[show abstract][hide abstract] ABSTRACT: The limb girdle and congenital muscular dystrophies (LGMD and CMD) are characterized by skeletal muscle weakness and dystrophic muscle changes. The onset of symptoms in CMD is within the first few months of life, whereas in LGMD they can occur in late childhood, adolescence or adult life. We have recently demonstrated that the fukutin-related protein gene (FKRP) is mutated in a severe form of CMD (MDC1C), characterized by the inability to walk, leg muscle hypertrophy and a secondary deficiency of laminin 2 and -dystroglycan. Both MDC1C and LGMD2I map to an identical region on chromosome 19q13.3. To investigate whether these are allelic disorders, we undertook mutation analysis of FKRP in 25 potential LGMD2I families, including some with a severe and early onset phenotype. Mutations were identified in individuals from 17 families. A variable reduction of -dystroglycan expression was observed in the skeletal muscle biopsy of all individuals studied. In addition, several cases showed a deficiency of laminin 2 either by immunocytochemistry or western blotting. Unexpectedly, affected individuals from 15 families had an identical C826A (Leu276Ileu) mutation, including five that were homozygous for this change. Linkage analysis identified at least two possible haplotypes in linkage disequilibrium with this mutation. Patients with the C826A change had the clinically less severe LGMD2I phenotype, suggesting that this is a less disruptive FKRP mutation than those found in MDC1C. The spectrum of LGMD2I phenotypes ranged from infants with an early presentation and a Duchenne-like disease course including cardiomyopathy, to milder phenotypes compatible with a favourable long-term outcome.
Human Molecular Genetics 01/2002; · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders presenting in infancy with muscle weakness, contractures, and dystrophic changes on skeletal-muscle biopsy. Structural brain defects, with or without mental retardation, are additional features of several CMD syndromes. Approximately 40% of patients with CMD have a primary deficiency (MDC1A) of the laminin alpha2 chain of merosin (laminin-2) due to mutations in the LAMA2 gene. In addition, a secondary deficiency of laminin alpha2 is apparent in some CMD syndromes, including MDC1B, which is mapped to chromosome 1q42, and both muscle-eye-brain disease (MEB) and Fukuyama CMD (FCMD), two forms with severe brain involvement. The FCMD gene encodes a protein of unknown function, fukutin, though sequence analysis predicts it to be a phosphoryl-ligand transferase. Here we identify the gene for a new member of the fukutin protein family (fukutin related protein [FKRP]), mapping to human chromosome 19q13.3. We report the genomic organization of the FKRP gene and its pattern of tissue expression. Mutations in the FKRP gene have been identified in seven families with CMD characterized by disease onset in the first weeks of life and a severe phenotype with inability to walk, muscle hypertrophy, marked elevation of serum creatine kinase, and normal brain structure and function. Affected individuals had a secondary deficiency of laminin alpha2 expression. In addition, they had both a marked decrease in immunostaining of muscle alpha-dystroglycan and a reduction in its molecular weight on western blot analysis. We suggest these abnormalities of alpha-dystroglycan are caused by its defective glycosylation and are integral to the pathology seen in MDC1C.
The American Journal of Human Genetics 01/2002; 69(6):1198-209. · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: beta-Dystrobrevin is a dystrophin-related and -associated protein that is highly expressed in brain, kidney, and liver. Recent studies with the kidneys of the mdx3Cv mouse, which lacks all dystrophin isoforms, suggest that beta-dystrobrevin, and not the dystrophin isoforms, may be the key component in the assembly of complexes similar to the muscle dystrophin-associated protein complexes (DPC) in nonmuscle tissues. To understand the role of beta-dystrobrevin in the function of nonmuscle tissues, we generated beta-dystrobrevin-deficient (dtnb(-/-)) mice by gene targeting. dtnb(-/-) mice are healthy, fertile, and normal in appearance. No beta-dystrobrevin was detected in these mice by Western blotting or immunocytochemistry. In addition, the levels of several beta-dystrobrevin-interacting proteins, namely Dp71 isoforms and the syntrophins, were greatly reduced from the basal membranes of kidney tubules and liver sinusoids and on Western blots of crude kidney and liver microsomes of beta-dystrobrevin-deficient mice. However, no abnormality was detected in the ultrastructure of membranes of kidney and liver cells or in the renal function of these mice. beta-Dystrobrevin may therefore be an anchor or scaffold for Dp71 and syntrophin isoforms, as well as other associating proteins at the basal membranes of kidney and liver, but is not necessary for the normal function of these mice.
