RESUMO
Visual pigment in photoreceptors is activated by light. Activated visual pigment (R*) is believed to be inactivated by phosphorylation of R* with subsequent binding of arrestin. There are two types of photoreceptors, rods and cones, in the vertebrate retina, and they express different subtypes of arrestin, rod and cone type. To understand the difference in the function between rod- and cone-type arrestin, we first identified the subtype of arrestins expressed in rods and cones in carp retina. We found that two rod-type arrestins, rArr1 and rArr2, are co-expressed in a rod and that a cone-type arrestin, cArr1, is expressed in blue- and UV-sensitive cones; the other cone-type arrestin, cArr2, is expressed in red- and green-sensitive cones. We quantified each arrestin subtype and estimated its concentration in the outer segment of a rod or a cone in the dark; they were â¼0.25 mm (rArr1 plus rArr2) in a rod and 0.6-0.8 mm (cArr1 or cArr2) in a cone. The effect of each arrestin was examined. In contrast to previous studies, both rod and cone arrestins suppressed the activation of transducin in the absence of visual pigment phosphorylation, and all of the arrestins examined (rArr1, rArr2, and cArr2) bound transiently to most probably nonphosphorylated R*. One rod arrestin, rArr2, bound firmly to phosphorylated pigment, and the other two, rArr1 and cArr2, once bound to phosphorylated R* but dissociated from it during incubation. Our results suggested a novel mechanism of arrestin effect on the suppression of the R* activity in both rods and cones.
Assuntos
Arrestina/metabolismo , Carpas/metabolismo , Proteínas de Peixes/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Pigmentos da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Arrestina/genética , Carpas/genética , Proteínas de Peixes/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Immunoblotting , Imuno-Histoquímica , Luz , Fosforilação , Ligação Proteica/efeitos da radiação , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Transducina/metabolismoRESUMO
On absorption of light by vertebrate visual pigment, the chromophore, 11-cis retinal, is isomerized to all-trans retinal to activate the phototransduction cascade, which leads to a hyperpolarizing light response. Activated pigment is inactivated by phosphorylation on the protein moiety, opsin. Isomerized all-trans retinal is ultimately released from opsin, and the pigment is regenerated by binding to 11-cis retinal. In this pigment regeneration cycle, the phosphates incorporated should be removed in order that the pigment regains the capability of activating the phototransduction cascade. However, it is not clear yet how pigment dephosphorylation takes place in the regeneration cycle. First in this study, we tried to estimate the dephosphorylation activity in living carp rods and cones and found that the activity, which is present mainly in the cytoplasm in both rods and cones, is three times higher in cones than in rods. Second, we examined at which stage the dephosphorylation takes place; before or after the release of all-trans retinal, during pigment regeneration, or after pigment regeneration. For this purpose we prepared three types of phosphorylated substrates in purified carp rod and cone membranes: phosphorylated bleaching intermediate, phosphorylated opsin, and phosphorylated and regenerated pigment. We also examined the effect of pigment regeneration on the dephosphorylation. The results showed that the dephosphorylation does not show substrate preference in the regeneration cycle and suggested that the dephosphorylation takes place constantly. The results also suggest that, under bright light, some of the regenerated visual pigment remains phosphorylated to reduce the light sensitivity in cones.
