RESUMO
Color vision is facilitated by distinct populations of cone photoreceptors in the retina. In rodents, cones expressing different opsin photopigments are sensitive to middle (M, 'green') and short (S, 'blue') wavelengths, and are differentially distributed across the retina. The mechanisms that control which opsin is expressed in a particular cone are poorly understood, but previous in vitro studies implicated thyroid hormone in cone differentiation. Thyroid hormone receptor beta 2 (TR beta 2) is a ligand-activated transcription factor that is expressed in the outer nuclear layer of the embryonic retina. Here we delete Thrb (encoding Tr beta 2) in mice, causing the selective loss of M-cones and a concomitant increase in S-opsin immunoreactive cones. Moreover, the gradient of cone distribution is disturbed, with S-cones becoming widespread across the retina. The results indicate that cone photoreceptors throughout the retina have the potential to follow a default S-cone pathway and reveal an essential role for Tr beta 2 in the commitment to an M-cone identity. Our findings raise the possibility that Thrb mutations may be associated with human cone disorders.
Assuntos
Receptores dos Hormônios Tireóideos/metabolismo , Células Fotorreceptoras Retinianas Cones/embriologia , Animais , Contagem de Células , Cor , Eletrorretinografia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Imuno-Histoquímica , Camundongos , Camundongos Knockout , RNA Mensageiro/análise , RNA Mensageiro/genética , Radioimunoensaio , Receptores dos Hormônios Tireóideos/química , Receptores dos Hormônios Tireóideos/deficiência , Receptores dos Hormônios Tireóideos/genética , Células Fotorreceptoras Retinianas Cones/anormalidades , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia , Opsinas de Bastonetes/genética , Hormônios Tireóideos/análise , Tireotropina/análiseRESUMO
In the chart accompanying Christopher Anderson's News & Comment article "Clinton asks for a greener DOE" (9 Apr., p. 153), the budget figures for Basic Energy Science were incorrect. The correct figures are $861 million for the 1993 appropriation and $802 million for the 1994 request.
Assuntos
Antígenos de Neoplasias/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas do Olho , Guanilato Ciclase/metabolismo , Lipoproteínas , Proteínas do Tecido Nervoso , Células Fotorreceptoras/metabolismo , Animais , Antígenos de Neoplasias/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , Bovinos , Ativação Enzimática , Hipocalcina , Proteínas Recombinantes , Recoverina , Retina/químicaRESUMO
A bovine retinal complementary DNA clone encoding the alpha subunit of transducin (T alpha) was isolated with the use of synthetic oligodeoxynucleotides as probes, and the complete nucleotide sequence of the insert was determined. THe predicted protein sequence of 354 amino acids includes the known sequences of four tryptic peptides and sequences adjacent to the residues that undergo adenosine diphosphate ribosylation by cholera toxin and pertussis toxin. On the basis of homologies to other proteins, such as the elongation factors of protein synthesis and the ras oncogene proteins, regions are identified that are predicted to be acylated and involved in guanine nucleotide binding and hydrolysis. Amino acid sequence similarity between T alpha and ras is confined to these regions of the molecules.