Molecular and Cellular Biology 12/2001; 21(21):7442-8. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: The dystrophin-associated protein complex (DPC) is required for the maintenance of muscle integrity during the mechanical stresses of contraction and relaxation. In addition to providing a membrane scaffold, members of the DPC such as the alpha-dystrobrevin protein family are thought to play an important role in intracellular signal transduction. To gain additional insights into the function of the DPC, we performed a yeast two-hybrid screen for dystrobrevin-interacting proteins. Here we describe the identification of a dysbindin, a novel dystrobrevin-binding protein. Dysbindin is an evolutionary conserved 40-kDa coiled-coil-containing protein that binds to alpha- and beta-dystrobrevin in muscle and brain. Dystrophin and alpha-dystrobrevin are co-immunoprecipitated with dysbindin, indicating that dysbindin is DPC-associated in muscle. Dysbindin co-localizes with alpha-dystrobrevin at the sarcolemma and is up-regulated in dystrophin-deficient muscle. In the brain, dysbindin is found primarily in axon bundles and especially in certain axon terminals, notably mossy fiber synaptic terminals in the cerebellum and hippocampus. These findings have implications for the molecular pathology of Duchenne muscular dystrophy and may provide an alternative route for anchoring dystrobrevin and the DPC to the muscle membrane.
Journal of Biological Chemistry 07/2001; 276(26):24232-41. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dystrophin coordinates the assembly of a complex of structural and signaling proteins that are required for normal muscle function. A key component of the dystrophin protein complex is alpha-dystrobrevin, a dystrophin-associated protein whose absence results in neuromuscular junction defects and muscular dystrophy. To gain further insights into the role of alpha-dystrobrevin in skeletal muscle, we used the yeast two-hybrid system to identify a novel alpha-dystrobrevin-binding partner called syncoilin. Syncoilin is a new member of the intermediate filament superfamily and is highly expressed in skeletal and cardiac muscle. In normal skeletal muscle, syncoilin is concentrated at the neuromuscular junction, where it colocalizes and coimmunoprecipitates with alpha-dystrobrevin-1. Expression studies in mammalian cells demonstrate that, while alpha-dystrobrevin and syncoilin associate directly, overexpression of syncoilin does not result in the self-assembly of intermediate filaments. Finally, unlike many components of the dystrophin protein complex, we show that syncoilin expression is up-regulated in dystrophin-deficient muscle. These data suggest that alpha-dystrobrevin provides a link between the dystrophin protein complex and the intermediate filament network at the neuromuscular junction, which may be important for the maintenance and maturation of the synapse.
Journal of Biological Chemistry 04/2001; 276(9):6645-55. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dystrophin coordinates the assembly of a complex of structural and signalling proteins that is required for normal muscle function. A key component of the dystrophin-associated protein complex (DPC) is alpha-dystrobrevin, a dystrophin-related and -associated protein whose absence results in muscular dystrophy and neuromuscular junction defects [1,2]. The current model of the DPC predicts that dystrophin and dystrobrevin each bind a single syntrophin molecule . The syntrophins are PDZ-domain-containing proteins that facilitate the recruitment of signalling proteins such as nNOS (neuronal nitric oxide synthase) to the DPC . Here we show, using yeast two-hybrid analysis and biochemical binding studies, that alpha-dystrobrevin in fact contains two independent syntrophin-binding sites in tandem. The previously undescribed binding site is situated within an alternatively spliced exon of alpha-dystrobrevin, termed the variable region-3 (vr3) sequence, which is specifically expressed in skeletal and cardiac muscle [5,6]. Analysis of the syntrophin-binding region of dystrobrevin reveals a tandem pair of predicted alpha helices with significant sequence similarity. These alpha helices, each termed a syntrophin-binding motif, are also highly conserved in dystrophin and utrophin. Together these data show that there are four potential syntrophin-binding sites per dystrophin complex in skeletal muscle: two on dystrobrevin and two on dystrophin or utrophin. Furthermore, alternative splicing of dystrobrevin provides a mechanism for regulating the stoichiometry of syntrophin association with the DPC. This is likely to have important consequences for the recruitment of specific signalling molecules to the DPC and ultimately for its function.
