Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain

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.     

Molecular basis of dystrobrevin interaction with kinesin heavy chain: structural determinants of their binding

Dystrobrevins are a family of widely expressed dystrophin-associated proteins that comprises alpha and beta isoforms and displays significant sequence homology with several protein-binding domains of the dystrophin C-terminal region. The complex distribution of the multiple dystrobrevin isoforms suggests that the variability of their composition may be important in mediating their function. We have recently identified kinesin as a novel dystrobrevin-interacting protein and localized the dystrobrevin-binding site on the cargo-binding domain of neuronal kinesin heavy chain (Kif5A). In the present study, we assessed the kinetics of the dystrobrevin-Kif5A interaction by quantitative pull-down assay and surface plasmon resonance (SPR) analysis and found that beta-dystrobrevin binds to kinesin with high affinity (K(D) approximately 40 nM). Comparison of the sensorgrams obtained with alpha and beta-dystrobrevin at the same concentration of analyte showed a lower affinity of alpha compared to that of beta-dystrobrevin, despite their functional domain homology and about 70% sequence identity. Analysis of the contribution of single dystrobrevin domains to the interaction revealed that the deletion of either the ZZ domain or the coiled-coil region decreased the kinetics of the interaction, suggesting that the tertiary structure of dystrobrevin may play a role in regulating the interaction of dystrobrevin with kinesin. In order to understand if structural changes induced by post-translational modifications could affect dystrobrevin affinity for kinesin, we phosphorylated beta-dystrobrevin in vitro and found that it showed reduced binding capacity towards kinesin. The interaction between the adaptor/scaffolding protein dystrobrevin and the motor protein kinesin may play a role in the transport and targeting of components of the dystrophin-associated protein complex to specific sites in the cell, with the differences in the binding properties of dystrobrevin isoforms reflecting their functional diversity within the same cell type. Phosphorylation events could have a regulatory role in this context.     

Dystrobrevin requires a dystrophin-binding domain to function in Caenorhabditis elegans.

Dystrobrevin is one of the intracellular components of the transmembrane dystrophin-glycoprotein complex (DGC). The functional role of this complex in normal and pathological situations has not yet been clearly established. Dystrobrevin disappears from the muscle membrane in Duchenne muscular dystrophy (DMD), which results from dystrophin mutations, as well as in limb girdle muscular dystrophies (LGMD), which results from mutations affecting other members of the DGC complex. These findings therefore suggest that dystrobrevin may play a pivotal role in the progression of these clinically related diseases. In this study, we used the Caenorhabditis elegans model to address the question of the relationship between dystrobrevin binding to dystrophin and dystrobrevin function. Deletions of the dystrobrevin protein were performed and the ability of the mutated forms to bind to dystrophin was tested both in vitro and in a two-hybrid assay, as well as their ability to rescue dystrobrevin (dyb-1) mutations in C. elegans. The deletions affecting the second helix of the Dyb-1 coiled-coil domain abolished the binding of dystrobrevin to dystrophin both in vitro and in the two-hybrid assay. These deletions also abolished the rescuing activity of a functional transgene in vivo. These results are consistent with a model according to which dystrobrevin must bind to dystrophin to be able to function properly.     

Alternative splicing of dystrobrevin regulates the stoichiometry of syntrophin binding to the dystrophin protein complex

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 [3]. The syntrophins are PDZ-domain-containing proteins that facilitate the recruitment of signalling proteins such as nNOS (neuronal nitric oxide synthase) to the DPC [4]. 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.     

Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans

Dystrobrevins are protein components of the dystrophin complex, whose disruption leads to Duchenne muscular dystrophy and related diseases. The Caenorhabditis elegans dystrobrevin gene (dyb-1) encodes a protein 38 % identical with its mammalian counterparts. The C. elegans dystrobrevin is expressed in muscles and neurons. We characterised C. elegans dyb-1 mutants and showed that: (1) their behavioural phenotype resembles that of dystrophin (dys-1) mutants; (2) the phenotype of dyb-1 dys-1 double mutants is not different from the single ones; (3) dyb-1 mutants are more sensitive than wild-type animals to reductions of acetylcholinesterase levels and have an increased response to acetylcholine; (4) dyb-1 mutations alone do not lead to muscle degeneration, but synergistically produce a progressive myopathy when combined with a mild MyoD/hlh-1 mutation. All together, these findings further substantiate the role of dystrobrevins in cholinergic transmission and as functional partners of dystrophin.     

