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2.
Neuroscience ; 137(1): 165-75, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16257493

RESUMO

Aquaporin-4 water channels and the inwardly rectifying potassium channels Kir4.1 are coexpressed in a highly polarized manner at the perivascular and subvitreal endfeet of retinal Müller cells and astrocytes. The present study was aimed at resolving the anchoring mechanisms responsible for the coexpression of these molecules. Both aquaporin-4 and Kir4.1 contain PDZ-domain binding motifs at their C-termini and it was recently shown that mice with targeted disruption of the dystrophin gene display altered distribution of aquaporin-4 and Kir4.1 in the retina. To test our hypothesis that alpha-syntrophin (a PDZ-domain containing protein of the dystrophin associated protein complex) is involved in aquaporin-4 and Kir4.1 anchoring in retinal cells, we studied the expression pattern of these molecules in alpha-syntrophin null mice. Judged by quantitative immunogold cytochemistry, deletion of the alpha-syntrophin gene causes a partial loss (by 70%) of aquaporin-4 labeling at astrocyte and Müller cell endfeet but no decrease in Kir4.1 labeling at these sites. These findings suggest that alpha-syntrophin is not involved in the anchoring of Kir4.1 and only partly responsible for the anchoring of aquaporin-4 in retinal endfeet membranes. Furthermore we show that wild type and alpha-syntrophin null mice exhibit strong beta1 syntrophin labeling at perivascular and subvitreal Müller cell endfeet, raising the possibility that beta1 syntrophin might be involved in the anchoring of Kir4.1 and the alpha-syntrophin independent pool of aquaporin-4.


Assuntos
Aquaporina 4/biossíntese , Proteínas de Ligação ao Cálcio/deficiência , Polaridade Celular , Proteínas de Membrana/deficiência , Proteínas Musculares/deficiência , Neuroglia/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Animais , Proteínas de Ligação ao Cálcio/genética , Polaridade Celular/genética , Imunofluorescência , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia Confocal , Proteínas Musculares/genética , Retina/citologia , Retina/metabolismo
3.
Neuromuscul Disord ; 13(6): 456-67, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12899872

RESUMO

The syntrophins and dystrobrevins are members of the dystrophin-associated protein complex, and are thought to function as modular adaptors for signalling proteins recruited to the sarcolemmal membrane. We have characterised the expression of the syntrophins (alpha-, beta1-, and beta2-) and alpha-dystrobrevin by immunohistochemistry in normal human muscle and in biopsies from 162 patients with myopathies of unknown aetiology (with normal staining for dystrophin and other dystrophin-associated proteins). Unlike mice, beta2-syntrophin is expressed at the sarcolemma in post-natal human skeletal muscle. Deficiency of alpha-dystrobrevin +/- beta2-syntrophin was present in 16/162 (10%) patients, compared to age-matched controls. All patients presented with congenital-onset hypotonia and weakness, although there was variability in clinical severity. Two major clinical patterns emerged: patients with deficiency of beta2-syntrophin and alpha-dystrobrevin presented with severe congenital weakness and died in the first year of life, and two patients with deficiency of alpha-dystrobrevin had congenital muscular dystrophy with complete external ophthalmoplegia. We have sequenced the coding regions of alpha-dystrobrevin and beta2-syntrophin in these patients, and identified a new isoform of dystrobrevin, but have not identified any mutations. This suggests that disease causing mutations occur outside the coding region of these genes, in gene(s) encoding other components of the syntrophin-dystrobrevin subcomplex, or in gene(s) responsible for their post-translational modification and normal localisation.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas Associadas à Distrofina , Proteínas de Membrana/genética , Músculo Esquelético/metabolismo , Distrofias Musculares/genética , Adulto , Processamento Alternativo , Western Blotting , Pré-Escolar , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/deficiência , Análise Mutacional de DNA , DNA Complementar , Feminino , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/deficiência , Músculo Esquelético/química , Músculo Esquelético/patologia , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Estudos Prospectivos , Estudos Retrospectivos
4.
Proc Natl Acad Sci U S A ; 98(24): 14108-13, 2001 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-11717465

RESUMO

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, beta-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking alpha-syntrophin (alpha-Syn(-/-)). The total level of AQP4 expression appears normal in brains of alpha-Syn(-/-) mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of alpha-Syn(-/-) mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in alpha-Syn(-/-) mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse-chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires alpha-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.


