RESUMO
Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.
Assuntos
Cílios/ultraestrutura , Doenças Retinianas/patologia , Segmento Externo da Célula Bastonete/ultraestrutura , Animais , Membrana Celular/metabolismo , Cílios/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Retina/química , Retina/metabolismo , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
Rhodopsin is a G protein-coupled receptor (GPCR) present in the rod outer segment (ROS) of photoreceptor cells that initiates the phototransduction cascade required for scotopic vision. Due to the remarkable advancements in technological tools, the chemistry of rhodopsin has begun to unravel especially over the past few decades, but mostly at the ensemble scale. Atomic force microscopy (AFM) is a tool capable of providing critical information from a single-molecule point of view. In this regard, to bolster our understanding of rhodopsin at the nanoscale level, AFM-based imaging, force spectroscopy, and nano-indentation techniques were employed on ROS disc membranes containing rhodopsin, isolated from vertebrate species both in normal and diseased states. These AFM studies on samples from native retinal tissue have provided fundamental insights into the structure and function of rhodopsin under normal and dysfunctional states. We review here the findings from these AFM studies that provide important insights on the supramolecular organization of rhodopsin within the membrane and factors that contribute to this organization, the molecular interactions stabilizing the structure of the receptor and factors that can modify those interactions, and the mechanism underlying constitutive activity in the receptor that can cause disease.
Assuntos
Rodopsina , Segmento Externo da Célula Bastonete , Rodopsina/análise , Rodopsina/química , Membrana Celular/química , Microscopia de Força Atômica , Espécies Reativas de Oxigênio , Segmento Externo da Célula Bastonete/químicaRESUMO
Rod and cone photoreceptor outer segment (OS) structural integrity is essential for normal vision; disruptions contribute to a broad variety of retinal ciliopathies. OSs possess many hundreds of stacked membranous disks, which capture photons and scaffold the phototransduction cascade. Although the molecular basis of OS structure remains unresolved, recent studies suggest that the photoreceptor-specific tetraspanin, peripherin-2/rds (P/rds), may contribute to the highly curved rim domains at disk edges. Here, we demonstrate that tetrameric P/rds self-assembly is required for generating high-curvature membranes in cellulo, implicating the noncovalent tetramer as a minimal unit of function. P/rds activity was promoted by disulfide-mediated tetramer polymerization, which transformed localized regions of curvature into high-curvature tubules of extended lengths. Transmission electron microscopy visualization of P/rds purified from OS membranes revealed disulfide-linked tetramer chains up to 100 nm long, suggesting that chains maintain membrane curvature continuity over extended distances. We tested this idea in Xenopus laevis photoreceptors, and found that transgenic expression of nonchain-forming P/rds generated abundant high-curvature OS membranes, which were improperly but specifically organized as ectopic incisures and disk rims. These striking phenotypes demonstrate the importance of P/rds tetramer chain formation for the continuity of rim formation during disk morphogenesis. Overall, this study advances understanding of the normal structure and function of P/rds for OS architecture and biogenesis, and clarifies how pathogenic loss-of-function mutations in P/rds cause photoreceptor structural defects to trigger progressive retinal degenerations. It also introduces the possibility that other tetraspanins may generate or sense membrane curvature in support of diverse biological functions.
