RESUMO
Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity-purified from complex CAR antisera and used for EM-immunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinity-purified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure.
Assuntos
Proteínas de Ligação ao Cálcio/análise , Proteínas do Olho/análise , Síndromes Paraneoplásicas/metabolismo , Retina/química , Degeneração Retiniana/metabolismo , Sequência de Aminoácidos , Animais , Autoanticorpos/imunologia , Western Blotting , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Bovinos , Cromatografia por Troca Iônica , Proteínas do Olho/química , Proteínas do Olho/imunologia , Soros Imunes , Imuno-Histoquímica , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Síndromes Paraneoplásicas/imunologia , Recoverina , Retina/imunologia , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/imunologia , Homologia de Sequência do Ácido NucleicoRESUMO
Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/química , Rodopsina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Membrana Celular/química , Cristalografia por Raios X , Ligação de Hidrogênio , Luz , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Retinaldeído/química , Retinaldeído/metabolismo , Rodopsina/metabolismo , Bases de Schiff , Estereoisomerismo , Visão OcularRESUMO
Rhodopsin kinase and the beta-adrenergic receptor kinase (beta ARK) catalyse the phosphorylation of the activated forms of the G-protein-coupled receptors, rhodopsin and the beta 2-adrenergic receptor (beta 2AR), respectively. The interaction between receptor and kinase is independent of second messengers and appears to involve a multipoint attachment of kinase and substrate with the specificity being restricted by both the primary amino acid sequence and conformation of the substrate. Kinetic, functional and sequence information reveals that rhodopsin kinase and beta ARK are closely related, suggesting they may be members of a family of G-protein-coupled receptor kinases.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Proteínas do Olho , Proteínas Quinases/fisiologia , Receptores Adrenérgicos beta/metabolismo , Rodopsina/metabolismo , Animais , Receptor Quinase 1 Acoplada a Proteína G , Proteínas de Ligação ao GTP/fisiologia , Humanos , Fosforilação , Quinases de Receptores Adrenérgicos betaRESUMO
G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.
Assuntos
Rodopsina/química , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Citoplasma/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Fótons , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Doenças Retinianas/metabolismo , Transdução de SinaisRESUMO
The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.
Assuntos
Antígenos/fisiologia , Proteínas do Olho/fisiologia , Luz , Proteínas Quinases/fisiologia , Segmento Externo da Célula Bastonete/fisiologia , Visão Ocular/fisiologia , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Animais , Arrestina , Bovinos , Diálise , Condutividade Elétrica , Receptor Quinase 1 Acoplada a Proteína G , Lagartos , Fosforilação , Ácido Fítico/farmacologia , Inibidores de Proteínas Quinases , Nucleosídeos de Pirimidina/farmacologia , Rodopsina/metabolismoRESUMO
The rod photoresponse is triggered by an enzyme cascade that stimulates cGMP hydrolysis. The resulting fall in cGMP leads to a decrease in Ca2+, which promotes photoresponse recovery by activating guanylate cyclase, causing cGMP resynthesis. In vitro biochemical studies suggest that Ca2+ activation of guanylate cyclase is medicated by recoverin, a 26 kd Ca(2+)-binding protein. To evaluate this, exogenous bovine recoverin and two other homologous Ca(2+)-binding proteins from chicken and Gecko retina were dialyzed into functionally intact Gecko rods using whole-cell recording. All three proteins prolonged the rising phase of the photoresponse without affecting the kinetics of response recovery. These results suggest that recoverin-like proteins affect termination of the transduction cascade, rather than mediate Ca(2+)-sensitive activation of guanylate cyclase.
Assuntos
Antígenos de Neoplasias/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , Proteínas do Olho , Luz , Lipoproteínas , Proteínas do Tecido Nervoso , Células Fotorreceptoras/efeitos dos fármacos , Proteína G de Ligação ao Cálcio S100/farmacologia , Animais , Eletrofisiologia , Hipocalcina , Lagartos , Peso Molecular , Células Fotorreceptoras/fisiologia , Células Fotorreceptoras/efeitos da radiação , Recoverina , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.
