RESUMEN
Many aspects of photoreceptor metabolism are regulated as diurnal or circadian rhythms. The nature of the signals that drive rhythms in mouse photoreceptors is unknown. Dopamine amacrine cells in mouse retina express core circadian clock genes, leading us to test the hypothesis that dopamine regulates rhythms of protein phosphorylation in photoreceptor cells. To this end we investigated the phosphorylation of phosducin, an abundant photoreceptor-specific phosphoprotein. In mice exposed to a daily light-dark cycle, robust daily rhythms of phosducin phosphorylation and retinal dopamine metabolism were observed. Phospho-phosducin levels were low during the daytime and high at night, and correlated negatively with levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid. The effect of light on phospho-phosducin levels was mimicked by pharmacological activation of dopamine D4 receptors. The amplitude of the diurnal rhythm of phospho-phosducin was reduced by > 50% in D4 receptor-knockout mice, due to higher daytime levels of phospho-phosducin. In addition, the daytime level of phospho-phosducin was significantly elevated by L-745,870, a dopamine D4 receptor antagonist. These data indicate that dopamine and other light-dependent processes cooperatively regulate the diurnal rhythm of phosducin phosphorylation. Under conditions of constant darkness a circadian rhythm of phosducin phosphorylation was observed, which correlated negatively with the circadian rhythm of 3,4-dihydroxyphenylacetic acid levels. The circadian fluctuation of phospho-phosducin was completely abolished by constant infusion of L-745,870, indicating that the rhythm of phospho-phosducin level is driven by dopamine. Thus, dopamine release in response to light and circadian clocks drives daily rhythms of protein phosphorylation in photoreceptor cells.
Asunto(s)
Ritmo Circadiano/genética , Dopamina/metabolismo , Proteínas del Ojo/metabolismo , Reguladores de Proteínas de Unión al GTP/metabolismo , Fosfoproteínas/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/efectos de la radiación , Adaptación a la Oscuridad/efectos de los fármacos , Adaptación a la Oscuridad/genética , Adaptación a la Oscuridad/efectos de la radiación , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Luz , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/efectos de los fármacos , Estimulación Luminosa , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/efectos de la radiación , Receptores de Dopamina D4/efectos de los fármacos , Receptores de Dopamina D4/genética , Receptores de Dopamina D4/efectos de la radiación , Retina/citologíaRESUMEN
PURPOSE: To characterize the structure of cone betagamma-transducin (Tbeta3gamma8) and its interaction with phosducin (pdc). METHODS: The Tgamma8 subunit of Tbeta3gamma8 was isolated by column chromatography for peptide mapping with mass spectrometry. Tbeta3gamma8 was compared with rod betagamma-transducin (Tbeta1gamma1) in terms of the electrophoretic mobility, pdc binding affinity, and the effects of phosphorylation and methylation, and then the correlation to the crystal structures and functional domains of Tbeta1gamma1 was determined. RESULTS: The mature Tgamma8 is a 65-amino-acid peptide encoded by the Ggamma8 gene with an acetylated and a farnesylated-methylated N- and C-terminus, respectively. Purified Tbeta3gamma8 is similar to Tbeta1gamma1 in that (1) both are heterogeneous, containing methylated and demethylated Tgamma subunits; (2) each demethylated dimer migrates faster than its methylated counterpart during native gel electrophoresis, and the methylation-associated mobility differential is masked by pdc binding; and (3) both dimers bind pdc with the same affinity, and the affinity is reduced threefold by PKA phosphorylation of pdc and twofold by demethylation at the C-terminus of Tgamma. Tbeta3gamma8 differs from Tbeta1gamma1 in exhibiting lower intrinsic electrophoretic mobility, and the difference is unaffected by either pdc binding or the status of Tgamma methylation. CONCLUSIONS: Tbeta3gamma8 is identical with Tbeta1gamma1 in Tgamma isoprenylation, the spatial organization, and the mode of pdc binding, indicating that its interaction with pdc does not play an important role in the specialization of cones. Changes in Tbetagamma characteristics by Tgamma methylation reveal conformational changes on a surface domain that is essential for Tbetagamma functions and support a regulatory role for reversible methylation.