Assuntos
Aquaporinas/genética , Distrofina/análogos & derivados , Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Animais , Aquaporina 4 , Aquaporinas/biossíntese , Aquaporinas/metabolismo , Células CHO , Proteínas de Ligação ao Cálcio , Linhagem Celular , Cricetinae , Proteínas do Citoesqueleto/metabolismo , Cães , Distroglicanas , Distrofina/genética , Distrofina/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Ratos , Ratos Sprague-Dawley , Equilíbrio Hidroeletrolítico
5.
J Cell Biol ; 155(1): 113-22, 2001 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-11571312

RESUMO

alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.


Assuntos
Aquaporinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Óxido Nítrico Sintase/metabolismo , Sarcolema/metabolismo , Animais , Aquaporina 4 , Proteínas de Ligação ao Cálcio/metabolismo , Distrofina/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/química , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Óxido Nítrico Sintase Tipo I , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Sarcolema/enzimologia , Canais de Sódio/metabolismo
6.
EMBO J ; 20(15): 4013-23, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11483505

RESUMO

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain.


Assuntos
Autoantígenos/metabolismo , Calpaína/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/biossíntese , Inibidores de Cisteína Proteinase/farmacologia , Proteínas do Citoesqueleto/metabolismo , Dipeptídeos/farmacologia , Proteínas Associadas à Distrofina , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Secreção de Insulina , Proteínas de Membrana/biossíntese , Dados de Sequência Molecular , Ácido Okadáico/farmacologia , Fosforilação , Ligação Proteica , Precursores de Proteínas/biossíntese , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Vesículas Secretórias/metabolismo , Células Tumorais Cultivadas , Utrofina
7.
Eur J Cell Biol ; 79(9): 621-30, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11043403

RESUMO

Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting beta-cells of the pancreas. Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds beta2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS). The cDNA isolated by two-hybrid screening corresponded to a novel beta2-syntrophin isoform with a predicted molecular mass of 28 kDa. This isoform included the PDZ domain, but not the C-terminal region, which in full-length beta2-syntrophin is responsible for binding dystrophin-related proteins. In vitro binding of the beta2-syntrophin PDZ domain to ICA512 required both ICA512's C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain. Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-beta2-syntrophin complexes in vivo. ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells. Finally, we show that INS-1 cells, like muscle cells, contain beta2-syntrophin-utrophin oligomers. Thus, we propose that ICA512, through beta2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.


Assuntos
Ilhotas Pancreáticas/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Óxido Nítrico Sintase/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Alelos , Processamento Alternativo/fisiologia , Sequência de Aminoácidos , Animais , Autoantígenos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Sequência Consenso , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Distrofina/metabolismo , Proteínas Associadas à Distrofina , Expressão Gênica/fisiologia , Insulinoma , Ilhotas Pancreáticas/citologia , Proteínas de Membrana/química , Dados de Sequência Molecular , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase Tipo I , Estrutura Terciária de Proteína , Ratos , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido
8.
J Cell Biol ; 150(6): 1385-98, 2000 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-10995443

RESUMO

The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated alpha-syntrophin null mice by targeted gene disruption to test the function of this association. The alpha-Syn(-/)- mice show no evidence of myopathy, despite reduced levels of alpha-dystrobrevin-2. Neuronal nitric oxide synthase, a component of the dystrophin protein complex, is absent from the sarcolemma of the alpha-Syn(-/)- mice, even where other syntrophin isoforms are present. alpha-Syn(-/)- neuromuscular junctions have undetectable levels of postsynaptic utrophin and reduced levels of acetylcholine receptor and acetylcholinesterase. The mutant junctions have shallow nerve gutters, abnormal distributions of acetylcholine receptors, and postjunctional folds that are generally less organized and have fewer openings to the synaptic cleft than controls. Thus, alpha-syntrophin has an important role in synapse formation and in the organization of utrophin, acetylcholine receptor, and acetylcholinesterase at the neuromuscular synapse.