Assuntos
Proteínas de Membrana/genética , Oncogenes , Fatores de Alongamento de Peptídeos/genética , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/metabolismo , Sequência de Bases , Bovinos , Toxina da Cólera/metabolismo , Clonagem Molecular , DNA/genética , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Toxina Pertussis , Transducina , Fatores de Virulência de BordetellaRESUMO
Recoverin, a calcium ion (Ca2+)-binding protein of vertebrate photoreceptors, binds to photoreceptor membranes when the Ca2+ concentration is greater than 1 micromolar. This interaction requires a fatty acyl residue covalently linked to the recoverin amino (NH2)-terminus. Removal of the acyl residue, either by proteolytic cleavage of the NH2-terminus or by production of nonacylated recoverin, prevented recoverin from binding to membranes. The acylated recoverin NH2-terminus could be cleaved by trypsin only when Ca2+ was bound to recoverin. These results suggest that the hydrophobic NH2-terminus is constrained in Ca(2+)-free recoverin and liberated by Ca2+ binding. The hydrophobic acyl moiety of recoverin may interact with the membrane only when recoverin binds Ca2+.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas do Olho , Lipoproteínas , Proteínas de Membrana/metabolismo , Ácidos Mirísticos/metabolismo , Proteínas do Tecido Nervoso , Segmento Externo da Célula Bastonete/metabolismo , 1,2-Dipalmitoilfosfatidilcolina , Acilação , Animais , Antígenos de Neoplasias/isolamento & purificação , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/isolamento & purificação , Bovinos , Membrana Celular/metabolismo , Ácido Egtázico/farmacologia , Eletroforese em Gel de Poliacrilamida , Hipocalcina , Cinética , Lipossomos , Proteínas de Membrana/isolamento & purificação , Peso Molecular , Ácido Mirístico , Fragmentos de Peptídeos/isolamento & purificação , Fosfatidilserinas , Ligação Proteica , RecoverinaRESUMO
Transducin is a guanyl nucleotide-binding protein that couples rhodopsin photolysis to hydrolysis of guanosine 3',5'-monophosphate in rod photoreceptor cells of vertebrate retinas. Several complementary DNA clones encoding transducin subunits have recently been characterized. One clone, isolated from a bovine retina complementary DNA library, encodes a previously unidentified polypeptide with an amino acid sequence 78% identical to the sequence of the alpha subunit of bovine rod outer segment transducin. Antibodies to a synthetic peptide with amino acid sequence derived specifically from this novel polypeptide recognize a 41-kilodalton polypeptide in homogenates of bovine retina. Localization of this polypeptide in bovine retina by indirect immunofluorescence demonstrates that it is expressed only in cone outer segments. Antibodies to specific sequences found only in the rod transducin alpha subunit recognize a polypeptide localized only in the rod outer segment. Therefore, bovine rod and cone cells each express structurally related yet significantly different forms of transducin.
Assuntos
Proteínas de Membrana/fisiologia , Células Fotorreceptoras/metabolismo , Animais , Sequência de Bases , Bovinos , DNA/genética , Imunofluorescência , Proteínas de Membrana/genética , TransducinaRESUMO
The guanosine triphosphate-binding proteins (G proteins) found in a variety of tissues transduce signals generated by ligand binding to cell surface receptors into changes in intracellular metabolism. Amino acid sequences of peptides prepared by partial proteolysis of the alpha subunit of a bovine brain G protein and the alpha subunit of rod outer-segment transducin were determined. The two proteins show regions of sequence identity as well as regions of diversity. A portion of the amino-terminal peptide sequence of each protein is highly homologous with the corresponding region in the ras protein (a protooncogene product). These similarities suggest that G proteins and ras proteins may have analogous functions.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Oncogenes , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras) , Transdução GenéticaRESUMO