Current Biology 11/2000; 10(20):1295-8. · 9.49 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dystrophin is the key component in the assembly and maintenance of the dystrophin-associated protein complex (DPC) in skeletal muscle. In kidney, dystroglycan, an integral component of the DPC, is involved in kidney epithelial morphogenesis, suggesting that the DPC is important in linking the extracellular matrix to the internal cytoskeleton of kidney epithelia. Here, we have investigated the molecular architecture of dystrophin-like protein complexes in kidneys from normal and dystrophin-deficient mice. Using isoform-specific antibodies, we show that the different cell types that make up the kidney maintain different dystrophin-like complexes. These complexes can be broadly grouped according to their dystrobrevin content: beta-dystrobrevin containing complexes are present at the basal region of renal epithelial cells, whilst alpha-dystrobrevin-1 containing complexes are found in endothelial and smooth muscle cells. Furthermore, these complexes are maintained even in the absence of all dystrophin isoforms. Thus our data suggest that the functions and assembly of the dystrophin-like complexes in kidney differ from those in skeletal muscle and implicate a protein other than dystrophin as the primary molecule in the assembly and maintenance of kidney complexes. Our findings also provide a possible explanation for the lack of kidney pathology in Duchenne muscular dystrophy patients and mice lacking all dystrophin isoforms.
[show abstract][hide abstract] ABSTRACT: alpha-Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a dystrophin-associated and dystrophin-related protein. Knockout of the gene in the mouse results in muscular dystrophy. The control of the alpha-dystrobrevin gene in the various tissues is therefore of interest. Multiple dystrobrevin isoforms differing in their domain content are generated by alternative splicing of a single gene. The data presented here demonstrate that expression of alpha-dystrobrevin from three promoters, that are active in a tissue-selective manner, also plays a role in the function of the protein in different tissues. The most proximal promoter A is active in brain and to a lesser extent in lung, whereas the most distal promoter B, which possesses several Sp1 binding sites, is restricted to brain. Promoter C, which contains multiple consensus myogenic binding sites, is up-regulated during in vitro myoblast differentiation. Interestingly, the organization and the activity of the alpha-dystrobrevin promoters is reminiscent of those in the dystrophin gene. Taken together we suggest that the multipromoter system, distributed over a region of 270 kilobases at the 5'-end of the alpha-dystrobrevin gene, has been developed to allow the regulation of this gene in different cell types and/or different developmental stages.
Journal of Biological Chemistry 04/1999; 274(10):6250-8. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: beta-Dystrobrevin, a dystrophin-related protein that is expressed in non-muscle tissues, is highly homologous to alpha-dystrobrevin, a member of the dystrophin-associated protein complex (DPC). beta-Dystrobrevin associates with Dp71 and syntrophin and is believed to have a role in non-muscle DPCs. Here we report the characterization and mapping of the mouse beta-dystrobrevin gene. The mouse beta-dystrobrevin gene is organized into 21 exons spanning over 130 kb of DNA. We provide evidence that this gene is transcribed from at least two promoter regions but appears to utilize a common translation initiation site. We show that the similarity between beta-dystrobrevin and alpha-dystrobrevin is reflected in the conservation of their exon-intron junctions. beta-Dystrobrevin has been localized to proximal mouse Chromosome (Chr) 12 by backcross mapping. A database search revealed that two mouse genetic diseases involving tissues expressing beta-dystrobrevin have been mapped to this region, namely, congenital polycystic kidneys (cpk) and fatty liver dystrophy (fld). However, refined mapping analysis has excluded beta-dystrobrevin as a candidate gene for either disease.
[show abstract][hide abstract] ABSTRACT: Dystrophin-related and associated proteins are important for the formation and maintenance of the mammalian neuromuscular junction. Initial studies in the electric organ of Torpedo californica showed that the dystrophin-related protein dystrobrevin (87K) co-purifies with the acetylcholine receptors and other postsynaptic proteins. Dystrobrevin is also a major phosphotyrosine-containing protein in the postsynaptic membrane. Since inhibitors of tyrosine protein phosphorylation block acetylcholine receptor clustering in cultured muscle cells, we examined the role of alpha-dystrobrevin during synapse formation and in response to agrin. Using specific antibodies, we show that C2 myoblasts and early myotubes only produce alpha-dystrobrevin-1, the mammalian orthologue of Torpedo dystrobrevin, whereas mature skeletal muscle expresses three distinct alpha-dystrobrevin isoforms. In myotubes, alpha-dystrobrevin-1 is found on the cell surface and also in acetylcholine receptor-rich domains. Following agrin stimulation, alpha-dystrobrevin-1 becomes re-localised beneath the cell surface into macroclusters that contain acetylcholine receptors and another dystrophin-related protein, utrophin. This redistribution is not associated with tyrosine phosphorylation of alpha-dystrobrevin-1 by agrin. Furthermore, we show that alpha-dystrobrevin-1 is associated with both utrophin in C2 cells and dystrophin in mature skeletal muscle. Thus alpha-dystrobrevin-1 is a component of two protein complexes in muscle, one with utrophin at the neuromuscular junction and the other with dystrophin at the sarcolemma. These results indicate that alpha-dystrobrevin-1 is not involved in the phosphorylation-dependent, early stages of receptor clustering, but rather in the stabilisation and maturation of clusters, possibly via an interaction with utrophin.
