Direct binding of Torpedo syntrophin to dystrophin and the 87 kDa dystrophin homologue

282

266

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 ␣-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 dystrobrevinbinding protein. Dysbindin is an evolutionary conserved 40-kDa coiled-coil-containing protein that binds to ␣-and ␤-dystrobrevin in muscle and brain. Dystrophin and ␣-dystrobrevin are co-immunoprecipitated with dysbindin, indicating that dysbindin is DPC-associated in muscle. Dysbindin co-localizes with ␣-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.

Section: Resultsmentioning

confidence: 99%

Dysbindin, a Novel Coiled-coil-containing Protein That Interacts with the Dystrobrevins in Muscle and Brain

Benson

Newey

Martin‐Rendon

et al. 2001

282

266

“…In Vivo and in Vitro Assays of Binding with Dystrophin-The ␣1-, ␤1-, and ␤2-syntrophins have been shown to bind dystrophin, utrophin, and dystrobrevins in vitro (34,35,39,40,55). To verify whether ␥1-and ␥2-syntrophins can also bind dystrophin or related proteins, assays of protein-protein interaction were used.…”

Section: Two Novel Dystrophin-binding Proteins In Neuronal Cellsmentioning

confidence: 99%

γ1- and γ2-Syntrophins, Two Novel Dystrophin-binding Proteins Localized in Neuronal Cells

Piluso¹,

Mirabella²,

Ricci³

et al. 2000

123

104

Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3 terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (␣1) and two basic (␤1 and ␤2) proteins of ϳ57-60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named ␥1-and ␥2-syntrophins. The human ␥1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human ␥2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the ␥1-syntrophin gene to human chromosome 8q11 and the ␥2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system.Since the identification of dystrophin, the product of the Duchenne muscular dystrophy gene at Xp21, molecular genetics has moved quickly (1, 2). The deficiency of dystrophin in Duchenne muscular dystrophy (DMD) 1 and its first animal model, the mdx mouse, leads to a dramatic reduction in a group of previously unknown proteins identified as the dystrophinassociated protein complex.In the last few years, the dystrophin-associated protein complex proteins have been isolated; their genes have been cloned; and the following model of the complex has been hypothesized (3-5). Dystrophin is a large rod-shaped protein, primarily localized beneath the muscle cell membrane. Its actinin-like N terminus binds F-actin (6), whereas its C terminus is anchored to the transmembrane protein, ␤-dystroglycan, which is linked through ␣-dystroglycan to the extracellular merosin (laminin-2) (7). Then, this complex bridges the muscle membrane from the cytoskeleton to the extracellular matrix. In addition, dystroglycan is the receptor for agrin, a protein with a pivotal role in the clustering of acetylcholine receptors at the neuromuscular junction (8 -10) and a fundamental element of the basal lamina (11). At the muscle membrane, this complex is associated with the hydrophobic sarcospan DAP25 (dystrophinassociated protein; A5) (12) and the sarcoglycan complex, which is composed of at least four interacting transmembrane glycoproteins: ␣-sarcoglycan (DAG50 (dystrophin-associated glycoprotein), A2, adhalin) (13, 14), ␤-sarcoglycan (DAG43, A3b) (15, 16), ␥-sarcoglycan (DAG35, A4) (17), and ␦-sarcoglycan (18). Mutations in the laminin-␣2 gene are responsible for congenital muscular dystrophy (19); mutations in the ␥-, ␣-, ␤-, and ␦-sarcoglycan genes cause limb-girdle mus...

“…Several ␣-dystrobrevin-associated proteins have been described including the syntrophins (17)(18)(19)(20), syncoilin (21), desmuslin (22), and dysbindin (23). Of these, the syntrophins are the best characterized and are involved in signaling by recruiting neuronal nitric-oxide synthase (1,24), ion channels (6), and kinases (3,5) to the dystrophin complex.…”

mentioning

confidence: 99%

DAMAGE, a Novel α-Dystrobrevin-associated MAGE Protein in Dystrophin Complexes

Albrecht

Froehner

2004

Self Cite

Mice rendered null for ␣-dystrobrevin, a component of the dystrophin complex, have muscular dystrophy, despite the fact that the sarcolemma remains relatively intact (Grady, R. M., Grange, R. W., Lau, K. S., Maimone, M. M., Nichol, M. C., Stull, J. T., and Sanes, J. R. (1999) Nat. Cell Biol. 1, 215-220) Thus, ␣-dystrobrevin may serve a signaling function that is important for the maintenance of muscle integrity. We have identified a new dystrobrevin-associated protein, DAMAGE, that may play a signaling role in brain, muscle, and peripheral nerve. In humans, DAMAGE is encoded by an intronless gene located at chromosome Xq13.1, a locus that contains genes involved in mental retardation. DAM-AGE associates directly with ␣-dystrobrevin, as shown by yeast two-hybrid, and co-immunoprecipitates with the dystrobrevin-syntrophin complex from brain. This co-immunoprecipitation is dependent on the presence of ␣-dystrobrevin but not ␤-dystrobrevin. The DAMAGE protein contains a potential nuclear localization signal, 30 12-amino acid repeats, and two MAGE homology domains. The domain structure of DAMAGE is similar to that of NRAGE, a MAGE protein that mediates p75 neurotrophin receptor signaling and neuronal apoptosis (Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000) Neuron 27, 279 -288). DAMAGE is highly expressed in brain and is present in the cell bodies and dendrites of hippocampal and Purkinje neurons. In skeletal muscle, DAMAGE is at the postsynaptic membrane and is associated with a subset of myonuclei. DAMAGE is also expressed in peripheral nerve, where it localizes along with other members of the dystrophin complex to the perineurium and myelin. These results expand the role of dystrobrevin and the dystrophin complex in membrane signaling and disease.The dystrophin glycoprotein complex links the actin cytoskeleton to the extracellular matrix in skeletal muscle. In addition, recent studies (1-7) have shown that the dystrophin complex is critical for the localization of a variety of signaling molecules to the skeletal muscle sarcolemma. Thus, the dystrophin complex functions as both a structural link to the extracellular matrix and as a scaffold that concentrates and stabilizes functionally inter-related signaling proteins at the muscle cell membrane.Part of the signaling function of the dystrophin complex may be mediated by the dystrobrevins. The role of the dystrobrevins in signaling is not fully understood; however, targeted disruption of the ␣-dystrobrevin gene in mice results in muscle pathology without the membrane fragility characteristic of dystrophin-deficient muscular dystrophy (8). These findings suggest that ␣-dystrobrevin is important for muscle function but in a non-structural way. Dystrobrevin has no enzymatic activity of its own; therefore, the involvement of dystrobrevin in signaling pathways is dependent on its interaction with other proteins.The dystrobrevins are transcribed from two separate genes, designated ␣ and ␤, that shar...