Assuntos
Proteínas do Citoesqueleto/deficiência , Proteínas Associadas à Distrofina , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas Musculares/genética , Junção Neuromuscular/anormalidades , Sinapses/metabolismo , Acetilcolinesterase/metabolismo , Animais , Southern Blotting , Proteínas de Ligação ao Cálcio , Distrofina/metabolismo , Imunofluorescência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Proteínas Musculares/metabolismo , Músculo Esquelético/anormalidades , Músculo Esquelético/enzimologia , Junção Neuromuscular/química , Junção Neuromuscular/ultraestrutura , Neuropeptídeos/metabolismo , Óxido Nítrico Sintase/metabolismo , Receptores Colinérgicos/análise , Receptores Colinérgicos/metabolismo , Sarcolema/metabolismo , Sinapses/química , Utrofina
9.
J Cell Biol ; 150(6): 1399-410, 2000 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-10995444

RESUMO

Dystrophin is a multidomain protein that links the actin cytoskeleton to laminin in the extracellular matrix through the dystrophin associated protein (DAP) complex. The COOH-terminal domain of dystrophin binds to two components of the DAP complex, syntrophin and dystrobrevin. To understand the role of syntrophin and dystrobrevin, we previously generated a series of transgenic mouse lines expressing dystrophins with deletions throughout the COOH-terminal domain. Each of these mice had normal muscle function and displayed normal localization of syntrophin and dystrobrevin. Since syntrophin and dystrobrevin bind to each other as well as to dystrophin, we have now generated a transgenic mouse deleted for the entire dystrophin COOH-terminal domain. Unexpectedly, this truncated dystrophin supported normal muscle function and assembly of the DAP complex. These results demonstrate that syntrophin and dystrobrevin functionally associate with the DAP complex in the absence of a direct link to dystrophin. We also observed that the DAP complexes in these different transgenic mouse strains were not identical. Instead, the DAP complexes contained varying ratios of syntrophin and dystrobrevin isoforms. These results suggest that alternative splicing of the dystrophin gene, which naturally generates COOH-terminal deletions in dystrophin, may function to regulate the isoform composition of the DAP complex.


Assuntos
Proteínas Associadas à Distrofina , Distrofina , Distrofias Musculares/metabolismo , Actinas/metabolismo , Animais , Sítios de Ligação/fisiologia , Núcleo Celular/patologia , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Distrofina/química , Distrofina/genética , Distrofina/metabolismo , Éxons , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Microssomos/química , Microssomos/metabolismo , Contração Muscular/genética , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Distrofias Musculares/patologia , Neuropeptídeos/análise , Neuropeptídeos/metabolismo , Estrutura Terciária de Proteína , Utrofina
10.
Am J Med Genet ; 92(2): 122-7, 2000 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-10797436

RESUMO

The autosomal dominant hereditary spastic paraplegias (AD-HSP) are a heterogeneous group of degenerative disorders of the central motor system, characterized by progressive spasticity of the lower limbs. Five loci for pure AD-HSP have been identified to date: SPG3 at 14q, SPG4 at 2p, SPG6 at 15q, SPG8 at 8q, and more recently SPG10 at 12q. We have analyzed a Brazilian family with 16 affected individuals by pure AD-HSP who developed progressive gait disturbance with onset at age 18-26 years. Linkage analysis performed with 13 relatives (6 affected and 7 normal) excluded SPG3, SPG4, and SPG6 as candidate regions. However, positive LOD scores were obtained with markers flanking the candidate region for the SPG8 locus [maximum two point Lod score (Zmax) = 3.3 at theta = 0 for D8S1804]. In this region lies the syntrophin beta 1 gene (SNT2B1), a widely expressed dystrophin-associated protein and therefore a good positional and functional candidate for this disease. Immunohistochemical and Western Blot (WB) studies showed that the distribution, expression, and apparent molecular weight of the beta 1 syntrophin protein were comparable to those of normal control individuals. Therefore, it is unlikely that defects in this protein are related to SPG8, at least in the present family.