[show abstract][hide abstract] ABSTRACT: Utrophin is normally present exclusively in synaptic regions of skeletal muscle fibers, although it is expressed extrasynaptically in certain pathological situations, where it has been proposed to compensate for the absence of dystrophin in Duchenne muscular dystrophy patients and mdx mice. Recently there have been conflicting reports regarding the preferential expression of utrophin mRNA at the neuromuscular junction. Using in situ hybridization with RNA probes, we show a clear accumulation of autoradiographic labeling at more than 90% of neuromuscular junctions (identified by histochemical demonstration of cholinesterase activity). The intensity of this labeling is proportional to the number of junctional myonuclei in the section. Some clusters of labeling were found associated with nonmuscle nuclei (e.g., blood vessels, nerves), where utrophin is present. In addition, labeling for utrophin mRNA was associated with about 25% of extrajunctional myonuclei, where the protein is not present. The mean labeling per nucleus at junctional myonuclei was at least 10 times greater than at extrajunctional myonuclei. We discuss the possible regulatory mechanisms involved in the heterogeneous expression of utrophin mRNA in skeletal muscle.
Molecular and Cellular Neuroscience 05/1998; 10(5-6):229-42. · 3.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: The importance of dystrophin and its associated proteins in normal muscle function is now well established. Many of these proteins are expressed in nonmuscle tissues, particularly the brain. Here we describe the characterization of beta-dystrobrevin, a dystrophin-related protein that is abundantly expressed in brain and other tissues, but is not found in muscle. beta-dystrobrevin is encoded by a 2.5-kb alternatively spliced transcript that is found throughout the brain. In common with dystrophin, beta-dystrobrevin is found in neurons of the cortex and hippocampal formation but is not found in the brain microvasculature. In the brain, beta-dystrobrevin coimmunoprecipitates with the dystrophin isoforms Dp71 and Dp140. These data provide evidence that the composition of the dystrophin-associated protein complex in the brain differs from that in muscle. This finding may be relevant to the cognitive dysfunction affecting many patients with Duchenne muscular dystrophy.
Proceedings of the National Academy of Sciences 02/1998; 95(1):241-6. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mutations in the genes encoding dystrophin or dystrophin-associated proteins are responsible for Duchenne muscular dystrophy or various forms of limb-girdle muscular dystrophies respectively. We have recently cloned the gene for the murine 87 kDa postsynaptic protein dystrobrevin, a dystrophin-associated protein. Anti-dystrobrevin antibodies stain the sarcolemma in normal skeletal muscle indicating that dystrobrevin co-localises with dystrophin and the dystrophin-associated protein complex. By contrast, dystrobrevin membrane staining is severely reduced in muscles of Duchenne muscular dystrophy patients, consistent with dystrobrevin being a dystrophin-associated protein. Interestingly, dystrobrevin staining at the sarcolemma is dramatically reduced in patients with limb-girdle muscular dystrophy arising from the loss of one or all of the sarcoglycan components. Normal dystrobrevin staining is observed in patients with other forms of limb-girdle muscular dystrophy where dystrophin and the rest of the dystrophin-associated protein complex are normally expressed and in other neuromuscular disorders. Our results show that dystrobrevin-deficiency is a generic feature of dystrophies linked to dystrophin and the dystrophin-associated proteins. This is the first indication that a cytoplasmic component of the dystrophin-associated protein complex may be involved in the pathogenesis of limb-girdle muscular dystrophy.
Human Molecular Genetics 08/1997; 6(7):1185-91. · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: PDZ (also called DHR or GLGF) domains are found in diverse membrane-associated proteins including members of the MAGUK family of guanylate kinase homologues, several protein phosphatases and kinases, neuronal nitric oxide synthase, and several dystrophin-associated proteins, collectively known as syntrophins. Many PDZ domain-containing proteins appear to be localised to highly specialised submembranous sites, suggesting their participation in cellular junction formation, receptor or channel clustering, and intracellular signalling events. PDZ domains of several MAGUKs interact with the C-terminal polypeptides of a subset of NMDA receptor subunits and/or with Shaker-type K+ channels. Other PDZ domains have been shown to bind similar ligands of other transmembrane receptors. Recently, the crystal structures of PDZ domains, with and without ligand, have been determined. These demonstrate the mode of ligand-binding and the structural bases for sequence conservation among diverse PDZ domains.