Assuntos
Aminoácido Oxirredutases/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas do Olho , Guanilato Ciclase/metabolismo , Lipoproteínas , Receptores de Detecção de Cálcio , Segmento Externo da Célula Bastonete/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Proteínas de Ligação ao Cálcio/química , Calmodulina/química , Bovinos , Clonagem Molecular , Sondas de DNA/química , Ativação Enzimática , Hipocalcina , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Neurocalcina , Óxido Nítrico Sintase , Fragmentos de Peptídeos/química , Células Fotorreceptoras/metabolismo , Filogenia , Ranidae , Recoverina , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de FluorescênciaRESUMO
Detailed biochemical, structural and physiological studies of the role of Ca2(+)-binding proteins in mammalian retinal neurons have yielded new insights into the function of these proteins in normal and pathological states. In phototransduction, a biochemical process that is responsible for the conversion of light into an electrical impulse, guanylate cyclases (GCs) are regulated by GC-activating proteins (GCAPs). These regulatory proteins respond to changes in cytoplasmic Ca2+ concentrations. Disruption of Ca2+ homeostasis in photoreceptor cells by genetic and environmental factors can result ultimately in degeneration of these cells. Pathogenic mutations in GC1 and GCAP1 cause autosomal recessive Leber congenital amaurosis and autosomal dominant cone dystrophy, respectively. This report provides a recent account of the advances, challenges, and possible future prospects of studying this important step in visual transduction that transcends to other neuronal Ca2+ homeostasis processes.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase/fisiologia , Guanilato Ciclase/metabolismo , Doenças Retinianas/fisiopatologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sinalização do Cálcio/fisiologia , Motivos EF Hand , Ativação Enzimática , Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/genética , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Células Fotorreceptoras/fisiologia , Alinhamento de Sequência , Visão Ocular/fisiologiaRESUMO
To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol dehydrogenase knockout mice, the appearance of the fundus was normal and punctata typical of this human hereditary ocular disease were not present. A second typical symptom associated with this disease is delayed dark adaptation. Homozygous 11-cis-retinol dehydrogenase mutants showed normal rod and cone responses. 11-cis-Retinol dehydrogenase knockout mice were capable of dark adaptation. At bleaching levels under which patients suffering from fundus albipunctatus could be detected unequivocally, 11-cis-retinol dehydrogenase knockout animals displayed normal dark adaptation kinetics. However, at high bleaching levels, delayed dark adaptation in 11-cis-retinol dehydrogenase knockout mice was noticed. Reduced 11-cis-retinol oxidation capacity resulted in 11-cis-retinol/13-cis-retinol and 11-cis-retinyl/13-cis-retinyl ester accumulation. Compared with wild-type mice, a large increase in the 11-cis-retinyl ester concentration was noticed in 11-cis-retinol dehydrogenase knockout mice. In the murine retinal pigment epithelium, there has to be an additional mechanism for the biosynthesis of 11-cis-retinal which partially compensates for the loss of the 11-cis-retinol dehydrogenase activity. 11-cis-Retinyl ester formation is an important part of this adaptation process. Functional consequences of the loss of 11-cis-retinol dehydrogenase activity illustrate important differences in the compensation mechanisms between mice and humans. We furthermore demonstrate that upon 11-cis-retinol accumulation, the 13-cis-retinol concentration also increases. This retinoid is inapplicable to the visual processes, and we therefore speculate that it could be an important catabolic metabolite and its biosynthesis could be part of a process involved in regulating 11-cis-retinol concentrations within the retinal pigment epithelium of 11-cis-retinol dehydrogenase knockout mice.
Assuntos
Oxirredutases do Álcool/metabolismo , Retinoides/metabolismo , Oxirredutases do Álcool/genética , Animais , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Visão OcularRESUMO
A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins. Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand. Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria. Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand. Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal. The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures. Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently. The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements. For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.
Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Membrana/química , Células Fotorreceptoras de Vertebrados/química , Retinaldeído/metabolismo , Rodopsina/química , Animais , Bacteriorodopsinas/química , Bovinos , Cristalografia por Raios X , Isomerismo , Ligantes , Proteínas de Membrana/fisiologia , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Retinaldeído/química , Rodopsina/metabolismoRESUMO
Vertebrate photoreceptor cells can signal the absorption of a single photon and then modulate their response as the intensity of the light and the intensity of the background illumination vary, and it has long been recognized that Ca2+ ions contribute to the underlying processes. Recently, several Ca(2+)-binding proteins of the EF-hand family were identified that mediate the actions of Ca2+ during the response to light. Molecular interactions between these Ca(2+)-binding proteins and their cellular targets are amenable to study owing in part to the unique features of phototransduction. In addition, two of the proteins, recoverin and guanylate cyclase activating protein (GCAP), appear to be involved in separate degenerative diseases of the retina that arise in humans and in animal models of human disease. Information obtained from these studies should also be relevant to the growing number of homologous proteins found in other neural tissues.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Cálcio/fisiologia , Retina/patologia , Retina/fisiologia , Animais , Humanos , Retina/químicaRESUMO
Recent genetic, biochemical and electrophysiological evidence has provided insights into the molecular identity of the substance responsible for bleaching desensitization in vision. Studies examining the molecular defects that cause delayed dark adaptation suggest that the desensitizing substance is recognized by rhodopsin kinase and/or arrestin and, therefore, is probably a complex comprising all-trans-retinal and opsin.
Assuntos
Transdução de Sinais/fisiologia , Vias Visuais/fisiologia , Animais , Eletrofisiologia , Humanos , Rodopsina/fisiologiaRESUMO
Examination of the role of Ca(2+)-binding proteins (CaBPs) in mammalian retinal neurons has yielded new insights into the function of these proteins in normal and pathological states. In the last 8 years, studies on guanylate cyclase (GC) regulation by three GC-activating proteins (GCAP1-3) led to several breakthroughs, among them the recent biochemical analysis of GCAP1(Y99) mutants associated with autosomal dominant cone dystrophy. Perturbation of Ca(2+) homeostasis controlled by mutant GCAP1 in photoreceptor cells may result ultimately in degeneration of these cells. Here, detailed analysis of biochemical properties of GCAP1(P50L), which causes a milder form of autosomal dominant cone dystrophy than constitutive active Y99C mutation, showed that the P50L mutation resulted in a decrease of Ca(2+)-binding, without changes in the GC activity profile of the mutant GCAP1. In contrast to this biochemically well-defined regulatory mechanism that involves GCAPs, understanding of other processes in the retina that are regulated by Ca(2+) is at a rudimentary stage. Recently, we have identified five homologous genes encoding CaBPs that are expressed in the mammalian retina. Several members of this subfamily are also present in other tissues. In contrast to GCAPs, the function of this subfamily of calmodulin (CaM)-like CaBPs is poorly understood. CaBPs are closely related to CaM and in biochemical assays CaBPs substitute for CaM in stimulation of CaM-dependent kinase II, and calcineurin, a protein phosphatase. These results suggest that CaM-like CaBPs have evolved into diverse subfamilies that control fundamental processes in cells where they are expressed.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Oftalmopatias/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Calmodulina/biossíntese , Bovinos , Mapeamento Cromossômico , Clonagem Molecular , Ativação Enzimática , Escherichia coli/metabolismo , Guanilato Ciclase/biossíntese , Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Células Fotorreceptoras de Vertebrados/enzimologia , Retina/enzimologia , Alinhamento de SequênciaRESUMO
The crystal structure of rhodopsin has provided the first three-dimensional molecular model for a G-protein-coupled receptor (GPCR). Alignment of the molecular model from the crystallographic structure with the helical axes seen in cryo-electron microscopic (cryo-EM) studies provides an opportunity to investigate the properties of the molecule as a function of orientation and location within the membrane. In addition, the structure provides a starting point for modeling and rational experimental approaches of the cone pigments, the GPCRs in cone cells responsible for color vision. Homology models of the cone pigments provide a means of understanding the roles of amino acid sequence differences that shift the absorption maximum of the retinal chromophore in the environments of different opsins.