Vertebrate retinal photoreceptors recover from photoexcitation-induced hydrolysis of guanosine 3', 5'-monophosphate (cyclic GMP) by resynthesizing cyclic GMP, which reopens cation channels that have been closed by light. Activation of guanylate cyclase by light-induced depletion of cytosolic calcium is a key event in this recovery process. This cyclase has now been shown to be regulated by a 23-kilodalton calcium binding protein. The protein is present in both rod and cone photoreceptors and was named recoverin because it promotes recovery of the dark state. The amino acid sequence of recoverin exhibits three potential calcium binding sites (EF hands). That recoverin binds calcium was confirmed with calcium-45 and by observing calcium-induced changes in its tryptophan fluorescence. Recoverin activated guanylate cyclase when free calcium was lowered from 450 to 40 nM, an effect that was blocked by an antibody to recoverin. Thus, guanylate cyclase in retinal rods is stimulated during recovery by the calcium-free form of recoverin. A comparison of recoverin with other calcium binding proteins reveals that it may represent, along with the protein visinin, a family of proteins that are regulated by submicromolar calcium concentrations.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/farmacologia , Proteínas do Olho , Guanilato Ciclase/metabolismo , Lipoproteínas , Proteínas do Tecido Nervoso , Células Fotorreceptoras/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/isolamento & purificação , Bovinos , Ativação Enzimática , Hipocalcina , Cinética , Dados de Sequência Molecular , Peso Molecular , Especificidade de Órgãos , Recoverina , Retina/citologia , Homologia de Sequência do Ácido NucleicoRESUMO
Phototransduction in vertebrate rod and cone photoreceptor cells involves G protein-mediated light stimulation of cGMP hydrolysis. Enzymes of the cGMP hydrolysis cascades of rods and cones are products of different genes. Three different classes of cones in the human retina are maximally sensitive to either blue, green, or red light. Distinct opsin genes are expressed in each type of cone. The distribution of cone types in human retina was determined using anti-peptide antibodies that recognize specific amino acid sequences in green/red opsin and blue opsin. These antibodies together with an anti-peptide antibody against Tc alpha were used in double labeling experiments to demonstrate the presence of the Tc alpha peptide in all types of cones. cDNA clones corresponding to human rod and cone transducin alpha subunit (Tr alpha and Tc alpha) genes were isolated. Southern blot analyses of human genomic DNA suggest that there is only one rod T alpha gene but more than one cone T alpha gene. The multiple Tc alpha genes could be closely related genes or different Tc alpha alleles, or one could be a pseudogene.
Assuntos
Células Fotorreceptoras/análise , Transducina/análise , Sequência de Aminoácidos , Anticorpos/imunologia , Sequência de Bases , Southern Blotting , Percepção de Cores , DNA , Proteínas do Olho/imunologia , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , RNA Mensageiro/metabolismo , Opsinas de Bastonetes , Transducina/genéticaRESUMO
A G protein beta subunit gene (Gbe) is expressed only in the eyes of adult D. melanogaster. This gene was identified by probing a Drosophila head cDNA expression library with monoclonal antibodies to a previously characterized Drosophila G protein beta subunit (Gbb). Immunoblot and Northern analyses demonstrate that Gbe protein and mRNA is not present in Drosophila mutants that lack eyes. Immunocytochemical and in situ hybridization analyses further demonstrate that Gbe is expressed in the eyes but not in the brain, whereas Gbb is abundantly expressed in the brain. The Gbe product is approximately 45% identical to previously identified G beta subunits and defines a new G beta class. Its localization suggests a possible role in phototransduction.
Assuntos
Proteínas de Ligação ao GTP/química , Células Fotorreceptoras/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Northern Blotting , Western Blotting , Química Encefálica , Mapeamento Cromossômico , Clonagem Molecular , Drosophila melanogaster , Olho/química , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Alinhamento de SequênciaRESUMO
A human photoreceptor membrane guanylyl cyclase, RetGC, was recently cloned and expressed, but its localization and manner of regulation were not defined. We report here that RetGC is detected primarily in outer segments of human photoreceptor cells. Recombinant RetGC can be stimulated by a soluble retinal-specific factor. Ca2+ interferes with stimulation of RetGC by this factor with a cooperativity coefficient of 1.7 and EC50 near 200 nM. The Ca2+ sensitivities of recombinant RetGC and of guanylyl cyclase activity from rod outer segment membranes are very similar. Our findings indicate that RetGC is a photoreceptor-specific guanylyl cyclase which is stimulated by a retinal-specific activator and inhibited by physiologically relevant concentrations of free Ca2+. The Ca2+ sensitivity of RetGC may be responsible for some of the previously reported effects of Ca2+ on light adaptation and recovery of the dark state.
Assuntos
Fatores Biológicos/fisiologia , Cálcio/farmacologia , Guanilato Ciclase/metabolismo , Retina/fisiologia , Segmento Externo da Célula Bastonete/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos , Fatores Biológicos/isolamento & purificação , Bovinos , Membrana Celular/enzimologia , Clonagem Molecular , Humanos , Imuno-Histoquímica , Cinética , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/imunologia , Proteínas Recombinantes/metabolismo , Retinaldeído/farmacologia , TermodinâmicaRESUMO
Brain type II Ca2+/calmodulin-dependent protein kinase is a holoenzyme composed of several copies each of three subunits, alpha (50 kd), beta (60 kd), and beta' (58 kd), in varying proportions. The deduced amino acid sequences of alpha (reported here) and beta are highly similar but not identical. The major difference between them is the deletion from alpha of two short segments (residues 316-339 and 354-392 in beta). cDNAs that appear to encode beta' are identical to beta except for the deletion of a segment encoding residues 378-392. Thus, the structural differences among alpha, beta, and beta' arise primarily from deletions (or insertions) in a variable region lying immediately carboxyl to the protein kinase and calmodulin-binding domains. The alpha and beta subunits are encoded by distinct genes expressed primarily, if not exclusively, in brain. Rather than being encoded by a third gene, beta' may arise by alternative splicing of the beta gene transcript.
Assuntos
Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Dados de Sequência Molecular , Proteínas Quinases/análise , RatosRESUMO
Dark adaptation requires timely deactivation of phototransduction and efficient regeneration of visual pigment. No previous study has directly compared the kinetics of dark adaptation with rates of the various chemical reactions that influence it. To accomplish this, we developed a novel rapid-quench/mass spectrometry-based method to establish the initial kinetics and site specificity of light-stimulated rhodopsin phosphorylation in mouse retinas. We also measured phosphorylation and dephosphorylation, regeneration of rhodopsin, and reduction of all-trans retinal all under identical in vivo conditions. Dark adaptation was monitored by electroretinography. We found that rhodopsin is multiply phosphorylated and then dephosphorylated in an ordered fashion following exposure to light. Initially during dark adaptation, transduction activity wanes as multiple phosphates accumulate. Thereafter, full recovery of photosensitivity coincides with regeneration and dephosphorylation of rhodopsin.
Assuntos
Adaptação Ocular/fisiologia , Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/química , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Escuridão , Eletrorretinografia , Cinética , Luz , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Oxirredução , Fosforilação , Estimulação Luminosa , Retinaldeído/metabolismo , Retinoides/metabolismo , Serina , Fatores de Tempo , Visão Ocular/fisiologiaRESUMO
Mutations in the human CRALBP gene cause retinal pathology and delayed dark adaptation. Biochemical studies have not identified the primary physiological function of CRALBP. To resolve this, we generated and characterized mice with a non-functional CRALBP gene (Rlbp1(-/-) mice). The photosensitivity of Rlbp1(-/-) mice is normal but rhodopsin regeneration, 11-cis-retinal production, and dark adaptation after illumination are delayed by >10-fold. All-trans-retinyl esters accumulate during the delay indicating that isomerization of all-trans- to 11-cis-retinol is impaired. No evidence of photoreceptor degeneration was observed in animals raised in cyclic light/dark conditions for up to 1 year. Albino Rlbp(-/-) mice are protected from light damage relative to the wild type. These findings support a role for CRALBP as an acceptor of 11-cis-retinol in the isomerization reaction of the visual cycle.