[show abstract][hide abstract] ABSTRACT: Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a member of the dystrophin gene family with homology to the cysteine-rich carboxy-terminal domain of dystrophin. Torpedo dystrobrevin copurifies with the acetylcholine receptors and is thought to form a complex with dystrophin and syntrophin. This complex is also found at the sarcolemma in vertebrates and defines the cytoplasmic component of the dystrophin-associated protein complex. Previously we have cloned several dystrobrevin isoforms from mouse brain and muscle. Here we show that these transcripts are the products of a single gene located on proximal mouse chromosome 18. To investigate the diversity of dystrobrevin transcripts we have determined that the mouse dystrobrevin gene is organized into 24 coding exons that span between 130 and 170 kb at the genomic level. The gene encodes at least three distinct protein isoforms that are expressed in a tissue-specific manner. Interestingly, although there is only 27% amino acid identity between the homologous regions of dystrobrevin and dystrophin, the positions of 8 of the 15 exon-intron junctions are identical.
[show abstract][hide abstract] ABSTRACT: In the last decade, our knowledge of human diseases genes has been growing rapidly as a result of the availability of resources and techniques for mapping and sequencing the human genome. New disease genes are now reported almost weekly. This review illustrates how the identification of genes involved in neuromuscular disorders has led to the characterization of not only novel genes, but also of a variety of different types of genetic mutation. These observations, which include high deletion frequencies, unstable tandem repeat sequences, genomic duplications and triplet repeat expansions, have facilitated the identification of similar types of mutation in other genetic disorders.
Trends in Genetics 09/1996; 12(8):294-8. · 9.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: YACs from the complex repetitive human genomic region 5q13, spanning the spinal muscular atrophy (SMA) locus, have been searched for transcribed sequences using the method of End Ligation Coincident Sequence Cloning. Six transcripts (PT1-6) have been identified, three of which (PT4, PT5 and PT6) are novel. Five of these elements hybridise to multiple loci in 5q13, but PT5 is single copy and maps very close to markers that show linkage disequilibrium with SMA.
Biochimica et Biophysica Acta 09/1996; 1308(2):97-102. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have identified a novel mouse gene encoding a protein that shows high homology to the dual-specificity tyrosine/threonine phosphatase family of proteins. The gene encodes a 5 kb transcript which is expressed predominantly in brain and lung and contains a translated complex trinucleotide repeat within the coding region. Using interspecific mouse backcross analysis, the gene has been localised to distal mouse chromosome 7. In human, homologous sequences are located in the syntenic region on distal chromosome 11p as well as to chromosome 10q11.2 and 10q22. The presence of a CG-rich trinucleotide repeat in the coding region provides a target for mutation which might result in loss of function or altered properties of this phosphatase.
Human Molecular Genetics 06/1996; 5(5):675-84. · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dystrophin-related and -associated proteins are important in the formation and maintenance of the mammalian neuromuscular junction. We have characterized mouse cDNA clones encoding isoforms of the dystrophin-homologous 87-kDa postsynaptic protein, dystrobrevin. In Torpedo, the 87-kDa protein is multiply phosphorylated and closely associated with proteins in the postsynaptic cytoskeleton, including the acetylcholine receptor. In contrast to Torpedo, where only a single transcript is seen, the mouse expresses several mRNAs encoding different isoforms. A 6.0-kilobase transcript in brain encodes a 78-kDa protein (dystrobrevin-2) that has a different C terminus, lacking the putative tyrosine kinase substrate domain. In skeletal and cardiac muscle, transcripts of 1.7 and 3.3/3.5 kilobases predominate and encode additional isoforms. Alternative splicing within the coding region and differential usage of untranslated regions produce additional variation. Multiple dystrobrevin-immunoreactive proteins copurify with syntrophin from mouse tissues. In skeletal muscle, dystrobrevin immunoreactivity is restricted to the neuromuscular junction and sarcolemma. The occurrence of many dystrobrevin isoforms is significant because alternative splicing and phosphorylation often have profound effects upon the biological activity of synaptic proteins.
Journal of Biological Chemistry 04/1996; 271(13):7802-10. · 4.65 Impact Factor