Assuntos
Carpas/fisiologia , Fotodegradação , Células Fotorreceptoras Retinianas Cones/metabolismo , Pigmentos da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Membrana Celular/metabolismo , Citoplasma/metabolismo , Luz , Transdução de Sinal Luminoso , Fosfoproteínas Fosfatases/química , Fosforilação , Células Fotorreceptoras de Vertebrados , Pigmentação , Regeneração , Retina/patologia , Retinaldeído/metabolismo , Rodopsina/química , Opsinas de Bastonetes/metabolismo , Especificidade por SubstratoRESUMO
Cone photoreceptors require effective pigment regeneration mechanisms to maintain their sensitivity in the light. Our previous studies in carp cones suggested the presence of an unconventional and very effective mechanism to produce 11-cis retinal, the necessary component in pigment regeneration. In this reaction (aldehyde-alcohol redox coupling reaction, AL-OL coupling reaction), formation of 11-cis retinal, i.e. oxidation of 11-cis retinol is coupled to reduction of an aldehyde at a 1:1 molar ratio without exogenous NADP(H) which is usually required in this kind of reaction. Here, we identified carp retinol dehydrogenase 13-like (RDH13L) as an enzyme catalyzing the AL-OL coupling reaction. RDH13L was partially purified from purified carp cones, identified as a candidate protein, and its AL-OL coupling activity was confirmed using recombinant RDH13L. We further examined the substrate specificity, subcellular localization, and expression level of RDH13L. Based on these results, we concluded that RDH13L contributes to a significant part, but not all, of the AL-OL coupling activity in carp cones. RDH13L contained tightly bound NADP(+) which presumably functions as a cofactor in the reaction. Mouse RDH14, a mouse homolog of carp RDH13L, also showed the AL-OL coupling activity. Interestingly, although carp cone membranes, carp RDH13L and mouse RDH14 all showed the coupling activity at 15-37 °C, they also showed a conventional NADP(+)-dependent 11-cis retinol oxidation activity above 25 °C without addition of aldehydes. This dual mechanism of 11-cis retinal synthesis attained by carp RDH13L and mouse RDH14 probably contribute to effective pigment regeneration in cones that function in the light.
Assuntos
Carpas/metabolismo , Células Fotorreceptoras Retinianas Cones/enzimologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Retinaldeído/metabolismo , Vitamina A/metabolismo , Animais , Camundongos , OxirreduçãoRESUMO
Cones are less light-sensitive than rods. We showed previously in carp that more light (>100-fold) is required in cones than in rods to activate 50% of cGMP phosphodiesterase (PDE). The lower effectiveness of PDE activation in carp cones is due partly to the fact that the activation rate of transducin (Tr) by light-activated visual pigment (R*) is 5-fold lower in carp cones than in rods. In this study, we tried to explain the remaining difference. First, we examined the efficiency of activation of PDE by activated Tr (Tr*). By activating PDE with known concentrations of the active (guanosine 5'-Ο-(γ-thio)triphosphate (GTPγS)-bound) form of Tr*, we found that Tr* activated PDE at a similar efficiency in rods and cones. Next, we examined the contribution of R* and Tr* lifetimes. In a comparison of PDE activation in the presence (with GTP) and absence (with GTPγS) of Tr* inactivation, PDE activation required more light (and was therefore less effective) when Tr* was inactivated in both rod and cone membranes. This is probably because inactivation of Tr* shortened its lifetime, thereby reducing the number of activated PDE molecules. The effect of Tr* inactivation was larger in cones, probably because the lifetime of Tr* is shorter in cones than in rods. The shorter lifetimes of Tr* and R* in cones seem to explain the remaining difference in the effectiveness of PDE activation between rods and cones.
Assuntos
GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Células Fotorreceptoras Retinianas Cones/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Adaptação Ocular/fisiologia , Animais , Carpas , Adaptação à Escuridão/fisiologia , Ativação Enzimática/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transducina/metabolismo , Visão Ocular/fisiologiaRESUMO
Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.
Assuntos
Membranas Intracelulares/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Retinaldeído/metabolismo , Vitamina A/metabolismo , Álcoois/metabolismo , Animais , Benzaldeídos/metabolismo , Sítios de Ligação , Carpas , Oxirredução , Segmento Interno das Células Fotorreceptoras da Retina/metabolismo , Especificidade por Substrato , Vitamina A/análogos & derivadosRESUMO
Cone photoreceptors show lower light sensitivity and briefer light responses than rod photoreceptors. The light detection signal in these cells is amplified through a phototransduction cascade. The first step of amplification in the cascade is the activation of a GTP-binding protein, transducin (Tr), by light-activated visual pigment (R*). We quantified transducin activation by measuring the binding of GTPγS in purified carp rod and cone membrane preparations with the use of a rapid quench apparatus and found that transducin activation by an R* molecule is â¼5 times less efficient in cones than in rods. Transducin activation terminated in less than 1 s in cones, more quickly than in rods. The rate of GTP hydrolysis in Tr*, and thus the rate of Tr* inactivation, was â¼25 times higher in cones than in rods. This faster inactivation of Tr* ensures briefer light responses in cones. The expression level of RGS9 was found to be â¼20 times higher in cones than in rods, which explains higher GTP hydrolytic activity and, thus, faster Tr* inactivation in cones than in rods. Although carp rods and cones express rod- or cone-versions of visual pigment and transducin, these molecules themselves do not seem to induce the differences significantly in the transducin activation and Tr* inactivation in rods and cones. Instead, the differences seem to be brought about in a rod or cone cell-type specific manner.