Assuntos
Proteínas de Membrana/química , Receptores de Superfície Celular/química , Células Fotorreceptoras Retinianas Cones/química , Pigmentos da Retina/química , Rodopsina/química , Sequência de Aminoácidos , Animais , Anuros , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia , Citoplasma/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Opsinas de Bastonetes/químicaRESUMO
Recoverin is an EF-hand calcium-binding protein reportedly involved in the transduction of light by vertebrate photoreceptor cells. It also is an autoantigen in a cancer-associated degenerative disease of the retina. Measurements by circular dichroism presented here demonstrate that the binding of calcium to recoverin causes large structural changes. increasing the alpha-helical content of the protein and decreasing its beta-turn, beta-sheet and 'other' structures. The maximum helical content (67%) was observed at 100 microM free calcium and, unlike calmodulin, decreased as the calcium concentration was modulated in either direction from this value. Fluorescence measurements indicated that recoverin may aggregate or undergo structural changes independent of calcium binding as the calcium concentration is increased above 100 microM. EGTA also appeared to affect the structure of recoverin independent of its chelation of calcium. While calcium-induced conformational changes have been proposed to alter the membrane binding of recoverin through association of its myristoylated amino terminus, in the experiments presented here the partitioning of recoverin between the cytoplasmic and membrane compartments of the rod photoreceptor outer segment was unaffected by the concentration of calcium, therefore it appears unlikely that a calcium-myristoyl switch acts alone to anchor recoverin directly to the membrane. These experiments were conducted with native recoverin which is heterogeneously acylated, but mass spectrometry confirmed that simple chromatographic methods could be devised to isolate the different forms of recoverin for further studies.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas do Olho , Lipoproteínas , Proteínas do Tecido Nervoso , Estrutura Secundária de Proteína , Animais , Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Bovinos , Dicroísmo Circular , Ácido Egtázico/farmacologia , Hipocalcina , Membranas/química , Membranas/efeitos dos fármacos , Membranas/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Recoverina , Segmento Externo da Célula Bastonete/metabolismo , Espectrometria de Fluorescência , Triptofano , TirosinaRESUMO
Absorption of light by rhodopsin or cone pigments in photoreceptors triggers photoisomerization of their universal chromophore, 11-cis-retinal, to all-trans-retinal. This photoreaction is the initial step in phototransduction that ultimately leads to the sensation of vision. Currently, a great deal of effort is directed toward elucidating mechanisms that return photoreceptors to the dark-adapted state, and processes that restore rhodopsin and counterbalance the bleaching of rhodopsin. Most notably, enzymatic isomerization of all-trans-retinal to 11-cis-retinal, called the visual cycle (or more properly the retinoid cycle), is required for regeneration of these visual pigments. Regeneration begins in rods and cones when all-trans-retinal is reduced to all-trans-retinol. The process continues in adjacent retinal pigment epithelial cells (RPE), where a complex set of reactions converts all-trans-retinol to 11-cis-retinal. Although remarkable progress has been made over the past decade in understanding the phototransduction cascade, our understanding of the retinoid cycle remains rudimentary. The aim of this review is to summarize recent developments in our current understanding of the retinoid cycle at the molecular level, and to examine the relevance of these reactions to phototransduction.
Assuntos
Retina/metabolismo , Retinoides/metabolismo , Visão Ocular/fisiologia , Animais , Adaptação à Escuridão/fisiologia , Humanos , Luz , Retina/efeitos da radiação , Rodopsina/fisiologia , Rodopsina/efeitos da radiaçãoRESUMO
Transmembrane signal transductions in a variety of cell types that mediate signals as diverse as those carried by neurotransmitters, hormones, and sensory signals share basic biochemical mechanisms that include: (1) an extracellular perturbation (neurotransmitter, hormone, odor, light); (2) specific receptors; (3) coupling proteins, such as G proteins; and (4) effector enzymes or ion channels. Parallel to these amplification reactions, receptors are precisely inactivated by mechanisms that involve protein kinases and regulatory proteins called arrestins. The structure and functions of arrestins are the focus of this review.
Assuntos
Antígenos , Proteínas do Olho , Inibidores de Fosfodiesterase , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Arrestina , Humanos , Dados de Sequência Molecular , Rodopsina/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Arrestin is involved in the quenching of phototransduction by binding to photoactivated and phosphorylated rhodopsin (P-Rho*). To study its conformational changes and regions interacting with P-Rho*, arrestin was subjected to (1) differential acetylation at lysine residues in the presence and absence of P-Rho*, and (2) amide hydrogen/deuterium exchange. Labeled protein was proteolysed and analyzed by mass spectrometry. Three Lys residues, 28, 176, and 211, were protected from acetylation in native arrestin, although they were not located in regions exhibiting slow amide hydrogen exchange rates. The presence of P-Rho* protected lysine 201 from acetylation and partially protected 14 other lysyl residues, including (2, 5), (163, 166, 167), (232, 235, 236, 238), (267, 276), (298, 300), and 367, where parentheses indicate lysine residues found within the same peptide. In contrast, in the C-terminal region of arrestin, lysyl residues (386, 392, 395) were more exposed upon binding to P-Rho*. These data allowed us to identify functional regions in the arrestin molecule.
Assuntos
Antígenos/química , Deutério/metabolismo , Proteínas do Olho/química , Hidrogênio/metabolismo , Conformação Proteica , Rodopsina/metabolismo , Acetilação , Sequência de Aminoácidos , Antígenos/metabolismo , Arrestina , Proteínas do Olho/metabolismo , Heparina/metabolismo , Lisina/química , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fotólise , Rodopsina/efeitos da radiaçãoRESUMO
The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal 35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N- and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.
Assuntos
Antígenos/química , Proteínas do Olho/química , Segmento Externo da Célula Bastonete/química , Sequência de Aminoácidos , Animais , Antígenos/isolamento & purificação , Antígenos/metabolismo , Arrestina , Bovinos , Proteínas do Olho/isolamento & purificação , Proteínas do Olho/metabolismo , Immunoblotting , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Fotólise , Rodopsina/metabolismo , Tripsina/metabolismoRESUMO
An isoform of RGS9 was recently identified as the GTPase activating protein in bovine and mouse rod and cone photoreceptors. To explore the potential role of the RGS9 gene in human retinal disease, we determined its exon/intron arrangement, and investigated its expression in human retina. The results show that the gene, located on 17q24, consists of 19 exons and spans more than 75kb of genomic DNA. The entire gene was found to be contained on a single BAC clone with an insert size of 170kb. The major transcripts of the gene are alternatively spliced into a 9.5kb retina-specific transcript (RGS9-1) and a brain specific 2.5kb transcript (RGS9-2). Exons 1-16 are constitutive and present in both variants. Exon 17 contains the 3' end of the open reading frame and the 3'-UTR of the RGS9-1 variant. In RGS9-2, exon 17 is alternatively spliced and joined to exons 18 and 19 that are not present in the retina variant. Immunolocalization with a monoclonal antibody recognizing the retina and brain variants shows abundant expression in photoreceptors and possibly very low levels in cell types of the inner retina. Owing to the specific expression of RGS9-1 in photoreceptors the RGS9 gene is a candidate gene for RP17, a form of autosomal retinitis pigmentosa, located on the long arm of chromosome 17.