Dystrobrevin requires a dystrophin-binding domain to function in Caenorhabditis elegans Karine Grisoni, Kathrin Gieseler and Laurent Se ´ galat CGMC, CNRS-UMR, Universite ´ Lyon, Villeurbanne, France Dystrobrevin is one of the intracellular components of the transmembrane dystrophin–glycoprotein complex (DGC). The functional role of th is complex in normal an d patho- logical situations has not yet been clearly established. Dystrobrevin disappears from t he muscle m embrane in Duchenne muscular dystrophy (DMD), which results from dystrophin mutations, as well as in limb girdle muscular dystrophies (LGMD), which results f rom mutations affect- ing other members of the DGC complex. These findings therefore s uggest that dystrobrevin may play a pivotal role in the progression of these clinically related diseases. In this study, w e u sed t he Ca enorhabditis elegans mod el t o a ddress the question of the relationship between dystrobrevin binding to dystrophin and dystrobrevin function. Deletions of the dystrobrevin protein w ere performed and the ability of the mutated forms to bind t o dystrophin was tested both in vitro and in a two-hybrid assay, as well as their ability t o rescue dystrobrevin (dyb-1) mutations in C. elegans. The deletions affecting the second helix of the D yb-1 coiled-coil domain abolished the binding of dystrobrevin to dystrophin both in vitro and in the two-hybrid assay. These deletions also abolished t he rescuing activity of a functional transgene in vivo. These r esults are consistent with a model according to which dystrobrevin must bind to dystrophin to be able t o function properly. Keywords: dystrophin; dystrobrevin; nematode; Caeno- rhabditis elegans. Duchenne muscular dystrophy (DMD) is an inherited muscular disease in which the patients’ muscles gradually degenerate. So f ar, n o t reatment exists for DMD. The disease i s c aused b y mutations affecting t he dystrophin gene, which encodes a 3685-amino-acid protein (reviewed in [1]). Dystrophin is a submembrane protein associated with a transmembrane dystrophin–glycoprotein complex (DGC) comprising dystroglycans, sarcoglycans, sarcospan, syntro- phins and dystrobrevins [1–3]. DGC pro teins have attracted an increasing amount of attention over the last few years, because they might help to explain the physiopathology of the disease, and may also provide therapeutic clues. Dystrobrevins form a family of proteins that are uniqu e in that they are both dystrophin-associated proteins, and homologous to the C-terminal region of dystrophin. Alpha- dystrobrevin was originally identified as a molecule that copurifies with nicotinic acetylcholine receptors in sucrose gradients [4,5]. I t was later recognized as one of the proteins, which associates with dystrophin to form the dystrophin– glycoprotein complex (DGC) [4,6,7]. A second dystro- brevin, b-dystrobrevin, is mainly expressed in n erve tissues [8,9]. Mice carrying a knockout mutation of the a-dystrobrevin gene (adbn mice) suffer from a cardiac and skeletal muscle myopathy reminiscent o f dystrophin (mdx ) mutations [10]. a-Dystrobrevin binds to dystrophin via a coiled-coiled motif present in both proteins, and to the PDZ domain containing syntrophins [11,12]. Indirect evidence suggests that dystrobrevin may also bind to other members of the DGC [13]. Although no enzymatic activity has yet been assigned to dystrobrevins, there a re several i ndications that they may play a role in signalling mechanisms. First, dystrobrevins are tyrosine-phosphorylated proteins [5,14]. Secondly, in the absence of a-dystrobrevin, the signalling molecule, neuronal nitric o xide synthase (nNO S) disappears from the muscular membrane [10]. In addition, two lines of evidence suggest that dystro- brevin may p lay a key role i