Assuntos
Células Fotorreceptoras Retinianas Cones/metabolismo , Transducina/metabolismo , Trifosfato de Adenosina/química , Animais , Carpas , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Hidrólise , Luz , Modelos Químicos , Modelos Teóricos , Pigmentação , Pigmentos da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Cone photoreceptor subtypes having different spectral sensitivities exhibit different recovery kinetics in their photoresponses in some vertebrates. Phosphorylation by G protein-coupled receptor kinase (GRK) is essential for the rapid inactivation of light-activated visual pigment, which is the rate-limiting step of the cone photoresponse recovery in salamander. In this study we compared the rate of light-dependent phosphorylation by GRK7 of carp green- and blue-sensitive cone visual pigments. Blue pigment was phosphorylated significantly less effectively than green pigment, suggesting that the difference in the pigment phosphorylation rate is responsible for the difference in photoresponse kinetics among cone photoreceptor subtypes.
Assuntos
Opsinas dos Cones/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Animais , Carpas , Clonagem Molecular , Opsinas dos Cones/genética , Células HEK293 , Humanos , FosforilaçãoRESUMO
Cones show briefer light responses than rods and do not saturate even under very bright light. Using purified rod and cone homogenates, we measured the activity of guanylate cyclase (GC), an enzyme responsible for cGMP synthesis and therefore recovery of a light response. The basal GC activity was 36 times higher in cones than in rods: It was mainly caused by higher expression levels of GC in cones (GC-C) than in rods (GC-R). With identification and quantification of GC-activating protein (GCAP) subtypes expressed in rods and cones together with determination of kinetic parameters of GC activation in the presence and absence of GCAP, we estimated the in situ GC activity in rods and cones at low and high Ca(2+) concentrations. It was revealed that the GC activity would be >10 times higher in cones than in rods in both the dark-adapted and the light-adapted states. Electrophysiological estimation of the GC activity measured in the truncated preparations of rod and cone outer segments gave consistent results. Our estimation of the in situ GC activity reasonably explained the rapid recovery and nonsaturating behavior of cone light responses.
Assuntos
Carpas/metabolismo , GMP Cíclico/biossíntese , Guanilato Ciclase/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Adaptação Ocular/fisiologia , Animais , Sequência de Bases , Primers do DNA/genética , Eletrofisiologia , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Immunoblotting , Cinética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
In the vertebrate retina, rods and cones both detect light, but they differ in functional aspects such as light sensitivity and temporal resolution, and in some cell biological aspects. For functional aspects, both types of photoreceptors use a phototransduction cascade, consisting of a series of enzymatic reactions, to convert photon capture to an electrical signal. To understand the mechanisms underlying the functional differences between rods and cones at the molecular level, we compared biochemically, each of the reactions in the phototransduction cascades of rods and cones using the cells isolated and purified from carp retina. Although the cascade proteins are identical or are functionally similar between rods and cones, their activities together with their expression levels are mostly different. In general, reactions that generate a response are somewhat less effective in cones than in rods, but each of the reactions for termination and recovery of a response are much more effective in cones. These findings explain lower light sensitivity and briefer light responses in cones than in rods. In addition, our considerations suggest that a Ca2+-binding protein, S-modulin or recoverin, has a currently unnoticed role in shaping light responses. Upon comparison of the expression levels of proteins and/or mRNAs using purified cells, several proteins were found to be specifically or predominantly expressed in cones. These proteins will be of interest in future studies aimed at characterizing the differences between rods and cones.