Immunohistochemical detection of dysbindin at the astroglial endfeet around the capillaries of mouse brain

Dysbindin was first identified by the yeast two hybrid assay as a binding partner of dystrobrevin which is a cytoplasmic member of dystrophin glycoprotein complex. Immunolocalization of dystrobrevin in the astrocyte endfeet and endothelial cells in the rat cerebellum was reported. Therefore, we were interested in the expression and localization of dystrobrevin binding protein dysbindin in the mouse brain capillary wall and its surrounding astroglial endfeet. We examined whether the dysbindin expression is present in astroglial endfeet and/or capillary endothelial cells at light and electron microscopic levels. Using brain samples from five normal mice (C57BL/6ScSn), we prepared the anti-dysbindin antibody stained brain samples with immunoperoxidase method at light microscopic level and with immunogold method at ultrastructural level. Immunohistochemistry showed that dysbindin was located in the brain capillary at light microscopic level. Immunogold electron microscopy revealed that dysbindin signal was observed at the inside surface of plasma membrane of glial endfeet which surrounded the brain capillary endothelial cells and pericytes.     

Tyrosine and Serine Phosphorylation of Dystrophin and the 58-kDa Protein in the Postsynaptic Membrane of Torpedo Electric Organ

Abstract: Dystrophin associates with a 58-kDa and an 87-kDa protein in the postsynaptic membrane of the Torpedo electric organ. We have previously shown that the 87-kDa protein is a major phosphotyrosine-containing protein in these membranes. Immunoprecipitation of the 87-kDa protein from phosphorylated postsynaptic membranes results in coimmunoprecipitation of additional phosphoproteins. These phosphoproteins are identified as dystrophin and the 58-kDa protein. Monoclonal antibodies to dystrophin and the 58-kDa protein immunoprecipitate phosphorylated forms of these proteins from postsynaptic membranes phosphorylated in vitro. Phosphoamino acid analysis reveals that dystrophin and the 58-kDa protein are phosphorylated on serine and tyrosine residues. In addition, both dystrophin and the 58-kDa protein are shown to be phosphorylated on tyrosine residues in vivo. These results suggest that the synaptic function of dystrophin and its associated proteins, the 58-kDa and 87-kDa proteins, may be modulated by tyrosine and serine protein Phosphorylation.     

Tissue-selective expression of alpha-dystrobrevin is determined by multiple promoters

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.     

Reinvestigation of the dysbindin subunit of BLOC-1 (biogenesis of lysosome-related organelles complex-1) as a dystrobrevin-binding protein

Dysbindin was identified as a dystrobrevin-binding protein potentially involved in the pathogenesis of muscular dystrophy. Subsequently, genetic studies have implicated variants of the human dysbindin-encoding gene, DTNBP1, in the pathogeneses of Hermansky-Pudlak syndrome and schizophrenia. The protein is a stable component of a multisubunit complex termed BLOC-1 (biogenesis of lysosome-related organelles complex-1). In the present study, the significance of the dystrobrevin-dysbindin interaction for BLOC-1 function was examined. Yeast two-hybrid analyses, and binding assays using recombinant proteins, demonstrated direct interaction involving coiled-coil-forming regions in both dysbindin and the dystrobrevins. However, recombinant proteins bearing the coiled-coil-forming regions of the dystrobrevins failed to bind endogenous BLOC-1 from HeLa cells or mouse brain or muscle, under conditions in which they bound the Dp71 isoform of dystrophin. Immunoprecipitation of endogenous dysbindin from brain or muscle resulted in robust co-immunoprecipitation of the pallidin subunit of BLOC-1 but no specific co-immunoprecipitation of dystrobrevin isoforms. Within BLOC-1, dysbindin is engaged in interactions with three other subunits, named pallidin, snapin and muted. We herein provide evidence that the same 69-residue region of dysbindin that is sufficient for dystrobrevin binding in vitro also contains the binding sites for pallidin and snapin, and at least part of the muted-binding interface. Functional, histological and immunohistochemical analyses failed to detect any sign of muscle pathology in BLOC-1-deficient, homozygous pallid mice. Taken together, these results suggest that dysbindin assembled into BLOC-1 is not a physiological binding partner of the dystrobrevins, likely due to engagement of its dystrobrevin-binding region in interactions with other subunits.     