Asunto(s)
Proteínas del Ojo/metabolismo , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Células Fotorreceptoras Retinianas Conos/fisiología , Transducina/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Reguladores de Proteínas de Unión al GTP , Datos de Secuencia Molecular , Mapeo Peptídico , Mapeo de Interacción de Proteínas , Células Fotorreceptoras Retinianas Bastones/fisiología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
PURPOSE: During the course of our investigations on cyclic nucleotide dependent-phosphorylation of membrane proteins, we observed that phosducin was present in both the cytosolic and nuclear fractions. It has been suggested that phosducin might have a role in regulating transcription, but its presence in the nucleus has not been previously reported. We therefore attempted to purify nuclei from bovine retina and determine whether the purified preparation contained phosducin. Cyclic nucleotide-dependent phosphorylation of the protein was also investigated in the homogenate and subcellular fractions of bovine retina. METHODS: Freshly obtained bovine retinas were homogenized and fractionated in an isotonic buffer. The homogenate and subcellular fractions were subjected to phosphorylation in the presence of gamma-32P ATP and the presence and absence of cyclic GMP. The phosphorylated proteins were identified by 1- or 2-dimensional electrophoresis and autoradiography. Phosducin was detected in the fractions by western blotting. A nuclear preparation was obtained from the homogenate by sucrose gradient centrifugation, its purity was determined with a nuclear stain, and the presence and phosphorylation of phosducin was investigated as above. RESULTS: Phosducin was found in the cytosol, 100,000x g membrane fraction, as well as in the 120x g pellet of a fresh, isotonic retinal homogenate, but cyclic nucleotide-dependent phosphorylation of the protein was observed only in the cytosolic fraction. Western blotting on a highly purified nuclear fraction showed that phosducin was present in the nuclei. The protein could be phosphorylated in a cyclic GMP-dependent fashion in a nuclear preparation permeabilized by freezing and thawing. CONCLUSIONS: It has been suggested that a C-terminal fragment of phosducin might be transported into the nucleus where it might have a role in the regulation of transcription. Phosducin localization in the nucleus was shown in COS cells cotransfected with genes of phosducin and a transcription factor. However, the presence of phosducin in the nuclei of retina has not been demonstrated hitherto. The present results show that phosducin is present in the nuclei in bovine retina. It could be phosphorylated in the nuclei in a cyclic GMP-dependent fashion. These results support a possible role for phosducin in the regulation of transcription.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas del Ojo/metabolismo , Fosfoproteínas/metabolismo , Retina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Autorradiografía , Western Blotting , Bovinos , GMP Cíclico/metabolismo , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Reguladores de Proteínas de Unión al GTP , Microscopía Fluorescente , Fosforilación , Proteínas Quinasas/metabolismo , Retina/citología , Fracciones SubcelularesRESUMEN
G proteins (Galphabetagamma) are essential signaling molecules, which dissociate into Galpha and Gbetagamma upon activation by heptahelical membrane receptors. We have identified the betagamma subunit complex of the photoreceptor-specific G protein, transducin (T), as a target of the ubiquitin-proteasome pathway. Ubiquitylated species of the transducin gamma-subunit (Tgamma) but not the alpha- or beta-subunits were assembled de novo in bovine photoreceptor preparations. In addition, Tgamma was exclusively ubiquitylated when Tbetagamma was dissociated from Talpha. Ubiquitylation of Tbetagamma on Tgamma was selectively catalyzed by human ubiquitin-conjugating enzymes UbcH5 and UbcH7 and was coincident with degradation of the entire Tbetagamma subunit complex in vitro by a mechanism requiring ATP and the proteasome. We also show that Tbetagamma association with phosducin, a photoreceptor-specific protein of unknown physiological function, blocks Tbetagamma ubiquitylation and subsequent degradation. Phosphorylation of phosducin by Ca(2+)/calmodulin-dependent protein kinase II, which inhibits phosducin-Tbetagamma complex formation, completely restored Tbetagamma ubiquitylation and degradation. We conclude that Tbetagamma is a substrate of the ubiquitin-proteasome pathway and suggest that phosducin serves to protect Tbetagamma following the light-dependent dissociation of Talphabetagamma.
Asunto(s)
Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Transducina/química , Transducina/metabolismo , Ubiquitina/metabolismo , Animales , Bovinos , Citosol/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Reguladores de Proteínas de Unión al GTP , Fosforilación , Unión Proteica , Isoformas de Proteínas , Células Fotorreceptoras Retinianas Bastones/metabolismoRESUMEN
PURPOSE: To report novel immunoreactivity in a patient with melanoma-associated retinopathy. DESIGN: Retrospective case report and experimental study. METHODS: A 32-year-old woman with a history of metastatic melanoma presented with bilateral decreased visual acuity. Electroretinography, Goldmann perimetry, immunohistochemistry, and Western blotting of her serum were performed. RESULTS: Electroretinography showed a "negative" B-wave. Paracentral and central scotomas were observed on Goldmann perimetry. Antibodies to a retinal transducin were demonstrated by Western blotting. No immunoreactivity to retinal bipolar cells was detected by immunohistochemistry. CONCLUSION: Melanoma-associated retinopathy can be related to a variety of antiretinal antibodies. Recognition of transducin, a novel melanoma-associated retinopathy antigen, may be important for identifying and treating patients with night blindness and melanoma.