Assuntos
Fotofobia , Células Fotorreceptoras Retinianas Bastonetes , Animais , Humanos , Transdução de Sinal Luminoso , Fotofobia/metabolismo , Retina , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologiaRESUMO
Retinal photoreceptor cells, rods and cones, convert photons of light into chemical and electrical signals as the first step of the visual transduction cascade. Although the chemical processes in the phototransduction system are very similar to each other in these photoreceptors, the light sensitivity and time resolution of the photoresponse in rods are functionally different than those in the photoresponses of cones. To systematically investigate how photoresponses are divergently regulated in rods and cones, we have developed a detailed mathematical model on the basis of the Hamer model. The current model successfully reconstructed light intensity-, ATP- and GTP-dependent changes in concentrations of phosphorylated visual pigments (VPs), activated transducins (Tr*s) and phosphodiesterases (PDEs) in rods and cones. In comparison to rods, the lower light sensitivity of cones was attributed not only to the lower affinity of activated VPs for Trs but also to the faster desensitization of the VPs. The assumption of an intermediate inactive state, MIIi, in the thermal decay of activated VPs was essential for inducing faster inactivation of VPs in rods, and possibly also in cones.
Assuntos
Fotofobia , Células Fotorreceptoras Retinianas Bastonetes , Humanos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Transdução de Sinal Luminoso/fisiologia , Transducina/metabolismoRESUMO
This paper presents room temperature nanoseconds to milliseconds time-resolved spectra and kinetics of the intermediate states and species of bovine and carp fish rhodopsin visual pigments, which also contained ~5% cone pigments. The nanoseconds to milliseconds range cover all the major intermediates in the visual phototransduction process except the formation of bathorhodopsin intermediate which occurs at the femtosecond time scale. The dynamics of these visual pigment intermediates are initiated by excitation with a 532 nm nanosecond laser pulse. The recorded differences between bovine and carp rhodopsin time-resolved spectra of the formation and decay kinetics of their intermediates are presented and discussed. The data show that the carp samples batho intermediate decays faster, nearly by a factor of three, compared to the bovine samples. The formation and decay spectra and kinetics of rhodopsin outer segments and extracted rhodopsin inserted in buffer solution were found to be identical, with very small differences between them in the decay lifetimes of bathorhodopsin and formation of lumirhodopsin.
Assuntos
Carpas , Rodopsina , Bovinos , Animais , Temperatura , Espectrofotometria , FotóliseRESUMO
After bleaching of visual pigment in vertebrate photoreceptors, all-trans retinal is reduced to all-trans retinol by retinol dehydrogenases (RDHs). We investigated this reaction in purified carp rods and cones, and we found that the reducing activity toward all-trans retinal in the outer segment (OS) of cones is >30 times higher than that of rods. The high activity of RDHs was attributed to high content of RDH8 in cones. In the inner segment (IS) in both rods and cones, RDH8L2 and RDH13 were found to be the major enzymes among RDH family proteins. We further found a previously undescribed and effective pathway to convert 11-cis retinol to 11-cis retinal in cones: this oxidative conversion did not require NADP(+) and instead was coupled with reduction of all-trans retinal to all-trans retinol. The activity was >50 times effective than the oxidizing activity of RDHs that require NADP(+). These highly effective reactions of removal of all-trans retinal by RDH8 and production of 11-cis retinal by the coupling reaction are probably the underlying mechanisms that ensure effective visual pigment regeneration in cones that function under much brighter light conditions than rods.
