Isoform Diversity of Dystrobrevin, the Murine 87-kDa Postsynaptic Protein

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-1) that is very similar to the Torpedo sequence. A second transcript encodes a 59-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

The genetic basis of neuromuscular disorders

In the last decade, our knowledge of human disease 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

Genomic organization of the mouse dystrobrevin gene: comparative analysis with the dystrophin gene

Dystrobrevin, the mammalian orthologue of the Torpedo87-kDa postsynaptic protein, is a member of the dystrophin gene family with homology to the cysteine-rich carboxy-terminal domain of dystrophin. Torpedodystrobrevin 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

Binding of gephyrin to microtubules is regulated by its phosphorylation at Ser270

Gephyrin is a multifunctional scaffolding protein anchoring glycine- and subtypes of GABA type A- receptors at inhibitory postsynaptic membrane specializations by binding to the microtubule (MT) and/or the actin cytoskeleton. However, the conditions under which gephyrin can bind to MTs and its regulation are currently unknown. Here, we demonstrate that during the purification of MTs from rat brain by sedimentation of polymerized tubulin using high-speed centrifugation a fraction of gephyrin was bound to MTs, whereas gephyrin phosphorylated at the CDK5-dependent site Ser270 was detached from MTs and remained in the soluble protein fraction. Moreover, after collybistin fostered phosphorylation at Ser270 the binding of a recombinant gephyrin to MTs was strongly reduced in co-sedimentation assays. Correspondingly, upon substitution of wild-type gephyrin with recombinant gephyrin carrying alanine mutations at putative CDK5 phosphorylation sites the binding of gephyrin to MTs was increased. Furthermore, the analysis of cultured HEK293T and U2OS cells by immunofluorescence-microscopy disclosed a dispersed and punctuated endogenous gephyrin immunoreactivity co-localizing with MTs which was evidently not phosphorylated at Ser270. Thus, our study provides additional evidence for the binding of gephyrin to MTs in brain tissue and in in vitro cell systems. More importantly, our findings indicate that gephyrin-MT binding is restricted to a specific gephyrin fraction and depicts phosphorylation of gephyrin as a regulatory mechanism of this process by showing that soluble gephyrin detached from MTs can be detected specifically with the mAb7a antibody, which recognizes the Ser270 phosphorylated- version of gephyrin.publishe

β-dystrobrevin, a member of the dystrophin-related protein family

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 β-dystrobrevin, a dystrophin-related protein that is abundantly expressed in brain and other tissues, but is not found in muscle. β-dystrobrevin is encoded by a 2.5-kb alternatively spliced transcript that is found throughout the brain. In common with dystrophin, β-dystrobrevin is found in neurons of the cortex and hippocampal formation but is not found in the brain microvasculature. In the brain, β-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

β-dystrobrevin, a member of the dystrophin-related protein family

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 β-dystrobrevin, a dystrophin-related protein that is abundantly expressed in brain and other tissues, but is not found in muscle. β-dystrobrevin is encoded by a 2.5-kb alternatively spliced transcript that is found throughout the brain. In common with dystrophin, β-dystrobrevin is found in neurons of the cortex and hippocampal formation but is not found in the brain microvasculature. In the brain, β-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

Tissue-selective Expression of alpha-Dystrobrevin is Determined by Multiple Promoters

α-Dystrobrevin, the mammalian orthologue of theTorpedo 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 α-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 α-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 α-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 α-dystrobrevin gene, has been developed to allow the regulation of this gene in different cell types and/or different developmental stages

Genomic organization and refined mapping of the mouse β-dystrobrevin gene

β-Dystrobrevin, a dystrophin-related protein that is expressed in non-muscle tissues, is highly homologous to α-dystrobrevin, a member of the dystrophin-associated protein complex (DPC). β-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 β-dystrobrevin gene. The mouse β-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 β-dystrobrevin and α-dystrobrevin is reflected in the conservation of their exon-intron junctions. β-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 β-dystrobrevin have been mapped to this region, namely, congenital polycystic kidneys (cpk) and fatty liver dystrophy (fld). However, refined mapping analysis has excluded β-dystrobrevin as a candidate gene for either disease