RESUMO
In Drosophila photoreceptor cells, Ca2+ exerts regulatory functions that control the shape, duration, and amplitude of the light response. Ca2+ also orchestrates light adaptation allowing Drosophila to see in light intensity regimes that span several orders of magnitude ranging from single photons to bright sunlight. The prime source for Ca2+ elevation in the cytosol is Ca2+ influx from the extracellular space through light-activated TRP channels. This Ca2+ influx is counterbalanced by constitutive Ca2+ extrusion via the Na+/Ca2+ exchanger, CalX. The light-triggered rise in intracellular Ca2+ exerts its regulatory functions through interaction with about a dozen well-characterized Ca2+ and Ca2+/CaM binding proteins. In this review we will discuss the dynamic changes in Ca2+ concentration upon illumination of photoreceptor cells. We will present the proteins that are known to interact with Ca2+ (/CaM) and elucidate the physiological functions of these interactions.
Assuntos
Cálcio , Drosophila , Células Fotorreceptoras de Invertebrados , Transdução de Sinais , Animais , Antiporters/metabolismo , Cálcio/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/metabolismo , Luz , Células Fotorreceptoras de Invertebrados/fisiologiaRESUMO
Drosophila photoreceptors respond to oscillating light of high frequency (â¼100 Hz), while the detected maximal frequency is modulated by the light rearing conditions, thus enabling high sensitivity to light and high temporal resolution. However, the molecular basis for this adaptive process is unclear. Here, we report that dephosphorylation of the light-activated transient receptor potential (TRP) ion channel at S936 is a fast, graded, light-dependent, and Ca2+-dependent process that is partially modulated by the rhodopsin phosphatase retinal degeneration C (RDGC). Electroretinogram measurements of the frequency response to oscillating lights in vivo revealed that dark-reared flies expressing wild-type TRP exhibited a detection limit of oscillating light at relatively low frequencies, which was shifted to higher frequencies upon light adaptation. Strikingly, preventing phosphorylation of the S936-TRP site by alanine substitution in transgenic Drosophila (trpS936A ) abolished the difference in frequency response between dark-adapted and light-adapted flies, resulting in high-frequency response also in dark-adapted flies. In contrast, inserting a phosphomimetic mutation by substituting the S936-TRP site to aspartic acid (trpS936D ) set the frequency response of light-adapted flies to low frequencies typical of dark-adapted flies. Light-adapted rdgC mutant flies showed relatively high S936-TRP phosphorylation levels and light-dark phosphorylation dynamics. These findings suggest that RDGC is one but not the only phosphatase involved in pS936-TRP dephosphorylation. Together, this study indicates that TRP channel dephosphorylation is a regulatory process that affects the detection limit of oscillating light according to the light rearing condition, thus adjusting dynamic processing of visual information under varying light conditions.SIGNIFICANCE STATEMENTDrosophila photoreceptors exhibit high temporal resolution as manifested in frequency response to oscillating light of high frequency (≤â¼100 Hz). Light rearing conditions modulate the maximal frequency detected by photoreceptors, thus enabling them to maintain high sensitivity to light and high temporal resolution. However, the precise mechanisms for this process are not fully understood. Here, we show by combination of biochemistry and in vivo electrophysiology that transient receptor potential (TRP) channel dephosphorylation at a specific site is a fast, light-activated and Ca2+-dependent regulatory process. TRP dephosphorylation affects the detection limit of oscillating light according to the adaptation state of the photoreceptor cells by shifting the detection limit to higher frequencies upon light adaptation. This novel mechanism thus adjusts dynamic processing of visual information under varying light conditions.