Assuntos
Células Fotorreceptoras Retinianas Cones/metabolismo , Retinaldeído/metabolismo , Oxirredutases do Álcool/metabolismo , Animais , Carpas , Proteínas do Olho/metabolismo , Dados de Sequência Molecular , Oxirredução , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
In the carp retina, visual pigment kinase, GRK1 (G-protein coupled receptor kinase 1) in rods and GRK7 in cones, is inhibited by a photoreceptor neuronal Ca(2+)-sensor protein, S-modulin (or recoverin) in rods and visinin (formerly named s26) in cones. Here, we compared Ca(2+)-dependent inhibition of GRK1 by S-modulin and that of GRK7 by visinin. First, the concentrations of S-modulin and visinin in the outer segment were estimated: the concentration of visinin (1.2 mM) was 20 times higher than that of S-modulin (53 µM). Based on the determined concentrations of the Ca(2+)-sensor proteins and the known dark Ca(2+) concentrations, we estimated that in situ Ca(2+)-dependent inhibition on GRK in cones would be 2.5 times higher than that in rods at the Ca(2+) concentration in the dark. Because GRK activity is approximately 100 times higher in cones than in rods [Proc. Natl Acad. Sci. USA 102 (2005) 21359], the range of Ca(2+)-dependent inhibition on GRK activity is more than 100 times wider in cones than in rods. The inhibitory effects of S-modulin and visinin on photoreceptor GRKs were indistinguishable, although these Ca(2+)-sensor proteins are expressed in a cell-type specific manner. The inhibition by these Ca(2+)-sensor proteins was slightly higher on GRK7 than GRK1 probably because of a characteristic specific to GRK7.
Assuntos
Carpas/metabolismo , Células Fotorreceptoras Retinianas Cones/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Algoritmos , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Imunofluorescência , Receptor Quinase 1 Acoplada a Proteína G/antagonistas & inibidores , Membranas/efeitos dos fármacos , Membranas/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/farmacologia , Proteínas do Tecido Nervoso/fisiologia , Fosforilação , Recoverina/biossíntese , Recoverina/metabolismo , Recoverina/farmacologia , Recoverina/fisiologiaRESUMO
Activation of cGMP phosphodiesterase (PDE) by activated transducin α subunit (Tα*) is a necessary step to generate a light response in vertebrate photoreceptors. PDE in rods is a heterotetramer composed of two catalytic subunits, PDEα and PDEß, and two inhibitory PDEγ subunits, each binding to PDEα or PDEß. Activation of PDE is achieved by relief of the inhibitory constraint of PDEγ on the catalytic subunit. In this activation mechanism, it is widely believed that Tα* binds to PDEγ still bound to the catalytic subunit, and removes or displaces PDEγ from the catalytic subunit. However, recent structural analysis showed that the binding of Tα* to PDEγ still bound to PDEα or PDEß seems to be difficult because the binding site of PDEγ to PDEα or PDEß overlaps with the binding site to Tα*. In the present study, we propose a novel activation mechanism of PDE, the trapping mechanism, in which Tα* activates PDE by trapping PDEγ released reversibly and spontaneously from the catalytic subunit. This mechanism well explains PDE activation by Tα* in solution. Our further analysis with this mechanism suggests that more effective PDE activation in disk membranes is highly dependent on the membrane environment.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Anuros/metabolismo , Transducina/metabolismo , Animais , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Subunidades Proteicas/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Segmento Externo da Célula Bastonete/metabolismoRESUMO
PURPOSE: Rods and cones differ in their photoresponse characteristics, morphology, and susceptibilities to certain diseases. To contribute to the studies at the molecular level of these differences, we tried to identify genes expressed preferentially in rods or cones. METHODS: From purified carp rods and cones, we extracted their RNA and obtained corresponding cDNA pools (rod cDNA and cone cDNA). We employed the suppression subtractive hybridization method to identify the genes expressed preferentially in rods or cones. Cone cDNA was subtracted from rod cDNA to obtain cDNA, which ideally contained cDNA expressed preferentially in rods (R/c cDNA). Similarly, rod cDNA was subtracted from cone cDNA to obtain C/r cDNA. With differential array screening, we screened candidate genes that were expressed mainly or exclusively in rods or cones. The nucleotide sequences of the positive genes were determined. In some of them, their mRNA localizations were confirmed by in situ hybridization. RESULTS: R/c cDNA contained genes already known to code rod specific proteins, such as cGMP gated channel, transducin beta1, and rhodopsin. In sharp contrast, C/r cDNA contained genes that code proteins of which functions are mostly unknown. Among them, N-myc downregulated gene 1-like (NDRG1L) and aryl hydrocarbon receptor 2 (AhR2) were most abundant, and by in situ hybridization, they were proven to be expressed specifically in cones. CONCLUSIONS: Using purified rods and cones, we identified mRNAs expressed preferentially in rods or cones. Of particular interest is the specific expression of NDRG1L and AhR2 in cones.
