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

Dystrobrevin is one of the intracellular components of thetransmembrane dystrophin–glycoprotein complex (DGC).The functional role of this complex in normal and patho-logical situations has not yet been clearly established.Dystrobrevin disappears from the muscle membrane inDuchenne muscular dystrophy (DMD), which results fromdystrophin mutations, as well as in limb girdle musculardystrophies (LGMD), which results from mutations affect-ing other members of the DGC complex. These findingsthereforesuggestthatdystrobrevinmayplayapivotalroleinthe progression of these clinically related diseases. In thisstudy, we used the Caenorhabditis elegans model to addressthe question of the relationship between dystrobrevinbinding to dystrophin and dystrobrevin function. Deletionsof the dystrobrevin protein were performed and the abilityof the mutated forms to bind to dystrophin was tested bothin vitro and in a two-hybrid assay, as well as their ability torescue dystrobrevin (dyb-1) mutations in C. elegans. Thedeletions affecting the second helix of the Dyb-1 coiled-coildomainabolishedthebindingofdystrobrevintodystrophinboth in vitro and in the two-hybrid assay. These deletionsalsoabolishedtherescuingactivityofafunctionaltransgeneinvivo.Theseresultsareconsistentwithamodelaccordingtowhich dystrobrevin must bind to dystrophin to be able tofunction properly.Keywords: dystrophin; dystrobrevin; nematode; Caeno-rhabditis elegans.Duchenne muscular dystrophy (DMD) is an inheritedmuscular disease in which the patients’ muscles graduallydegenerate. So far, no treatment exists for DMD. Thediseaseiscausedbymutationsaffectingthedystrophingene,which encodes a 3685-amino-acid protein (reviewed in [1]).Dystrophin is a submembrane protein associated with atransmembrane dystrophin–glycoprotein complex (DGC)comprising dystroglycans, sarcoglycans, sarcospan, syntro-phinsand dystrobrevins[1–3].DGCproteinshaveattractedan increasing amount of attention over the last few years,because they might help to explain the physiopathology ofthe disease, and may also provide therapeutic clues.Dystrobrevins form a family of proteins that are uniquein that they are both dystrophin-associated proteins, andhomologous to the C-terminal region ofdystrophin. Alpha-dystrobrevin was originally identified as a molecule thatcopurifies with nicotinic acetylcholine receptors in sucrosegradients[4,5].Itwaslaterrecognizedasoneoftheproteins,which associates with dystrophin to form the dystrophin–glycoprotein complex (DGC) [4,6,7]. A second dystro-brevin, b-dystrobrevin, is mainly expressed in nerve tissues[8,9]. Mice carrying a knockout mutation of thea-dystrobrevin gene (adbn mice) suffer from a cardiac andskeletal muscle myopathy reminiscent of dystrophin ( mdx)mutations [10].a-Dystrobrevin binds to dystrophin via a coiled-coiledmotif present in both proteins, and to the PDZ domaincontaining syntrophins [11,12]. Indirect evidence suggeststhat dystrobrevin may also bind to other members of theDGC [13]. Although no enzymatic activity has yet beenassigned to dystrobrevins, there are several indications thatthey may play a role in signalling mechanisms. First,dystrobrevins are tyrosine-phosphorylated proteins [5,14].Secondly, in the absence of a-dystrobrevin, the signallingmolecule,neuronalnitricoxidesynthase(nNOS)disappearsfrom the muscular membrane [10].In addition, two lines of evidence suggest that dystro-brevin may play a key role in the muscle degenerationobserved in DMD and sarcoglycanopathies; first, dystro-brevin immunostaining decreases greatly in DMD and inseveral sarcoglycanopathies [15]. Secondly, although theDGC components (with the exception of NOS) are notaffectedbytheabsenceofdystrobrevinin adbn mice,muscledegeneration occurs.The nematode Caenorhabditis elegans has homologues ofmost of the DGC proteins (L. Se´galat, unpublished results).There is one dystrophin- and one dystrobrevin-like gene inthegenomeofC. elegans (dys-1 and dyb-1, respectively)[16,17]. C. elegans dystrophin and dystrobrevin are able tobind to each other in vitro [18] in the same way as theirmammalian counterparts [12], and they also bind tosyntrophin [18]. dys-1 and dyb-1 mutants display a similarbehavioural phenotype consisting of hyperactivity, exagger-ated bending of the head when moving forward, and atendency to hypercontract [16,17]. In addition, progressivemuscle degeneration is observed when dys-1 or dyb-1