Molecular heterogeneity of the dystrophin-associated protein complex in the mouse kidney nephron: differential alterations in the absence of utrophin and dystrophin

The dystrophin-associated protein complex (DPC) consisting of syntrophin, dystrobrevin, and dystroglycan isoforms is associated either with dystrophin or its homolog utrophin. It is present not only in muscle cells, but also in numerous tissues, including kidney, liver, and brain. Using high-resolution immunofluorescence imaging and Western blotting, we have investigated the effects of utrophin and dystrophin gene deletion on the formation and membrane anchoring of the DPC in kidney epithelial cells, which co-express utrophin and low levels of the C-terminal dystrophin isoform Dp71. We show that multiple, molecularly distinct DPCs co-exist in the nephron; these DPCs have a segment-specific distribution and are only partially associated with utrophin in the basal membrane of tubular epithelial cells. In utrophin-deficient mice, a selective reduction of 2-syntrophin has been observed in medullary tubular segments, whereas 1-syntrophin and 1-syntrophin are retained, concomintant with an upregulation of -dystroglycan, -dystrobrevin, and Dp71. These findings suggest that 2-syntrophin is dependent on utrophin for association with the DPC, and that loss of utrophin is partially compensated by Dp71, allowing the preservation of the DPC in kidney epithelial cells. This hypothesis is confirmed by the almost complete loss of all DPC proteins examined in mice lacking full-length utrophin and all C-terminal dystrophin isoforms (utrophin^0/0/mdx^3Cv). The DPC thus critically depends on these proteins for assembly and/or membrane localization in kidney epithelial cells. This project was supported by the Swiss National Science Foundation (no. 31-63901.00 to J.M.F.).     

Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin

We have characterized a protein immunologically related to dystrophin, the protein product of the Duchenne muscular dystrophy gene. We identify this related protein as a fast-twitch glycolytic isoform (mouse extensor digitorum longus-specific) of myofibrillar alpha-actinin. This specific isoform of alpha-actinin exhibits a more restricted pattern of expression in skeletal muscle than fast-twitch-specific isoforms of both myosin and Ca2+-ATPase. Our results provide evidence that dystrophin and myofibrillar alpha-actinin are related proteins, reinforcing the previous data concerning the sequence homologies noted between nonmuscle cytoskeletal alpha-actinin and dystrophin. In addition, we describe the first antisera directed against a specific myofibrillar skeletal muscle isoform of alpha-actinin.     

Subcellular Localization of Dystrophin Isoforms in Cardiomyocytes and Phenotypic Analysis of Dystrophin-deficient Mice Reveal Cardiac Myopathy is Predominantly Caused by a Deficiency in Full-length Dystrophin

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive muscle degenerative disorder that causes dilated cardiomyopathy in the second decade of life in affected males. Dystrophin, the gene responsible for DMD, encodes full-length dystrophin and various short dystrophin isoforms. In the mouse heart, full-length dystrophin Dp427 and a short dystrophin isoform, Dp71, are expressed. In this study, we intended to clarify the functions of these dystrophin isoforms in DMD-related cardiomyopathy. We used two strains of mice: mdx mice, in which Dp427 was absent but Dp71 was present, and DMD-null mice, in which both were absent. By immunohistochemical staining and density-gradient centrifugation, we found that Dp427 was located in the cardiac sarcolemma and also at the T-tubules, whereas Dp71 was specifically located at the T-tubules. In order to determine whether T tubule-associated Dp71 was involved in DMD-related cardiac disruption, we compared the cardiac phenotypes between DMD-null mice and mdx mice. Both DMD-null mice and mdx mice exhibited severe necrosis, which was followed by fibrosis in cardiac muscle. However, we could not detect a significant difference in myocardial fibrosis between mdx mice and DMD-null mice. Based on the present results, we have shown that cardiac myopathy is caused predominantly by a deficiency of full-length dystrophin Dp427.     

A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells

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.     

Recognition deficits in mice carrying mutations of genes encoding BLOC‐1 subunits pallidin or dysbindin

Numerous studies have implicated DTNBP1, the gene encoding dystrobrevin‐binding protein or dysbindin, as a candidate risk gene for schizophrenia, though this relationship remains somewhat controversial. Variation in dysbindin, and its location on chromosome 6p, has been associated with cognitive processes, including those relying on a complex system of glutamatergic and dopaminergic interactions. Dysbindin is one of the seven protein subunits that comprise the biogenesis of lysosome‐related organelles complex 1 (BLOC‐1). Dysbindin protein levels are lower in mice with null mutations in pallidin, another gene in the BLOC‐1, and pallidin levels are lower in mice with null mutations in the dysbindin gene, suggesting that multiple subunit proteins must be present to form a functional oligomeric complex. Furthermore, pallidin and dysbindin have similar distribution patterns in a mouse and human brain. Here, we investigated whether the apparent correspondence of pallid and dysbindin at the level of gene expression is also found at the level of behavior. Hypothesizing a mutation leading to underexpression of either of these proteins should show similar phenotypic effects, we studied recognition memory in both strains using the novel object recognition task (NORT) and social novelty recognition task (SNRT). We found that mice with a null mutation in either gene are impaired on SNRT and NORT when compared with wild‐type controls. These results support the conclusion that deficits consistent with recognition memory impairment, a cognitive function that is impaired in schizophrenia, result from either pallidin or dysbindin mutations, possibly through degradation of BLOC‐1 expression and/or function.     