Assuntos
Cromossomos Humanos Par 8/genética , Proteínas Associadas à Distrofina , Genes Dominantes , Paraplegia/genética , Idade de Início , Brasil , Mapeamento Cromossômico , DNA/genética , Saúde da Família , Feminino , Imunofluorescência , Humanos , Masculino , Proteínas de Membrana/metabolismo , Repetições de Microssatélites , Proteínas Musculares/metabolismo , Músculos/química , Músculos/patologia , Paraplegia/metabolismo , Paraplegia/patologia , Linhagem
11.
J Neurochem ; 73(6): 2358-68, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10582594

RESUMO

Selective transcription of acetylcholine receptor (AChR) subunit genes by neuregulin is one of the mechanisms involved in the synaptic localization of AChRs to the neuromuscular junction. Neuregulin stimulates ErbB receptor tyrosine kinases and subsequently activates the Ras/ERK pathway, which is required for neuregulin-mediated induction of AChR subunit genes in muscle cells and synapse-specific expression in vivo. Here we investigated the neuregulin transduction mechanism that leads to ERK activation after ErbB receptor tyrosine phosphorylation. Neuregulin increases the association of the adaptor proteins Grb2 and Shc with both ErbB2 and ErbB3 in C2C12 muscle cells. Dephosphorylation of the tyrosine-phosphorylated ErbB proteins abolished their association with both Grb2 and Shc, suggesting a tyrosine phosphorylation-dependent interaction. The interaction of Shc with the ErbB receptors is mediated by Shc's phosphotyrosine-binding domain. In addition, neuregulin increased tyrosine phosphorylation of Shc. Mutagenesis approaches demonstrated that tyrosine phosphorylation of Shc is required for neuregulin induction of AChR subunit gene expression. Taken together, these data indicate that the interaction of ErbB receptors with Grb2 alone is insufficient for neuregulin-activated transcription, but that ErbB receptor signaling via Shc is necessary and important.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Músculo Esquelético/efeitos dos fármacos , Neuregulina-1/farmacologia , Proteínas/fisiologia , Receptor ErbB-2/fisiologia , Receptor ErbB-3/fisiologia , Receptores Colinérgicos/genética , Substituição de Aminoácidos , Animais , Células COS , Células Cultivadas/efeitos dos fármacos , Chlorocebus aethiops , Dimerização , Proteína Adaptadora GRB2 , Genes Reporter , Genes erbB-2 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Substâncias Macromoleculares , Camundongos , Músculo Esquelético/citologia , Mutagênese Sítio-Dirigida , Junção Neuromuscular/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptor ErbB-2/química , Receptor ErbB-3/química , Receptores Colinérgicos/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Transfecção
12.
J Cell Biol ; 145(2): 391-402, 1999 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-10209032

RESUMO

Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes.


Assuntos
Membrana Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteínas Associadas à Distrofina , Células Epiteliais/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Musculares/fisiologia , Neuropeptídeos/fisiologia , Animais , Anticorpos Monoclonais , Linhagem Celular , Membrana Celular/ultraestrutura , Polaridade Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Cães , Células Epiteliais/ultraestrutura , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Modelos Biológicos , Proteínas Musculares/química , Proteínas Musculares/genética , Neuropeptídeos/química , Neuropeptídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transfecção , Utrofina , Domínios de Homologia de src
14.
J Cell Biol ; 142(5): 1269-78, 1998 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-9732287

RESUMO

alpha-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length alpha-dystrobrevin-1 (84 kD), and COOH-terminal truncated alpha-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that alpha-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. alpha-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, alpha-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro-translated proteins, alpha-dystrobrevin-2 binds dystrophin, whereas alpha-dystrobrevin-1 binds both dystrophin and utrophin. alpha-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, alpha-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of alpha-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the alpha-dystrobrevins.