Assuntos
Proteínas de Transporte/fisiologia , Adaptação à Escuridão , Visão Ocular/fisiologia , Animais , Proteínas de Transporte/genética , Eletrorretinografia , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras/metabolismo , Retina/fisiopatologia , Retinaldeído/metabolismo , Rodopsina/metabolismoRESUMO
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder caused by a CAG repeat expansion. To determine the mechanism of neurotoxicity, we produced transgenic mice and observed a cone-rod dystrophy. Nuclear inclusions were present, suggesting that the disease pathway involves the nucleus. When yeast two-hybrid assays indicated that cone-rod homeobox protein (CRX) interacts with ataxin-7, we performed further studies to assess this interaction. We found that ataxin-7 and CRX colocalize and coimmunoprecipitate. We observed that polyglutamine-expanded ataxin-7 can dramatically suppress CRX transactivation. In SCA7 transgenic mice, electrophoretic mobility shift assays indicated reduced CRX binding activity, while RT-PCR analysis detected reductions in CRX-regulated genes. Our results suggest that CRX transcription interference accounts for the retinal degeneration in SCA7 and thus may provide an explanation for how cell-type specificity is achieved in this polyglutamine repeat disease.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/fisiologia , Peptídeos/química , Transativadores/antagonistas & inibidores , Repetições de Trinucleotídeos , Fatores Etários , Animais , Ataxina-7 , Linhagem Celular , Núcleo Celular/ultraestrutura , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Perfilação da Expressão Gênica , Genes Sintéticos , Proteínas de Homeodomínio/fisiologia , Humanos , Substâncias Macromoleculares , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Príons/genética , Regiões Promotoras Genéticas , Ligação Proteica , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Transmissão Sináptica , Transativadores/fisiologia , Ativação Transcricional , Transfecção , Transgenes , Técnicas do Sistema de Duplo-HíbridoRESUMO
Rhodopsin dephosphorylation in Drosophila is a calcium-dependent process that appears to be catalyzed by the protein product of the rdgC gene. Two vertebrate rdgC homologs, PPEF-1 and PPEF-2, have been identified. PPEF-1 transcripts are present at low levels in the retina, while PPEF-2 transcripts and PPEF-2 protein are abundant in photoreceptors. To determine if PPEF-2 alone or in combination with PPEF-1 plays a role in rhodopsin dephosphorylation and to determine if retinal degeneration accompanies mutation of PPEF-1 and/or PPEF-2, we have produced mice carrying targeted disruptions in the PPEF-1 and PPEF-2 genes. Loss of either or both PPEFs has little or no effect on rod function, as mice lacking both PPEF-1 and PPEF-2 show little or no changes in the electroretinogram and PPEF-2-/- mice show normal single-cell responses to light in suction pipette recordings. Light-dependent rhodopsin phosphorylation and dephosphorylation are also normal or nearly normal as determined by (i) immunostaining of PPEF-2-/- retinas with the phosphorhodopsin-specific antibody RT-97 and (ii) mass spectrometry of C-terminal rhodopsin peptides from mice lacking both PPEF-1 and PPEF-2. Finally, PPEF-2-/- retinas show normal histology at 1 year of age, and retinas from mice lacking both PPEF-1 and PPEF-2 show normal histology at 3 months of age, the latest time examined. These data indicate that, in contrast to loss of rdgC function in Drosophila, elimination of PPEF function does not cause retinal degeneration in vertebrates.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Drosophila , Luz , Fosfoproteínas Fosfatases/genética , Retina/metabolismo , Retina/fisiologia , Rodopsina/metabolismo , Alelos , Animais , Clonagem Molecular , Primers do DNA/metabolismo , Vetores Genéticos , Immunoblotting , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Fosforilação , Fótons , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodopsina/química , Fatores de TempoRESUMO
Vertebrate and invertebrate photoreceptors respond with great speed and sensitivity to the onset of light; however, they also adapt quickly to constant light or a reduction of illumination. During the past year or so, new information has become available concerning the molecular mechanisms by which photoreceptors recover from and adapt to stimuli. These data have identified mechanisms that inactivate nearly every step of the vertebrate and invertebrate phototransduction pathways. Light-induced changes in the concentration of intracellular Ca2+ play an important role in photoreceptor recovery and adaptation. Recently, several proteins that may mediate the effects of Ca2+ on phototransduction have been identified.