Assuntos
Carpas/genética , Perfilação da Expressão Gênica , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Adaptação Fisiológica/efeitos da radiação , Animais , Células Clonais , DNA Complementar/genética , Regulação da Expressão Gênica/efeitos da radiação , Hibridização In Situ , Luz , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Análise de Sequência de DNARESUMO
GRK1 is a visual pigment kinase in rods and is essential for inactivation of light-activated rhodopsin. The GRK1 activity is inhibited by binding of the Ca(2+)-bound form of S-modulin/recoverin. We previously identified the S-modulin/recoverin site to interact with GRK1. In the present study, we identified its counterpart in GRK1. We synthesized 29 of GRK1 or GRK7 partial peptides that cover the entire sequence of GRK1/GRK7, and examined whether these peptides inhibit S-modulin/recoverin activity most probably by preoccupying the binding site for GRK1. The inhibition was the greatest with the N-terminal peptide (p1, aa 3-23 in GRK7). On mutation of each of eight amino acid residues highly conserved in the p1 region of more than 10 orthologs, the inhibition was significantly reduced in the mutation of Leu(6), Asn(12) and Tyr(15). We further examined the binding of the peptides, including mutated ones, to S-modulin/recoverin with a resonance mirror biosensor. The binding correlated well with the degree of the inhibition by a peptide. The inhibition, therefore, seemed to be due to a direct binding of the kinase peptide to the binding site of active S-modulin/recoverin. A GRK1 region close to its C-terminus also seemed to be the binding site for S-modulin/recoverin.
Assuntos
Receptor Quinase 1 Acoplada a Proteína G/química , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Quinases de Receptores Acoplados a Proteína G/química , Quinases de Receptores Acoplados a Proteína G/metabolismo , Recoverina/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Sítios de Ligação , Cálcio/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Recoverina/químicaRESUMO
Vertebrate retinal photoreceptors consist of two types of cells, the rods and cones. Rods are highly light-sensitive but their flash response time course is slow, so that they can detect a single photon in the dark but are not good at detecting an object moving quickly. Cones are less light-sensitive and their flash response time course is fast, so that cones mediate daylight vision and are more suitable to detect a moving object than rods. The phototransduction mechanism was virtually known by the mid 80s, and detailed mechanisms of the generation of a light response are now understood in a highly quantitative manner at the molecular level. However, most of these studies were performed in rods, but not in cones. Therefore, the mechanisms of low light-sensitivity or fast flash response time course in cones have not been known. The major reason for this slow progress in the study of cone phototransduction was due to the inability of getting a large quantity of purified cones to study them biochemically. We succeeded in its purification using carp retina, and have shown that each step responsible for generation of a light response is less effective in cones and that the reactions responsible for termination of a light response are faster in cones. Based on these findings, we speculated a possible mechanism of evolution of rods that diverged from cones.
Assuntos
Células Fotorreceptoras/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Visão Ocular , Animais , Cálcio/metabolismo , Carpas , Retroalimentação Fisiológica , Luz , Modelos Biológicos , Rodopsina/metabolismo , Sensibilidade e Especificidade , Urodelos , VertebradosRESUMO
In the vertebrate retina, rods mediate twilight vision and cones mediate daylight vision. Their photoresponse characteristics are different. The light-sensitivity of a cone is 10(2)-10(3) times lower than that of a rod. In addition, the photoresponse time course is much faster in cones. The mechanism characterizing cone photoresponses has not been known mainly because of the difficulty in isolating cones in large quantities to perform biochemistry. Recently, we developed a method to purify cones from carp retina using a density gradient, which made it possible to analyze the differences in the molecular mechanism of phototransduction between rods and cones. The results showed that signal amplification in cones is less effective, which explains the lower light-sensitivity of cones. The results also showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. The rapid phosphorylation in cones is attributed to a very high total kinase activity in cones. Because of this high activity, cone pigment is readily phosphorylated even at very high bleaching levels, which probably explains why cone photoresponses recover quickly. Based on these findings, the molecular mechanisms of the differences in the photoresponse characteristics between rods and cones are outlined.
Assuntos
Luz , Células Fotorreceptoras/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Visão Ocular/fisiologia , Animais , Carpas , Cinética , Fosforilação , Fotofobia , Proteínas Quinases/metabolismo , Pigmentos da Retina/metabolismoRESUMO
Rods and cones are both photoreceptors in the retina, but they are different in many aspects including the light response characteristics and, for example, cell morphology and metabolism. These differences would be caused by differences in proteins expressed in rods and cones. To understand the molecular bases of these differences between rods and cones, one of the ways is to compare proteins expressed in rods and cones, and to find those expressed specifically or dominantly. In the present study, we are interested in proteins in the outer segment (OS), the site responsible for generation of rod- or cone-characteristic light responses and also the site showing different morphology between rods and cones. For this, we established a method to purify the OS and the inner segment (IS) of rods and also of cones from purified carp rods and cones, respectively, using sucrose density gradient. In particular, we were interested in proteins tightly bound to the membranes of cone OS. To identify these proteins, we analyzed proteins in some selected regions of an SDS-gel of washed membranes of the OS and the IS obtained from both rods and cones, with Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using a protein database constructed from carp retina. By comparing the lists of the proteins found in the OS and the IS of both rods and cones, we found some proteins present in cone OS membranes specifically or dominantly, in addition to the proteins already known to be present specifically in cone OS.
Assuntos
Proteínas de Membrana/metabolismo , Proteômica , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , CarpasRESUMO
PURPOSE: Visual pigment is phosphorylated and inactivated after light stimulus. The responsible enzyme is known as rhodopsin kinase or G-protein-coupled receptor kinase 1 (GRK1) in rods. We recently showed that the kinase in cones (GRK7) has much higher activity than GRK1 in rods in carp retina. During the course of these studies, we realized that there are several subtypes of GRK1 and GRK7. In the present study, therefore, to identify the GRK1 and GRK7 subtypes expressed in carp photoreceptors, we determined their nucleotide sequences together with their expression patterns in carp retina. We also analyzed their relationships to other GRK1s and GRK7s phylogenetically. METHODS: Oligonucleotides corresponding to the amino acid sequences conserved in GRK1 or GRK7 were synthesized to screen the GRK subtypes in a carp retinal cDNA library. The isolated partial cDNAs were used to determine the full length of GRK subtypes. Genomic Southern hybridization was performed to learn whether each of the isolated GRKs is encoded by a single gene or it is an allelic variation. Tissue localization of the isolated GRKs was examined with in situ hybridization. RESULTS: A novel subtype of GRK1, GRK1B, was found in addition to the conventional GRK1 (called GRK1A subtype in this study) in carp retina. The GRK1A subtype, more specifically the GRK1A-1 subtype, which is related to the mammalian-type GRK1, was expressed in rods, while the GRK1B subtype, related to chicken GRK1, was expressed in cones. Since GRK7-1 was also expressed in cones, carp cones express both GRK7-1 and GRK1B. There were two paralogous genes in all of the GRK1 and GRK7 subtypes in carp retina: GRK1A-1a and 1A-1b, GRK1Ba and 1Bb, and GRK7-1a and 7-1b. Each of these genes was suggested to be encoded by a single gene in the carp genome, and each pair was found to be expressed in the same type of photoreceptors. CONCLUSIONS: Carp rods and cones express at least two kinds of visual pigment kinases. Phylogenetic analysis suggested that GRK7-1 together with GRK1A-1 and GRK1B appeared before divergence of vertebrates and that some of these genes were lost during evolution in a species-dependent manner. This evolutional process probably explains why the expression pattern of GRK1 and GRK7 is complex among vertebrate species.