Assuntos
Proteínas Associadas à Distrofina , Distrofina/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Neuropeptídeos/metabolismo , Processamento Alternativo/genética , Animais , Proteínas do Citoesqueleto/metabolismo , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos mdx , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas/genética , Receptores Colinérgicos/metabolismo , Utrofina
15.
J Biol Chem ; 273(34): 21980-7, 1998 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-9705339

RESUMO

Syntrophins, a family of intracellular peripheral membrane proteins of the dystrophin-associated protein complex (DAPC), each contain a single PDZ domain. Syntrophin PDZ domains bind C-terminal peptide sequences with the consensus R/K-E-S/T-X-V-COOH, an interaction that mediates association of skeletal muscle sodium channels with the DAPC. Here, we have isolated cyclic peptide ligands for syntrophin PDZ domains from a library of combinatorial peptides displayed at the N terminus of protein III of bacteriophage M13. Affinity selection from a library of X10C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-Psi-C, where Psi represents any hydrophobic amino acid. These peptides contain residues (underlined) similar to the C-terminal consensus sequence for binding to syntrophin PDZ domains and bind to the same site on syntrophin PDZ domains as C-terminal peptides, but do not bind to other closely related PDZ domains. PDZ binding is dependent on the formation of an intramolecular disulfide bond in the peptides, since treatment with dithiothreitol, or substitution of either of the two cysteines with alanines, eliminated this activity. Furthermore, amino acid replacements revealed that most residues in the phage-selected peptides are required for binding. Our results define a new mode of binding to PDZ domains and suggest that proteins containing similar conformationally constrained sequences may be ligands for PDZ domains.


Assuntos
Proteínas Associadas à Distrofina , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Peptídeos Cíclicos/metabolismo , Sequência de Aminoácidos , Bacteriófagos/metabolismo , Sítios de Ligação , Técnicas Biossensoriais , Proteínas de Ligação ao Cálcio , Encefalina Metionina/análogos & derivados , Encefalina Metionina/metabolismo , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Precursores de Proteínas/metabolismo , Canais de Sódio/metabolismo
16.
Curr Opin Neurobiol ; 8(3): 357-63, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9687350

RESUMO

High densities of acetylcholine receptors and sodium channels in the crests and troughs of the postsynaptic folds, respectively, ensure reliable neuromuscular signalling. Clustering of both ion channels is mediated by agrin. In the case of acetylcholine receptors, agrin activates the tyrosine kinase receptor muscle-specific kinase (MuSK), initiating a process requiring rapsyn and possibly also receptor phosphorylation. In many respects, the interactions between agrin and MuSK and their downstream effectors are atypical of conventional receptor tyrosine kinase signalling systems. A new understanding of the structural features of rapsyn involved in receptor clustering, as well as syntrophin's role in sodium channel targeting, has recently been revealed. Perhaps the most surprising result of the past year with regard to synaptogenesis is a negative one--mice lacking both dystrophin and utrophin have nearly normal neuromuscular junctions.


Assuntos
Junção Neuromuscular/química , Receptores Colinérgicos/fisiologia , Transdução de Sinais/fisiologia , Canais de Sódio/fisiologia , Junção Neuromuscular/fisiologia , Receptores Colinérgicos/química , Canais de Sódio/química
19.
J Neurosci ; 18(4): 1250-60, 1998 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-9454835