Assuntos
Células Fotorreceptoras/fisiologia , Adaptação Fisiológica/fisiologia , Animais , Humanos , Células Fotorreceptoras de Invertebrados/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Cyclic guanosine 3',5'-monophosphate phosphodiesterase in crude extracts from bovine rod outer segments can be activated by the addition of bleached rod outer segment membranes and GTP. In the absence of rhodopsin-containing membranes, the phosphodiesterase specific activity decreases with increasing concentration. A trypsin-sensitive inhibitor believed to be responsible for this phenomenon can be separated from the phosphodiesterase by DEAE-cellulose chromatography of the crude extract. Phosphodiesterase eluted from the DEAE-cellulose column shows considerably less concentration-dependence than in the crude extract. This partially purified phosphodiesterase was used as the substrate to assay for inhibitor. A GTPase which is active only in the presence of bleached rod outer segment membranes coelutes with the phosphodiesterase and is distinct from the phosphodiesterase inhibitor we have isolated.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Células Fotorreceptoras/enzimologia , Proteínas/isolamento & purificação , Animais , Bovinos , Estabilidade de Medicamentos , Ativação Enzimática , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/farmacologia , Temperatura Alta , Membranas/análise , Células Fotorreceptoras/análise , Proteínas/farmacologia , Tripsina/farmacologiaRESUMO
Cholera toxin- and pertussis toxin-catalyzed ADP-ribosylation were used to identify and localize G protein substrates in Drosophila melanogaster and in Manduca sexta. Cholera toxin catalyzes ADP-ribosylation of 37 kDa and 50 kDa polypeptides, but these polypeptides are also substrates for an ADP-ribosyltransferase (EC 2.4.2.30) activity endogenous to the Drosophila extracts. Pertussis toxin modifies 37 kDa and 39 kDa polypeptides in Drosophila homogenates. The pattern of proteolysis of the 39 kDa pertussis toxin substrate is similar to that of mammalian Go and is influenced by guanyl nucleotide binding. The 39 kDa Go-like Drosophila and Manduca pertussis toxin substrates are found primarily in neural tissues. These studies provide further evidence that G proteins are present in Drosophila and that this organism can therefore be used to investigate the physiological roles of these enzymes using advanced genetic manipulations.
Assuntos
Toxina da Cólera/metabolismo , Drosophila melanogaster/enzimologia , Toxina Pertussis , Poli(ADP-Ribose) Polimerases/metabolismo , Fatores de Virulência de Bordetella/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Proteínas de Ligação ao GTP/metabolismo , Concentração de Íons de Hidrogênio , Peso Molecular , Mariposas , SolubilidadeRESUMO
Pertussis toxin inactivates certain G-proteins by introducing an ADP-ribose group near the carboxyl-terminus of the alpha-subunit. The major pertussis toxin substrate in Drosophila tissues is Go alpha. We introduced a pertussis toxin gene under control of the hsp70 heat-shock promoter into the Drosophila genome. When heat-shocked, transformed flies produce active pertussis toxin which ADP-ribosylates endogenous Go alpha. Pertussis toxin is expressed in photoreceptors, in the lamina of the eye and in epithelial cells lining the gut. As expected from the absence of Go alpha in photoreceptors, pertussis toxin does not affect the photoreceptor component of the Drosophila visual response. However, it abolishes light on- and off-transients in the electroretinogram. These transients normally arise from the lamina, a tissue where Go alpha transcripts have been detected. Pertussis toxin expression also blocks embryonic development and shortens the lifetime of adult Drosophila. Following heat-shock, transformed adults are active, but they fail to take up nutrients because they stop eating. High energy metabolites are significantly depleted shortly after pertussis toxin expression is induced and the flies die within 48 h.
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , Visão Ocular/efeitos dos fármacos , Adenosina Difosfato Ribose/metabolismo , Animais , Sequência de Bases , Drosophila melanogaster/embriologia , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Temperatura Alta , Dados de Sequência Molecular , Transfecção , Fatores de Virulência de Bordetella/genéticaRESUMO
GCAP-2, a mammalian photoreceptor-specific protein, is a Ca2+-dependent regulator of the retinal membrane guanylyl cyclases (Ret-GCs). Sensing the fall in intracellular free Ca2+ after photo-excitation, GCAP-2 stimulates the activity of Ret-GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP-2 is a small N-myristoylated protein containing four EF-hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP-2 alters its conformation in response to Ca2+-binding as measured by a Ca2+-dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+-bound and Ca2+-free forms of GCAP-2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N-terminus of Ca2+-bound GCAP-2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N-terminus of GCAP-2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP-2 and recoverin, the structural consequences of Ca2+-binding for these two proteins are significantly dissimilar.