n the muscle degeneration observed in DMD and sarcoglycanopathies; first, dystro- brevin immunostaining decreases greatly in DMD and in several sarcoglycanopathies [15]. Secondly, although t he DGC components (with the exception of NOS) are not affected by the absence of dystrobrevin in adbn mice, musc le degeneration occurs. The nematode Caenorhabditis elegans has homologues of most of the DGC proteins (L. Se ´ galat, unpublished results). There is one dystrophin- and one dystrobrevin-like gene in thegenomeofC. elegans (dys-1 and dyb-1, respectively) [16,17]. C. elegans dystrophin and dystrobrevin are able t o bind to each other in vitro [18] in the same way as their mammalian counterparts [12], and they also bind to syntrophin [18]. dys-1 and dyb-1 mutants d isplay a similar behavioural phenotype c onsisting of hyperactivity, exagger- ated bending of the head when moving forward, and a tendency t o hypercontract [16,17]. In addition, progressive muscle degeneration is observed when dys-1 or dyb-1 Correspondence t o L. Se ´ galat, CGMC, Universit e ´ Lyon1,43blddu11 Novembre, 69622 Villeurbanne cedex, France. Fax: + 3 3 4 72 44 05 55, Tel.: + 33 4 72 43 29 51, E-mail: segalat@maccgmc.univ-lyon1.fr Abbreviations: DGC, dystrophin–glycoprotein complex; DMD, Duchenne muscular dystrophy; LGMD, limb girdle muscular dystrophy; nNOS, neuronal nitric oxide synthase; AD, activation domain; DNA-BD, DNA binding domain; SD, synthetic dropout medium; SBR, syntrophin binding region. (Received 1 6 October 200 1, revised 1 0 January 2002 , accepted 11 January 2002) Eur. J. Biochem. 269, 1607–1612 (2002) Ó FEBS 2002 mutations are introduced in a sensitized hlh-1(cc561) genetic background that makes C. elegans muscles fragile [19,20]. In this study, we addressed the questio n as to whether the ability o f dystrobrevin to function properly may depend on its association with dystrophin. First, we refined the dystrophin-binding region on dystrobrevin (Dyb-1) by performing deletion-mapping experiments in vitro.Wethen tested the ability of t he truncated Dyb-1 proteins to bind to dystrophin (Dys-1) in a yeast two-hybrid assay, as well as their ability to rescue dyb-1 mutants. EXPERIMENTAL PROCEDURES Construction of deleted forms of Dyb-1 for in vitro binding experiments Deletions were carried out on the dyb-1 coding sequence, using clone AN450 [encoding Dyb-1 amino acids 3 90–543 fused in frame to the GST coding sequen ce; plasmid pGEX 3X (Pharmacia)] [18]. AN450 DNA (500 ng) was cut with the restriction enzyme MfeI. The cut DNA was then distributed among several tubes incubated with 0.05 lLof BAL31 exonuclease for various times (typically 0–10 min). The r eactions were stopped by adding EGTA to 4 m M and heating a t 6 5 °C f or 10 min. DNA was purified on a Wizard column (Promega) and the action of BAL31 was checke d by loading an aliquot o f each tube onto a n agarose gel column. The DNA corresponding to the deletions required was treated b y applying T4 DNA polymerase in the presence of nucleotides to create blunt ends, w hich were ligated and the plasmids were transformed in Escherichia coli DH5. Clones were picked randomly and analysed using sequencing procedures. Any clones carrying a frame shift were rejected. Construct 6¢4 was built using similar procedures, but using the enzyme HindIII instead of MfeI. The amino acids removed in the deletions were 489–499 (clone 2¢5), 487–513 (clone 5 ¢1), 489–528 ( clone 5 ¢2B), 471–517 (clone 5 ¢5B), 478– 543 ( clone 2 ¢1), and 391–450 (clone 6¢4). Clones 2¢1and6¢4 have been described previously [18], but clo ne 2¢1was erroneously reported t o be deleted in amino a cids 478–521. This correction make s no difference to the interpretation of our previous data. In vitro interactions Constructs were transformed