Assuntos
Periferinas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Humanos , Periferinas/química , Periferinas/genética , Células Fotorreceptoras Retinianas Cones/química , Células Fotorreceptoras Retinianas Bastonetes/química , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/metabolismo , Xenopus laevisRESUMO
Vision begins when light is captured by the outer segment organelle of photoreceptor cells in the retina. Outer segments are modified cilia filled with hundreds of flattened disk-shaped membranes. Disk membranes are separated from the surrounding plasma membrane, and each membrane type has unique protein components. The mechanisms underlying this protein sorting remain entirely unknown. In this study, we investigated the outer segment delivery of the rod cyclic nucleotide-gated (CNG) channel, which is located in the outer segment plasma membrane, where it mediates the electrical response to light. Using Xenopus and mouse models of both sexes, we now show that the targeted delivery of the CNG channel to the outer segment uses the conventional secretory pathway, including protein processing in both ER and Golgi, and requires preassembly of its constituent α1 and ß1 subunits. We further demonstrate that the N-terminal glutamic acid-rich protein (GARP) domain of CNGß1 contains two distinct functional regions. The glutamic acid-rich region encodes specific information targeting the channel to rod outer segments. The adjacent proline-enriched region connects the CNG channel to photoreceptor disk rims, likely through an interaction with peripherin-2. These data reveal fine functional specializations within the structural domains of the CNG channel and suggest that its sequestration to the outer segment plasma membrane requires an interaction with peripherin-2.SIGNIFICANCE STATEMENT Neurons and other differentiated cells have a remarkable ability to deliver and organize signaling proteins at precise subcellular locations. We now report that the CNG channel, mediating the electrical response to light in rod photoreceptors, contains two specialized regions within the N terminus of its ß-subunit: one responsible for delivery of this channel to the ciliary outer segment organelle and another for subsequent channel sequestration into the outer segment plasma membrane. These findings expand our understanding of the molecular specializations used by neurons to populate their critical functional compartments.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos/fisiologia , Segmento Externo da Célula Bastonete/metabolismo , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Sítios de Ligação/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Feminino , Masculino , Proteínas de Membrana/química , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/química , Segmento Externo da Célula Bastonete/química , XenopusRESUMO
Retinal, the vitamin A aldehyde, is a potent photosensitizer that plays a major role in light-induced damage to vertebrate photoreceptors. 11-Cis retinal is the light-sensitive chromophore of rhodopsin, the photopigment of vertebrate rod photoreceptors. It is isomerized by light to all-trans, activating rhodopsin and beginning the process of light detection. All-trans retinal is released by activated rhodopsin, allowing its regeneration by fresh 11-cis retinal continually supplied to photoreceptors. The released all-trans retinal is reduced to all-trans retinol in a reaction using NADPH. We have examined the photooxidation mediated by 11-cis and all-trans retinal in single living rod photoreceptors isolated from mouse retinas. Photooxidation was measured with fluorescence imaging from the oxidation of internalized BODIPY C11, a fluorescent dye whose fluorescence changes upon oxidation. We found that photooxidation increased with the concentration of exogenously added 11-cis or all-trans retinal to metabolically compromised rod outer segments that lacked NADPH supply. In dark-adapted metabolically intact rod outer segments with access to NADPH, there was no significant increase in photooxidation following exposure of the cell to light, but there was significant increase following addition of exogenous 11-cis retinal. The results indicate that both 11-cis and all-trans retinal can mediate light-induced damage in rod photoreceptors. In metabolically intact cells, the removal of the all-trans retinal generated by light through its reduction to retinol minimizes all-trans retinal-mediated photooxidation. However, because the enzymatic machinery of the rod outer segment cannot remove 11-cis retinal, 11-cis-retinal-mediated photooxidation may play a significant role in light-induced damage to photoreceptor cells.
Assuntos
Células Fotorreceptoras/química , Retinaldeído/química , Segmento Externo da Célula Bastonete/química , Vitamina A/química , Animais , Camundongos , Camundongos Knockout , Estrutura Molecular , Imagem Óptica , Oxirredução , Processos FotoquímicosRESUMO
The visual photopigment rhodopsin (Rh) is a prototypical G protein-coupled receptor (GPCR) responsible for initiation of the phototransduction cascade in rod photoreceptors. Similar to other GPCRs, Rh can form dimers or even higher oligomers and tends to have a supramolecular organization that is likely important in the dim light response. Rh also exhibits high affinity for lipid rafts (i.e. raftophilicity) upon light-dependent binding with the cognate G protein transducin (Gt), suggesting the presence of lipid raft-like domains in the retinal disk membrane and their importance in phototransduction. However, the relationship between Rh oligomerization and lipid rafts in the disk membrane remains to be explored. Given previous findings that Gt binds to dimeric Rh and that Rh is posttranslationally modified with two highly raftophilic palmitoyl moieties, we hypothesized that Rh becomes raftophilic upon dimerization. Here, using biochemical assays, we found that Rh*-Gt complexes in the detergent-resistant membrane are partially resistant to cholesterol depletion by methyl-ß-cyclodextrin and that the Rh-to-Gt stoichiometry in this methyl-ß-cyclodextrin-resistant complex is 2:1. Next, we found that IgG-mediated Rh-Rh cross-linking renders Rh highly raftophilic, supporting the premise that Rh becomes raftophilic upon dimerization. Rh depalmitoylation via reduction of thioester linkages blocked the translocation of IgG-cross-linked Rh to the detergent-resistant membrane, highlighting that the two palmitoyl moieties are important for the dimerization-dependent raftophilicity of Rh. These results indicate that palmitoylated GPCRs such as Rh can acquire raftophilicity upon G protein-stabilized dimerization and thereby organize receptor-cluster rafts by recruiting raftophilic lipids.
Assuntos
Lipoilação , Microdomínios da Membrana/metabolismo , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Rana catesbeiana/fisiologia , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Cisteína/química , Cistina/química , Adaptação à Escuridão , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Cinética , Luz , Lipoilação/efeitos da radiação , Microdomínios da Membrana/química , Microdomínios da Membrana/efeitos da radiação , Oxirredução , Conformação Proteica/efeitos da radiação , Multimerização Proteica/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Estabilidade Proteica/efeitos da radiação , Rodopsina/química , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/efeitos da radiação , Transducina/química , Transducina/metabolismoRESUMO
The function of the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) gene is currently not known. However, mutations within the gene lead to Leber Congenital Amaurosis and autosomal recessive retinitis pigmentosa in human patients. In a previously described knockout mouse model of the long splice variant of Rpgrip1, herein referred to as Rpgrip1(tm1Tili) mice, mislocalization of key outer segment proteins and dysmorphogenesis of outer segment discs preceded subsequent photoreceptor degeneration. In this report, we describe a new mouse model carrying a splice acceptor site mutation in Rpgrip1, herein referred to as Rpgrip1(nmf247) that is phenotypically distinct from Rpgrip1(tm1Tili) mice. Photoreceptor degeneration in homozygous Rpgrip1(nmf247) mice is earlier in onset and more severe when compared with Rpgrip1(tm1Tili) mice. Also, ultrastructural studies reveal that whereas Rpgrip1(nmf247) mutants have a normal structure and number of connecting cilia, unlike Rpgrip1(tm1Tili) mice, they do not elaborate rod outer segments (OS). Therefore, in addition to its role in OS disc morphogenesis, RPGRIP1 is essential for rod OS formation. Our study indicates the absence of multiple Rpgrip1 isoforms in Rpgrip1(nmf247) mice, suggesting different isoforms may play different roles in photoreceptors and underscores the importance of considering splice variants when generating targeted null mutations.
Assuntos
Morfogênese , Proteínas/metabolismo , Retina/crescimento & desenvolvimento , Retinose Pigmentar/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Mutação , Transporte Proteico , Proteínas/química , Proteínas/genética , Retina/metabolismo , Retinose Pigmentar/genética , Segmento Externo da Célula Bastonete/química , Alinhamento de SequênciaRESUMO
The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.
Assuntos
GMP Cíclico/química , Guanilato Ciclase/química , Peptídeos/metabolismo , Receptores de Superfície Celular/química , Segmento Externo da Célula Bastonete/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Clonagem Molecular , Reagentes de Ligações Cruzadas/química , GMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Succinimidas/químicaRESUMO
The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.
Assuntos
Membrana Celular/metabolismo , Proteômica/métodos , Segmento Externo da Célula Bastonete/fisiologia , Proteínas SNARE/química , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Bovinos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Microscopia de Fluorescência , Modelos Biológicos , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/metabolismo , Retina/metabolismo , Segmento Externo da Célula Bastonete/química , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
A method for obtaining a free complex of transducin betagamma-subunits from bovine retinal rod outer segments in a highly purified state has been suggested.
Assuntos
Segmento Externo da Célula Bastonete/química , Transducina/isolamento & purificação , Animais , Bovinos , Complexos Multiproteicos/isolamento & purificação , Subunidades Proteicas/isolamento & purificaçãoRESUMO
The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.
Assuntos
Proteínas do Olho/análise , Proteínas de Filamentos Intermediários/análise , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Células Fotorreceptoras/química , Degeneração Retiniana/metabolismo , Segmento Externo da Célula Bastonete/química , Animais , Bovinos , Membrana Celular/química , Membrana Celular/ultraestrutura , Imunofluorescência , Imuno-Histoquímica , Microscopia Eletrônica , Morfogênese , Periferinas , Células Fotorreceptoras/ultraestrutura , Ratos , Degeneração Retiniana/patologia , Segmento Externo da Célula Bastonete/ultraestrutura , SciuridaeRESUMO
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
In spite of recent advances in identifying genes causing monogenic human disease, very little is known about the genes involved in polygenic disease. Three families were identified with mutations in the unlinked photoreceptor-specific genes ROM1 and peripherin/RDS, in which only double heterozygotes develop retinitis pigmentosa (RP). These findings indicate that the allelic and nonallelic heterogeneity known to be a feature of monogenic RP is complicated further by interactions between unlinked mutations causing digenic RP. Recognition of the inheritance pattern exemplified by these three families might facilitate the identification of other examples of digenic inheritance in human disease.