RESUMO

Specific isoforms of laminin (LN) are concentrated at neuromuscular junctions (NMJs) where they may participate in synaptic organization or function. In myotubes from C2 cells, LN is concentrated within the majority of spontaneous acetylcholine receptor (AChR) aggregates. Neural agrin substantially increases this colocalization, suggesting that agrin can recruit LN into AChR aggregates. Addition of LN to C2 myotubes induces a more than twofold increase in the number of AChR aggregates. These aggregates have a larger size and are more dense than are those induced by agrin, suggesting that LN is involved in the growth and/or stabilization of AChR aggregates. Consistent with this hypothesis, an antiserum to LN reduces the size of individual AChR aggregates but increases their number. In C2 myotubes, extracellular matrix receptors containing the integrin beta1 subunit are poorly colocalized with AChR aggregates, suggesting that integrins may not be involved in LN-induced aggregation. In contrast, almost all AChR aggregates are associated with dystroglycan immunoreactivity, and monoclonal antibody (mAb) IIH6 against alpha-dystroglycan (alpha-DG), a LN and agrin receptor, causes a concentration-dependent inhibition of LN-induced aggregation. Moreover, S27 cells, which lack a functional alpha-DG, and two C2-derived cell lines expressing antisense DG mRNA fail to aggregate AChRs in response to LN. Finally, LN-induced AChR aggregation does not involve the phosphorylation of the muscle-specific tyrosine kinase receptor (MuSK) or the AChR beta subunit. We hypothesize that the interaction of LN with alpha-DG contributes to the growth and/or stabilization of AChR microaggregates into macroaggregates at the developing NMJ via a MuSK-independent mechanism.


Assuntos
Proteínas do Citoesqueleto/fisiologia , Laminina/fisiologia , Glicoproteínas de Membrana/fisiologia , Músculos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Agregação de Receptores/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Colinérgicos/fisiologia , Agrina/farmacologia , Animais , Interações Medicamentosas , Distroglicanas , Laminina/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Receptores Colinérgicos/efeitos dos fármacos , Receptores Colinérgicos/metabolismo , Distribuição Tecidual
20.
J Neurosci ; 18(1): 128-37, 1998 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-9412493

RESUMO

Syntrophins are cytoplasmic peripheral membrane proteins of the dystrophin-associated protein complex (DAPC). Three syntrophin isoforms, alpha1, beta1, and beta2, are encoded by distinct genes. Each contains two pleckstrin homology (PH) domains, a syntrophin-unique (SU) domain, and a PDZ domain. The name PDZ comes from the first three proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens protein 1). PDZ domains in other proteins bind to the C termini of ion channels and neurotransmitter receptors containing the consensus sequence (S/T)XV-COOH and mediate the clustering or synaptic localization of these proteins. Two voltage-gated sodium channels (NaChs), SkM1 and SkM2, of skeletal and cardiac muscle, respectively, have this consensus sequence. Because NaChs are sarcolemmal components like syntrophins, we have investigated possible interactions between these proteins. NaChs copurify with syntrophin and dystrophin from extracts of skeletal and cardiac muscle. Peptides corresponding to the C-terminal 10 amino acids of SkM1 and SkM2 are sufficient to bind detergent-solubilized muscle syntrophins, to inhibit the binding of native NaChs to syntrophin PDZ domain fusion proteins, and to bind specifically to PDZ domains from alpha1-, beta1-, and beta2-syntrophin. These peptides also inhibit binding of the syntrophin PDZ domain to the PDZ domain of neuronal nitric oxide synthase, an interaction that is not mediated by C-terminal sequences. Brain NaChs, which lack the (S/T)XV consensus sequence, also copurify with syntrophin and dystrophin, an interaction that does not appear to be mediated by the PDZ domain of syntrophin. Collectively, our data suggest that syntrophins link NaChs to the actin cytoskeleton and the extracellular matrix via dystrophin and the DAPC.


Assuntos
Química Encefálica/fisiologia , Proteínas Associadas à Distrofina , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio , Citoesqueleto/fisiologia , Detergentes , Distrofina/análise , Distrofina/isolamento & purificação , Distrofina/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/isolamento & purificação , Músculo Esquelético/metabolismo , Junção Neuromuscular/química , Junção Neuromuscular/enzimologia , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Canais de Potássio/isolamento & purificação , Canais de Potássio/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Canais de Sódio/química , Canais de Sódio/isolamento & purificação
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