Résumé :
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Communication n° 348. b-Dystrobrevin is a member of the dystrophin-related and -associated protein family, highly expressed in brain, lung and kidney. In brain b-dystrobrevin is highly enriched at the postsynaptic membrane in hippocampal and Purkinje neurons. We have recently identified kinesin heavy chain 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 used recombinant b-dystrobrevin and Kif5A cargo-binding domain to localize the kinesin-binding site on b-dystrobrevin and determine the kinetic rate constant of their interaction through surface plasmon resonance (SPR). Pull-down experiments of dystrobrevin-deletion mutants with GST-Kif5A localized the binding site on the coiled-coil region of b-dystrobrevin. SPR analysis showed that the deletion mutants of dystrobrevin bind to kinesin with a lower binding affinity than full-length dystrobrevin thus indicating that regions other than the coiled-coil can affect the binding capacity of the protein. We found a second binding site in the N-terminal of b-dystrobrevin that comprises the EF-hands domains. a-Dystrobrevin the isoform primarily expressed in muscle tissues comprises, similarly to b-dystrobrevin, two binding sites for kinesin. Since dystrophin and dystrobrevin interact through their homologous coiled-coil regions, we investigated whether dystrophin could affect the dystrobrevin-kinesin interaction. We found that dystrophin does not directly bind to Kif5A but is able to associate with b-dystrobrevin-Kif5A in a ternary complex. Taken together our data suggest that the transport of dystrophin to specific sites in the cell may occur via b-dystrobrevin bound to kinesin.
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