Assuntos
Proteínas do Olho/genética , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso , Retinose Pigmentar/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Eletrorretinografia , Proteínas do Olho/química , Feminino , Genes Dominantes , Genes Recessivos , Ligação Genética , Heterozigoto , Humanos , Proteínas de Filamentos Intermediários/química , Masculino , Proteínas de Membrana/química , Dados de Sequência Molecular , Mutação , Linhagem , Periferinas , Segmento Externo da Célula Bastonete/química , TetraspaninasRESUMO
G-protein-coupled receptors (GPCRs) participate in virtually all physiological processes. They constitute the largest and most structurally conserved family of signaling molecules. Several class C GPCRs have been shown to exist as dimers in their active form and growing evidence indicates that many, if not all, class A receptors also form dimers and/or higher-order oligomers. High-resolution crystal structures are available only for the detergent-solubilized light receptor rhodopsin (Rho), the archetypal class A GPCR. In addition, Rho is the only GPCR for which the presumed higher-order oligomeric state has been demonstrated, by imaging native disk membranes using atomic force microscopy (AFM). Based on these data and the X-ray structure, an atomic model of Rho dimers has been proposed, a model that is currently scrutinized in various ways. AFM has also been used to measure the forces required to unfold single Rho molecules, thereby revealing which residues are responsible for Rho's stability. Recent functional analyses of fractions from solubilized disk membranes revealed that higher-order Rho oligomers are the most active species. These and other results have enhanced our understanding of GPCR structure and function.
Assuntos
Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Rodopsina/química , Animais , Cristalografia por Raios X , Dimerização , Camundongos , Microscopia de Força Atômica , Conformação Proteica , Dobramento de Proteína , Segmento Externo da Célula Bastonete/químicaRESUMO
After solubilization of frog rod outer segments (ROS) with mild detergents (digitonin, n-dodecyl-beta-D-maltoside, Chaps, Triton X-100) and subsequent one-dimensional blue native polyacrylamide gel electrophoresis (1D BN-PAGE), the position of rhodopsin (Rh) on the gradient gel does not match the monomer with molecular weight of 40 kDa but appears self-associated into aggregate of Rh (RhA) with molecular mass varying in different detergents from 85 to 125 kDa. Short-term treatment (~2 h) of the excised BN-PAGE strip containing RhA by denaturing detergent mixture (10% SDS + 1 mM dithiothreitol (DTT)) followed by 2D SDS-PAGE revealed dissociation of the RhA into opsin monomer and unidentified proteins. Long-term treatment (approximately 2 days) of RhA that included extraction, denaturation, concentration, and electrophoresis induced, along with dissociation of RhA into opsin monomer + unidentified proteins, also formation of opsin dimers, trimers, and higher oligomers owing to a secondary aggregation of opsin. Direct solubilization of the ROS by harsh SDS + DTT detergent mixture followed by 1D SDS-PAGE revealed only opsin monomer that upon heating disappeared, transforming into higher oligomers owing to secondary aggregation. The data show that degree of Rh oligomerization depends on specific conditions in which it stays. In the native state in the photoreceptor membrane as well as in mild detergents frog Rh exists mainly as dimers or higher oligomers. After solubilization with denaturing detergents, RhA can dissociate into monomers that then spontaneously self-associate into higher oligomers under the influence of various factors (for example, heating).