Dystrophin Gene Mutation Location and the Risk of Cognitive Impairment in Duchenne Muscular Dystrophy

Background

A significant component of the variation in cognitive disability that is observed in Duchenne muscular dystrophy (DMD) is known to be under genetic regulation. In this study we report correlations between standardised measures of intelligence and mutational class, mutation size, mutation location and the involvement of dystrophin isoforms.

Methods and Results

Sixty two male subjects were recruited as part of a study of the cognitive spectrum in boys with DMD conducted at the Sydney Children''s Hospital (SCH). All 62 children received neuropsychological testing from a single clinical psychologist and had a defined dystrophin gene (DMD) mutation; including DMD gene deletions, duplications and DNA point mutations. Full Scale Intelligence Quotients (FSIQ) in unrelated subjects with the same mutation were found to be highly correlated (r = 0.83, p = 0.0008), in contrast to results in previous publications. In 58 cases (94%) it was possible to definitively assign a mutation as affecting one or more dystrophin isoforms. A strong association between the risk of cognitive disability and the involvement of groups of DMD isoforms was found. In particular, improvements in the correlation of FSIQ with mutation location were identified when a new classification system for mutations affecting the Dp140 isoform was implemented.

Significance

These data represent one of the largest studies of FSIQ and mutational data in DMD patients and is among the first to report on a DMD cohort which has had both comprehensive mutational analysis and FSIQ testing through a single referral centre. The correlation between FSIQ results with the location of the dystrophin gene mutation suggests that the risk of cognitive deficit is a result of the cumulative loss of central nervous system (CNS) expressed dystrophin isoforms, and that correct classification of isoform involvement results in improved estimates of risk.     

Suppression of Nonsense Mutations in the Dystrophin Gene by a Suppressor tRNA Gene

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10–15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing -galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.     

The dystrophin / utrophin homologues in Drosophila and in sea urchin

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene containing at least 79 introns, some of which are extremely large. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least two additional non-muscle full length dystrophin isoforms transcribed from different promoters located in the 5'-end region of the gene, and four smaller proteins transcribed from internal promoters located further downstream, and lack important domains of dystrophin. Several other genes, encoding evolutionarily related proteins, have been identified. To study the evolution of the DMD gene and the significance of its various products, we have searched for genes encoding dystrophin-like proteins in sea urchin and in Drosophila. We previously reported on the characterization of a sea urchin gene encoding a protein which is an evolutionary homologue of Dp116, one of the small products of the mammalian DMD gene, and on the partial sequencing of a large product of the same gene. Here we describe the full-length product which shows strong structural similarity and sequence identity to human dystrophin and utrophin. We also describe a Drosophila gene closely related to the human dystrophin gene. Like the human gene, the Drosophila gene encodes at least three isoforms of full length dystrophin-like proteins (dmDLP1, dmDLP2 and dmDLP3,), regulated by different promoters located at the 5' end of the gene, and a smaller product regulated by an internal promoter (dmDp186). As in mammals, dmDp186 and the dmDLPs share the same C-terminal and cysteine-rich domains which are very similar to the corresponding domains in human dystrophin and utrophin. In addition, dmDp186 contains four of the spectrin-like repeats of the dmDLPs and a unique N-terminal region of 512 amino acids encoded by a single exon. The full length products and the small product have distinct patterns of expression. Thus, the complex structure of the dystrophin gene, encoding several large dystrophin-like isoforms and smaller truncated products with different patterns of expression, existed before the divergence between the protostomes and deuterostomes. The conservation of this gene structure in such distantly related organisms, points to important distinct functions of the multiple products.     

Cognitive flexibility deficits in a mouse model for the absence of full‐length dystrophin

Duchenne muscular dystrophy (DMD) is a progressive muscle‐wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full‐length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full‐length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full‐length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients.