta gamma 12 associated with Go alpha more tightly than the unphosphorylated form. Exposure of Swiss 3T3 and aortic smooth muscle cells to phorbol 12-myristate 13-acetate and NaF induced phosphorylation of gamma 12. Stimulation of aortic smooth muscle cells with natural vasoactive agents such as angiotensin II and vasopressin also induced phosphorylation of gamma 12. The extent of phosphorylation of beta gamma 12 in vitro was suppressed by a complex formation with Go alpha, which was relieved by the addition of guanosine 5'-O-(3-thiotriphosphate) or aluminum fluoride. These results strongly suggest that gamma 12 is phosphorylated by protein kinase C during activation of receptor(s) and G protein(s) in living cells.

Amino Acid Sequence↗

Role of heterogeneous N-terminal acylation of recoverin in rhodopsin phosphorylation.

Recoverin, a new member of the EF-hand superfamily, plays a critical role in the light/dark adaptation of retinal rods by regulating rhodopsin phosphorylation in a Ca(2+)-dependent manner. Recoverin is composed of four isoforms, each of which is modified at its N terminus by myristate (C14:0) or its structurally related fatty acid (C12:0, C14:2, or C14:1). Although the N-fatty acylation is implicated in protein-membrane and protein-protein interactions, the functional difference among the recoverin isoforms and the significance of the heterogeneous acylation have not been defined. Here we separated the heterogeneous recoverin into three fractions, C14:0-recoverin, C14:1-recoverin, and a mixture of C14:2- and C12:0- (C14:2/C12:0-) recoverin to evaluate the individual properties. Recoverin in every fraction bound Ca2+ as assessed by fluorescence spectroscopy and inhibited the light-dependent rhodopsin phosphorylation in the same range of free Ca2+ concentration (0.3-0.8 microM). However, the magnitude of the inhibition at higher Ca2+ concentration was different among the isoforms and ranked in the same order of the hydrophobicity of the N-fatty acyl groups: C14:0 > C14:1 > C14:2/C12:0. These results indicate that the diverged hydrophobicity of the recoverin N terminus plays an important role in the interaction with the membranes and/or its target protein but not with Ca2+.

Acylation↗

Purification and low temperature spectroscopy of gecko visual pigments green and blue.

We purified two kinds of visual pigments, gecko green and gecko blue, from retinas of Tokay geckos (Gekko gekko) by two steps of column chromatography, and investigated their photobleaching processes by means of low temperature spectroscopy. Absorption maxima of gecko green and blue solubilized in a mixture of 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS) and phosphatidylcholine were 522 and 465 nm, respectively, which are close to those observed in the photoreceptor cells. Low temperature spectroscopy identified six intermediates in the photobleaching process of gecko green; batho (lambda max = 569 nm), BL (lambda max = 519 nm), lumi (507 nm), meta I (approximately 486 nm), meta II (approximately 384 nm), and meta III intermediates (approximately 500 nm). In contrast to the high similarity in amino acid sequence between gecko green and iodopsin [Kojima, D., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6841-6845], the batho-green did not revert thermally to original gecko green but converts to the next intermediate. The photobleaching process of gecko blue was investigated by low temperature spectroscopy, and three intermediates, meta I (lambda max = approximately 470 nm), meta II (lambda max = approximately 370 nm) and meta III (lambda max = approximately 475 nm), were identified. A comparative study on the thermal behavior of meta intermediates revealed that the thermal stability of meta II intermediate of both of the gecko visual pigments is lower than that of metarhodopsin II. The result supports the idea that both the gecko visual pigments are cone-type ones.

Animals↗

Molecular basis for tetrachromatic color vision.

Determination of the primary structures of six kinds of vertebrate visual pigments enabled us to classify them into four groups of cone-type pigments. The phylogenetic tree demonstrated that an ancestor of vertebrate visual pigments evolved into four kinds of cone-type pigments, from one of which rhodopsins diverged. Tetrachromatic color vision of chicken is discussed on the basis of both the absorption spectra of purified cone pigments and the filtering effect of colored oil-droplets.

Amino Acid Sequence↗

Characterization of interactions between transducin alpha/beta gamma-subunits and lipid membranes.

The gamma-subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are isoprenylated and alpha-carboxyl methylated at their COOH-terminal cysteine residues. These modifications are necessary for membrane attachment of the beta gamma complex, but a requirement of an additional factor has been proposed for the stable binding. We explored a possible contribution of the blocked amino terminus of beta-subunits of bovine photoreceptor G-protein, transducin (T alpha/T beta gamma = Gt alpha/beta 1 gamma 1), and of three beta gamma complexes (beta 1 gamma 2, beta 1 gamma 3, and beta 1 gamma 7) purified from bovine brains. Structural analyses revealed that every beta 1-subunit has an N-acetylated serine, which is unlikely to contribute to the membrane association. Since neither protease nor heat treatment of photoreceptor membranes affected the membrane binding of T beta gamma, it seems unlikely that rhodopsin (or other membrane proteins) serves as an anchor protein for accepting T beta gamma. In fact, T beta gamma bound to phospholipid large unilamellar vesicles (LUVs), of which the polar head groups strongly influenced the binding: T beta gamma alone showed 2-fold higher binding for negatively charged phosphatidylserine-LUVs than for neutral phosphatidylcholine (PC)-LUVs, while the affinity of T alpha/T beta gamma complex for the phosphatidylserine-LUVs was lower than that for the PC-LUVs. These results indicate that 1) an ionic interaction between T beta gamma and membrane surface plays an important role in the stable membrane association, and 2) the domain(s) of T beta gamma responsible for the association would be different between trimeric and dissociated states. We also found that synthetic peptides corresponding to the COOH-terminal region of T gamma inhibited T alpha-T beta gamma interaction only when the peptides were isoprenylated. This suggests that the isoprenyl moiety is located at the contact site between the subunits, not at the membrane-binding domain, when T beta gamma is complexed with T alpha.

Adenosine Diphosphate Ribose↗

Pinopsin is a chicken pineal photoreceptive molecule.

In avian pinealocytes, an environmental light signal resets the phase of the endogenous circadian pacemaker that controls the rhythmic production of melatonin. Investigation of the pineal phototransduction pathway should therefore reveal the molecular mechanism of the biological clock. The presence of rhodopsin-like photoreceptive pigment, transducin-like immunoreaction, and cyclic GMP-dependent cation-channel activity in the avian pinealocytes suggests that there is a similarity between retinal rod cells and pinealocytes in the phototransduction pathway. We have now cloned chicken pineal cDNA encoding the photoreceptive molecule, which is 43-48% identical in amino-acid sequence to vertebrate retinal opsins. Pineal opsin, produced by transfection of complementary DNA into cultured cells, was reconstituted with 11-cis-retinal, resulting in formation of a blue-sensitive pigment (lambda max approximately 470 nm). In the light of this functional evidence and because the gene is specifically expressed only in the pineal gland, we conclude that it is a pineal photosensor and name it pinopsin.

Amino Acid Sequence↗

Is chicken green-sensitive cone visual pigment a rhodopsin-like pigment? A comparative study of the molecular properties between chicken green and rhodopsin.

Chicken green is a visual pigment present in chicken green-sensitive cones and has an amino acid sequence more similar than any other cone visual pigments to the rod visual pigments, rhodopsins. Here we have investigated the molecular properties of chicken green and compared them with those of rhodopsin to elucidate whether or not chicken green is a rhodopsin-like pigment. While chicken green has a molecular extinction coefficient and a photosensitivity very similar to those of rhodopsin, it displays faster regeneration from 11-cis-retinal and opsin and faster formation and decay of the physiologically active meta II intermediate than rhodopsin. These differences correlate with the physiological difference between cones and rods. Thus in spite of the similarity in amino acid sequence, chicken green displays molecular properties required for a cone visual pigment that are clearly different from those of rhodopsin.

Animals↗

Circular dichroism of metaiodopsin II and its binding to transducin: a comparative study between meta II intermediates of iodopsin and rhodopsin.

Through low-temperature absorption and circular dichroism (CD) spectroscopies, and G-protein (transducin) binding experiments, we have investigated molecular properties of the meta II intermediate of iodopsin, a cone visual pigment present in chicken red-sensitive cones. The meta II intermediate of iodopsin (metaiodopsin II, lambda max = 390 nm) displayed a positive CD band at about 390 nm and a large negative CD band below 300 nm. It dissociated into all-trans-retinal and the protein moiety. A long-lived intermediate corresponding to the meta III intermediate of rhodopsin was not observed in iodopsin, under our experimental conditions. Decay of metaiodopsin II was significantly suppressed in the presence of transducin, but not in the presence of both transducin and GTP, indicating that metaiodopsin II can interact with transducin and activate it. Both metaiodopsin II and metarhodopsin II displayed a large negative CD band below 300 nm. This fact suggested that during the formation of both meta II intermediates, some aromatic amino acid residues and/or a disulfide bond are rearranged, which may be important for expression of catalytic activity for exchange of GDP to GTP on transducin. On the other hand, metaiodopsin II decayed more than 10 times faster than metarhodopsin II. This fact may be one of the reasons why cones are less photosensitive than rods.

Animals↗

What makes red visual pigments red? A resonance Raman microprobe study of retinal chromophore structure in iodopsin.

We have obtained resonance Raman spectra of iodopsin, a red-sensitive (lambda max 571 nm) pigment from chicken cone cells, to investigate the molecular mechanism of the opsin shift in visual pigments. Detergent-solubilized iodopsin samples were examined with a Raman microprobe to obtain spectra from a 77-K photostationary steady-state mixture composed of 11-cis-iodopsin and its 9-cis-isoiodopsin and all-trans-bathoiodopsin photoproducts. The vibrational modes of these species have been assigned by comparison with spectra of the corresponding bovine pigments. The single bond stretching frequencies of the bovine, toad, and chicken pigments are found to exhibit a regular correlation as a function of the pigment absorption maxima that is consistent with the expected effects of increased electron delocalization. The C = NH stretching frequencies of iodopsin and bathoiodopsin are at 1644 and 1638 cm-1, respectively, and shift down to 1621 and 1617 cm-1, respectively, when the nitrogen is deuterated. The C = ND stretching frequencies of the various pigments are found to decrease linearly with increasing absorption maxima, suggesting that at least part of the opsin shift in visual pigments results from weakened electrostatic interaction between the retinal chromophore and its protein counterion. The Raman data are inconsistent with the idea that a charged protein residue is shifted along the chromophore to regulate the opsin shift. Taken together with the mutagenesis and model compound results, these resonance Raman data suggest that the opsin shift between the green and red cone visual pigment arises from two effects. First, Tyr-274 provides increased electrostatic stabilization of the Schiff base-counterion ion pair. Second, the opsin shift is enhanced by the dipolar residues Ser-177 and Thr-282 that interact with the chromophore near the ionone ring to preferentially stabilize the highly dipolar charge distribution of the electronically excited retinal chromophore [Mathies, R., & Stryer, L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2169-2173].

Animals↗