Assuntos
Proteínas de Anfíbios/química , Rodopsina/química , Segmento Externo da Célula Bastonete/química , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/metabolismo , Animais , Detergentes , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Ligação Proteica , Desnaturação Proteica , Multimerização Proteica , Rana temporaria , Agregação de Receptores , Rodopsina/isolamento & purificação , Rodopsina/metabolismoRESUMO
Membrane proteins play an integral role in cellular communication. They are often organized within the crowded cell membrane into nanoscale domains (i.e., nanodomains), which facilitates their function in cell signaling processes. The visualization of membrane proteins and nanodomains within biological membranes under physiological conditions presents a challenge and is not possible using conventional microscopy methods. Atomic force microscopy (AFM) provides an opportunity to study the organization of membrane proteins within biological membranes with sub-nanometer resolution. An example of a membrane protein organized into nanodomains is rhodopsin. Rhodopsin is expressed in photoreceptor cells of the retina and upon photoactivation initiates a series of biochemical reactions called phototransduction, which represents the first steps of vision. AFM has provided an opportunity to directly visualize the packing of rhodopsin in native retinal membranes and the quantitative analysis of AFM images is beginning to reveal insights about the nanodomain organization of rhodopsin in the membrane. In this report, we outline procedures for imaging rhodopsin nanodomains by AFM and the quantitative analysis of those AFM images.
Assuntos
Membrana Celular/química , Microscopia de Força Atômica , Rodopsina/química , Animais , Membrana Celular/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Microscopia de Força Atômica/métodos , Células Fotorreceptoras/metabolismo , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/metabolismoRESUMO
The cyclic nucleotide-gated channel from rod photoreceptors is composed of two distinct subunits (alpha and beta). The properties of the alpha subunit, which can form functional channels by itself, are modified by coexpression with a homologous polypeptide, designated the beta subunit. However, the alpha subunit from rod photoreceptor membranes copurifies with a 240 kDa protein that is significantly larger than this putative beta subunit. We now demonstrate by peptide sequencing and by cloning and functional expression of cDNA that the 240 kDa protein represents the complete beta subunit with an unusual bipartite structure. The N-terminal part is essentially identical to a glutamic acid-rich protein (GARP), whereas the C-terminal part is highly homologous to the previously cloned human "beta subunit." Expression of the complete beta subunit in HEK 293 cells results in a polypeptide with the same apparent molecular weight as the 240 kDa protein of the native rod channel. Coexpression of the alpha subunit with the full-length beta subunit yields hetero-oligomeric channels with properties characteristic of the native channel.
Assuntos
Proteínas do Olho/química , Canais Iônicos/química , Segmento Externo da Célula Bastonete/química , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Bovinos , Linhagem Celular , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Eletrofisiologia , Proteínas do Olho/genética , Expressão Gênica , Humanos , Canais Iônicos/genética , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Proteínas do Tecido Nervoso , Células Fotorreceptoras/metabolismo , Proteínas/química , RNA Mensageiro/análise , Retina/química , TransfecçãoRESUMO
Mice homozygous for the retinal degeneration slow (rds) mutation exhibit abnormal development of photoreceptor cells, followed by their slow degeneration. We have recently cloned the rds gene and determined the structure of the wild-type rds mRNA. Here we show that the gene is expressed exclusively in photoreceptor cells. We demonstrate that it encodes a 39 kd membrane-associated glycoprotein that is restricted to photoreceptor outer segments. By electron microscopy, we show that the rds protein is distributed uniformly within outer segment discs. The developmental appearance of the rds protein coincides with outer segment disc formation. We propose that the rds protein functions as an adhesion molecule for stabilization of the outer segment discs.
Assuntos
Proteínas do Olho/genética , Proteínas de Filamentos Intermediários , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/genética , Sequência de Aminoácidos , Animais , Membrana Celular/química , DNA/genética , Proteínas do Olho/análise , Expressão Gênica , Glicosilação , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Mutantes , Microscopia Eletrônica , Dados de Sequência Molecular , Periferinas , Células Fotorreceptoras/química , Retina/química , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/ultraestruturaRESUMO
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
A cadherin family member, prCAD, was identified in retina cDNA by subtractive hybridization and high throughput sequencing. prCAD is expressed only in retinal photoreceptors, and the prCAD protein is localized to the base of the outer segment of both rods and cones. In prCAD(-/-) mice, outer segments are disorganized and fragmented, and there is progressive death of photoreceptor cells. prCAD is unlikely to be involved in protein trafficking between inner and outer segments, since phototransduction proteins appear to be correctly localized and the light responses of both rods and cones are only modestly compromised in prCAD(-/-) mice. These experiments imply a highly specialized cell biological function for prCAD and suggest that localized adhesion activity is essential for outer segment integrity.