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

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 ␣-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.

Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1)

“…We found interaction between dysbindin and pallidin and confirmed the interaction 8 between dysbindin and β-dystrobrevin (Fig. 4a).…”

“…We boiled extracts in Laemlli sample buffer with 5% mercaptoethanol and separated 40 μg of extract protein on 12% SDS-PAGE gels or 4-20% gradient gels (Gradipore) and blotted gels to a PVDF membrane. We probed the blots with rabbit polyclonal antisera to dysbindin 8 (1:250 dilution), muted 11 , pallidin 11 and dystrobrevin 8 . We purchased rabbit polyclonal antibody to Rab4 (D-20) from Santa Cruz Biotechnology and mouse monoclonal antibody to α-tubulin (Clone B512) from Sigma.…”

“…We transfected COS-7 cells with different combinations of expression constructs encoding dysbindin 8 , dysbindin lacking amino acids 120-171 (sdy/sdy dysbindin) in pCMVTag-4A, β-dystrobrevin 8 , pallidin in pCMV-Myc (Clontech), muted in pCMV-Myc and pearl (Ap3b1) in pCMVTag-3B (Stratagene) using Fugene-6 (Roche). Twenty-four hours after transfection we scraped the cells into 4 ml of RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% v/v Triton X-100, 0.5% w/v sodium deoxycholate, 1 mM EGTA), clarified the supernatant by centrifugation and precipitated proteins with 5 μg of antibody for 6 h at 4 °C.…”

“…This in-frame deletion creates a 1.5-kb mutant dysbindin transcript (Fig. 2b) and abolishes expression of the 51-kDa dysbindin 8 protein in sdy/sdy mice (Fig. 2c).…”

Genetic investigation of chromosome 5q GABAA receptor subunit genes in schizophrenia

“…For example, treatment of the rat hippocampus with NRG1 resulted in lower expression of GABA A receptor subunit transcripts, 66 while dysbindin likely colocalizes with GABA A receptor subunits in the hippocampus, cortex, and cerebellum through its association with beta-dystrobrevin in the dystrophin protein complex. [67][68][69] Despite the evidence for GABA system alterations in schizophrenia patients, there has been little investigation of association between GABA receptor genes and schizophrenia, although association with the GABRB2 gene was recently reported in a Chinese patient sample. 70 We performed an association study of the 5q GABA A receptor subunit gene cluster in schizophrenia based on: (1) strong meta-analytic evidence for linkage between schizophrenia and chromosome 5q, (2) linkage to 5q in our Portuguese schizophrenia families, (3) evidence for involvement of the GABA system in schizophrenia, and (4) location of the GABA A receptor subunit gene cluster in the 5q risk locus.…”