Assuntos
Adaptação Ocular/fisiologia , Proteínas de Drosophila/metabolismo , Estimulação Luminosa/métodos , Células Fotorreceptoras de Invertebrados/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila melanogaster , Eletrorretinografia/métodos , Feminino , Masculino , Fosforilação/fisiologiaRESUMO
The Drosophila phototransduction cascade terminates in the opening of an ion channel, designated transient receptor potential (TRP). TRP has been shown to become phosphorylated in vitro, suggesting regulation of the ion channel through posttranslational modification. However, except for one phosphorylation site, Ser(982), which was analyzed by functional in vivo studies (Popescu, D. C., Ham, A. J., and Shieh, B. H. (2006) J. Neurosci. 26, 8570-8577), nothing is known about the role of TRP phosphorylation in vivo. Here, we report the identification of 21 TRP phosphorylation sites by a mass spectrometry approach. 20 phosphorylation sites are located in the C-terminal portion of the channel, and one site is located near the N terminus. All 21 phosphorylation sites were also identified in the inaC(P209) mutant, indicating that phosphorylation of TRP at these sites occurred independently from the eye-enriched protein kinase C. Relative quantification of phosphopeptides revealed that at least seven phosphorylation sites were predominantly phosphorylated in the light, whereas one site, Ser(936), was predominantly phosphorylated in the dark. We show that TRP phosphorylated at Ser(936) was located in the rhabomere. Light-dependent changes in the phosphorylation state of this site occurred within minutes. The dephosphorylation of TRP at Ser(936) required activation of the phototransduction cascade.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos da radiação , Luz , Células Fotorreceptoras/efeitos da radiação , Canais de Potencial de Receptor Transitório/metabolismo , Sequência de Aminoácidos , Animais , Olho/enzimologia , Imunofluorescência , Immunoblotting , Técnicas Imunoenzimáticas , Imunoprecipitação , Transdução de Sinal Luminoso , Dados de Sequência Molecular , Fosforilação/efeitos da radiação , Células Fotorreceptoras/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In animals, visual pigments are essential for photoreceptor function and survival. These G-protein-coupled receptors consist of a protein moiety (opsin) and a covalently bound 11-cis-retinylidene chromophore. The chromophore is derived from dietary carotenoids by oxidative cleavage and trans-to-cis isomerization of double bonds. In vertebrates, the necessary chemical transformations are catalyzed by two distinct but structurally related enzymes, the carotenoid oxygenase beta-carotenoid-15,15'-monooxygenase and the retinoid isomerase RPE65 (retinal pigment epithelium protein of 65 kDa). Recently, we provided biochemical evidence that these reactions in insects are catalyzed by a single enzyme family member named NinaB. Here we show that in the fly pathway, carotenoids are mandatory precursors of the chromophore. After chromophore formation, the retinoid-binding protein Pinta acts downstream of NinaB and is required to supply photoreceptors with chromophore. Like ninaE encoding the opsin, ninaB expression is eye-dependent and is activated as a downstream target of the eyeless/pax6 and sine oculis master control genes for eye development. The requirement for coordinated synthesis of chromophore and opsin is evidenced by analysis of ninaE mutants. Retinal degeneration in opsin-deficient photoreceptors is caused by the chromophore and can be prevented by restricting its supply as seen in an opsin and chromophore-deficient double mutant. Thus, our study identifies NinaB as a key component for visual pigment production and provides evidence that chromophore in opsin-deficient photoreceptors can elicit retinal degeneration.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Opsinas/deficiência , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/metabolismo , Visão Ocular , beta-Caroteno 15,15'-Mono-Oxigenase/metabolismo , Animais , Carotenoides/metabolismo , Olho Composto de Artrópodes/crescimento & desenvolvimento , Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Olho/metabolismo , Regulação da Expressão Gênica , Larva/metabolismo , Larva/fisiologia , Mutação , Opsinas/genética , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/patologia , Pigmentos da Retina/biossíntese , Retinaldeído/farmacologia , Proteínas de Ligação ao Retinol/metabolismo , Xantofilas/metabolismo , Zeaxantinas , beta-Caroteno 15,15'-Mono-Oxigenase/química , beta-Caroteno 15,15'-Mono-Oxigenase/genéticaRESUMO
In animals, successful production of the visual chromophore (11-cis-retinal or derivatives thereof such as 11-cis-3-hydroxy-retinal) is essential for photoreceptor cell function and survival. These carotenoid-derived compounds must combine with a protein moiety (the opsin) to establish functional visual pigments. Evidence from cell culture systems has implicated that the retinal pigment epithelium protein of 65 kDa (RPE65) is the long-sought all-trans to 11-cis retinoid isomerase. RPE65 is structurally related to nonheme iron oxygenases that catalyze the conversion of carotenoids into retinoids. In vertebrate genomes, two carotenoid oxygenases and RPE65 are encoded, whereas in insect genomes only a single representative of this protein family, named NinaB (denoting neither inactivation nor afterpotential mutant B), is encoded. We here cloned and functionally characterized the ninaB gene from the great wax moth Galleria mellonella. We show that the recombinant purified enzyme combines isomerase and oxygenase (isomerooxygenase) activity in a single polypeptide. From kinetics and isomeric composition of cleavage products of asymmetrical carotenoid substrates, we propose a model for the spatial arrangement between substrate and enzyme. In Drosophila, we show that carotenoid-isomerooxygenase activity of NinaB is more generally found in insects, and we provide physiological evidence that carotenoids such as 11-cis-retinal can promote visual pigment biogenesis in the dark. Our study demonstrates that trans/cis isomerase activity can be intrinsic to this class of proteins and establishes these enzymes as key components for both invertebrate and vertebrate vision.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Insetos/metabolismo , Mariposas/enzimologia , Oxigenases/metabolismo , beta-Caroteno 15,15'-Mono-Oxigenase/metabolismo , cis-trans-Isomerases/metabolismo , Animais , Sítios de Ligação , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Humanos , Proteínas de Insetos/genética , Dados de Sequência Molecular , Estrutura Molecular , Mariposas/genética , Oxirredução , Oxigenases/genética , Células Fotorreceptoras de Invertebrados/química , Células Fotorreceptoras de Invertebrados/metabolismo , Pigmentos da Retina/metabolismo , Retinaldeído/química , Retinaldeído/metabolismo , Xantofilas/química , Xantofilas/metabolismo , Zeaxantinas , beta Caroteno/química , beta Caroteno/metabolismo , beta-Caroteno 15,15'-Mono-Oxigenase/genética , cis-trans-Isomerases/genéticaRESUMO
Drosophila retinal degeneration C (RDGC) is the founding member of the PPEF family of protein phosphatases. RDGC mediates dephosphorylation of the visual pigment rhodopsin and the TRP ion channel. From the rdgC locus, three protein isoforms, termed RDGC-S, -M, and -L, with different N-termini are generated. Due to fatty acylation, RDGC-M and -L are attached to the plasma membrane while RDGC-S is soluble. To assign physiological roles to these RDGC isoforms, we constructed flies that express various combinations of RDGC protein isoforms. Expression of the RDGC-L isoform alone did not fully prevent rhodopsin hyperphosphorylation and resulted in impaired photoreceptor physiology and in decelerated TRP dephosphorylation at Ser936. However, expression of RDGC-L alone as well as RDGC-S/M was sufficient to prevent degeneration of photoreceptor cells which is a hallmark of the rdgC null mutant. Membrane-attached RDGC-M displayed higher activity of TRP dephosphorylation than the soluble isoform RDGC-S. Taken together, in vivo activities of RDGC splice variants are controlled by their N-termini.
Assuntos
Processamento Alternativo , Proteínas de Ligação ao Cálcio , Proteínas de Drosophila , Proteínas de Membrana , Fosfoproteínas Fosfatases , Células Fotorreceptoras de Invertebrados/enzimologia , Acilação , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Domínios Proteicos , Rodopsina/genética , Rodopsina/metabolismoRESUMO
Protein phosphorylation is an abundant molecular switch that regulates a multitude of cellular processes. In contrast to other subfamilies of phosphoprotein phosphatases, the PPEF subfamily is only poorly investigated. Drosophila retinal degeneration C (RDGC) constitutes the founding member of the PPEF subfamily. RDGC dephosphorylates the visual pigment rhodopsin and the ion channel TRP.However, rdgC null mutant flies exhibit rhodopsin and TRP hyperphosphorylation, altered photoreceptor physiology, and retinal degeneration. Here, we report the identification of a third RDGC protein variant and show that the three RDGC isoforms harbor different N-termini that determine solubility and subcellular targeting due to fatty acylation. Taken together, solubility and subcellular targeting of RDGC splice variants are determined by their N-termini.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Acilação/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação ao Cálcio/química , Células Cultivadas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Espaço Intracelular/metabolismo , Mutação/fisiologia , Fosfoproteínas Fosfatases/química , Domínios Proteicos/genética , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/genética , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , SolubilidadeRESUMO
Drosophila photoreceptors respond to oscillating light of high frequency (â¼100 Hz), while increasing the oscillating light intensity raises the maximally detected frequency. Recently, we reported that dephosphorylation of the light-activated TRP ion channel at S936 is a fast, graded, light-, and Ca2+-dependent process. We further found that this process affects the detection limit of high frequency oscillating light. Accordingly, transgenic Drosophila, which do not undergo phosphorylation at the S936-TRP site (trpS936A), revealed a short time-interval before following the high stimulus frequency (oscillation-lock response) in both dark- and light-adapted flies. In contrast, the trpS936D transgenic flies, which mimic constant phosphorylation, showed a long-time interval to oscillation-lock response in both dark- and light-adapted flies. Here we extend these findings by showing that dark-adapted trpS936A flies reveal light-induced current (LIC) with short latency relative to trpWT or trpS936D flies, indicating that the channels are a limiting factor of response kinetics. The results indicate that properties of the light-activated channels together with the dynamic light-dependent process of TRP phosphorylation at the S936 site determine response kinetics.
Assuntos
Luz , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Drosophila , Cinética , FosforilaçãoRESUMO
Vitamin A derivatives (retinoids) are essential components in vision; they contribute to pattern formation during development and exert multiple effects on cell differentiation with important clinical implications. All naturally occurring vitamin A derives by enzymatic oxidative cleavage from carotenoids with pro-vitamin A activity. To become biologically active, these plant-derived compounds must first be absorbed, then delivered to the site of action in the body, and metabolically converted to the real vitamin. Recently, molecular players of this pathway were identified by the analysis of blind Drosophila mutants. Similar genome sequences were found in vertebrates. Subsequently, these homologous genes were cloned and their gene products were functionally characterized. This review will summarize the advanced state of knowledge about the vitamin A biosynthetic pathway and will discuss biochemical, physiological, developmental and medical aspects of carotenoids and their numerous derivatives.
Assuntos
Carotenoides/metabolismo , Oxigenases/metabolismo , Vitamina A/biossíntese , Animais , Proteínas de Transporte , Dioxigenases , Proteínas do Olho/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Oxigenases/genética , Receptores Imunológicos/metabolismo , Receptores Depuradores , Tretinoína/metabolismo , Vitamina A/metabolismo , beta-Caroteno 15,15'-Mono-Oxigenase , cis-trans-IsomerasesRESUMO
Drosophila inactivation no afterpotential D (INAD) is a PDZ domain-containing scaffolding protein that tethers components of the phototransduction cascade to form a supramolecular signaling complex. Here, we report the identification of eight INAD phosphorylation sites using a mass spectrometry approach. PDZ1, PDZ2, and PDZ4 each harbor one phosphorylation site, three phosphorylation sites are located in the linker region between PDZ1 and 2, one site is located between PDZ2 and PDZ3, and one site is located in the N-terminal region. Using a phosphospecific antibody, we found that INAD phosphorylated at Thr170/Ser174 was located within the rhabdomeres of the photoreceptor cells, suggesting that INAD becomes phosphorylated in this cellular compartment. INAD phosphorylation at Thr170/Ser174 depends on light, the phototransduction cascade, and on eye-Protein kinase C that is attached to INAD via one of its PDZ domains.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Proteínas do Olho/metabolismo , Transdução de Sinal Luminoso , Luz , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas do Olho/química , Proteínas do Olho/genética , Dados de Sequência Molecular , Mutação , Fosforilação , Proteína Quinase C/química , Proteína Quinase C/genéticaRESUMO
Transient receptor potential (TRP) channels constitute an ancient family of cation channels that have been found in many eukaryotic organisms from yeast to human. TRP channels exert a multitude of physiological functions ranging from Ca2+ homeostasis in the kidney to pain reception and vision. These channels are activated by a wide range of stimuli and undergo covalent post-translational modifications that affect and modulate their subcellular targeting, their biophysical properties, or channel gating. These modifications include N-linked glycosylation, protein phosphorylation, and covalent attachment of chemicals that reversibly bind to specific cysteine residues. The latter modification represents an unusual activation mechanism of ligand-gated ion channels that is in contrast to the lock-and-key paradigm of receptor activation by its agonists. In this review, we summarize the post-translational modifications identified on TRP channels and, when available, explain their physiological role.
RESUMO
Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Western Blotting , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Luz , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Fosforilação/efeitos da radiação , Células Fotorreceptoras de Invertebrados/metabolismo , Proteína Quinase C/metabolismo , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/genética , Triptofano/genética , Triptofano/metabolismoRESUMO
The Drosophila visual transduction cascade is embedded in the rhabdomeres of photoreceptor cells and culminates in the opening of the two ion channels, TRP and TRPL. TRPL translocates from the rhabdomeres to the cell body upon illumination and vice versa when flies are kept in the dark. Here, we studied the mechanisms underlying the light-dependent internalization of TRPL. Co-localization of TRPL and rhodopsin in endocytic particles revealed that TRPL is internalized by a vesicular transport pathway that is also utilized, at least partially, for rhodopsin endocytosis. TRPL internalization is attenuated under light conditions that result in a high rate of rhodopsin internalization and is highest in orange light that result in very little rhodopsin internalization. In line with a canonical vesicular transport pathway, we found that rab proteins, Rab5 and RabX4, are required for the internalization of TRPL into the cell body. Our results provide insight into stimulus-dependent internalization of a prominent member of the TRP superfamily.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Rodopsina/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Arrestinas/metabolismo , Imuno-Histoquímica , Células Fotorreceptoras de Invertebrados , Transporte Proteico/fisiologia , Transdução de Sinais , Visão OcularRESUMO
The blind Drosophila mutant ninaD lacks the visual chromophore. Genetic evidence that the molecular basis is a defect in carotenoid uptake which causes vitamin A deficiency exists. The ninaD gene encodes a scavenger receptor that is significantly homologous in sequence with the mammalian scavenger receptors SR-BI (scavenger receptor class B type I) and CD36 (cluster determinant 36), yet NinaD has not been characterized in functional detail. Therefore, we established a Drosophila S2 cell culture system for biochemically characterizing the ninaD gene products. We show that the two splice variant isoforms encoded by ninaD exhibit different subcellular localizations. NinaD-I, the long protein variant, is localized at the plasma membrane, whereas the short variant, NinaD-II, is localized at intracellular membranes. Only NinaD-I could mediate the cellular uptake of carotenoids from micelles in this cell culture system. Carotenoid uptake was concentration-dependent and saturable. By in vivo analyses of different mutant and transgenic fly strains, we provide evidence of an essential role of NinaD-I in the absorption of dietary carotenoids to support visual chromophore synthesis. Moreover, our analyses suggest a role of NinaD-I in tocopherol metabolism. Even though Drosophila is a sterol auxotroph, we found no evidence of a contribution of NinaD-I to the uptake of these compounds. Together, our study establishes an evolutionarily conserved connection between class B scavenger receptors and the numerous functions of fat soluble vitamins in animal physiology.