into the E. coli strain BL21 DE3 and the fusion proteins were produced as follows. After cell sonication and centrifugation, the supernatant was loaded onto glutathione–Sepharose beads (Pharma- cia). Approximately 20 lg of resin bound proteins were washed in binding buffer [Hepes 20 m M pH 7.4, KOAc 110 m M ,NaOAc5m M ,Mg(OAc) 2 2.5 m M , NP40 0.05%, dithiothreitol 1 m M , leupeptin 10 mgÆmL )1 , aprotinin 10 mg ÆmL )1 , pepstatin 10 mgÆmL )1 , phenylmethanesulfo- nyl fluoride (1 mM)]. The 35 S-labelled Dys-1 C-terminal end was synthesized using a coupled in vitro transcription and translation kit (Promega) with cDNA yk12c11 [16]. The p reparations were incub ated for 2 h at 4 °C. GST controls were performed using 1–2 times the amount of fusion protein. After five washes with binding buffer, the labelled proteins were e luted by boiling t he preparation f or 3 min in gel loading buffer. Gels were dried, exposed overnight and revealed using a radiographic analyser (Fuji BAS-1500). Band intensity was quantitated using the analyser software on at least three independent experiments. Constructs for the yeast two-hybrid assay The C-terminal end of Dys -1 (amino a cids 2857–3674) was fused t o t he DNA binding domain (DNA-BD) of t he Gal4 protein. For t his purpose, a 2,4 kb dys-1 cDNA fragment (yk12c11) was cloned into the polylinker of pAS2-1 (Clontech) with respect to the reading frame. Dyb-1 fragments and deletions were PCR a mplified using clones AN 450, 2¢5, 5¢1, 5¢2B, 5 ¢5B and 6¢4inpGEX3Xas templates (see b elow) and cloned into pACT2 (Clontech) in frame w ith the activation domain ( AD) of the Gal4 protein and the HA epitope. The resulting constructs were called AD-AN450, AD-2¢5, AD-5¢1, AD-5¢2B, AD-5¢5B, and AD-6¢4, respectively. All DNA constructs were checked by performing DNA sequencing. Yeast two-hybrid analysis Construct DNA-BD-Dys-1 was transformed into the yeast strain CG 1945 using the LiAc transformation procedure (Clontech, Yeast protocols Handbook, PT 3024-1). Trans- formants were selected on synthetic dropout (SD) media (Clontech) minus tryptophan. A DNA-BD-Dys-1 expressing yeast strain was selected and transformed with plasmids AD-AN450, AD-2¢5, AD-5¢1, AD-5¢2B, AD-5¢5B, or AD-6¢4. Transformants were selected on SD media minus tryptophan and leucin. Interactions between the DNA-BD-Dys-1 protein and t he various forms of the AD-Dyb-1 fusion proteins were analysed on the basis of transactivation of the HIS3 reporter gene a fter 3 days of growth on SD medium devoid of tryptophan, leucin and histidin. A strain carrying both the D NA-BD-Dys-1 p rote in a nd t he empty pAC T2 plasmid was used as a negative control. Western blots with yeast protein extracts For Western blot analysis, yeast protein extracts were prepared from strains carrying both DNA-BD-Dys-1 plasmids and AD-Dyb-1 plasmids (AN450, AD-2¢5, AD-5¢1, AD-5¢2B, AD-5¢5B, or AD-6¢4). Overnight cul- tures (5-mL) were prepared in SD media minus trypto- phan and leucin. The next day, 1 mL of o vernight culture was transferred into 10 mL of YPD medium. The diluted culture w as i ncubated f or several hours at 30 °C until D 600 ¼ 0.3 for 1 mL. Cells (3 D 600 units) were spun down and frozen at )70 °C for at least one hour. The yeast pellet was resuspended in 60 lLofsample buffer [21]. After boiling the mixture for 5 min, and centrifuging f or 30 s a t 1 3 000 g,10lL of supernatant was loaded onto each lane of a 0.1% SDS/10% polyacrylamide gel. Proteins were transferred onto a BA83 nitrocellulose membrane (Schleicher & Schuell) in transfer buffer (Tris 25 m M , glycine 190 m M , SDS 0.01%, ethanol 20%) for 1 h at 100 V. AD-Dyb-1 fusion proteins were detected using a rabbit polyclonal anti-(Dyb-1) Ig [19] at a dilution 1 : 500. Peroxidase-coupled anti-(rabbit IgG) Ig (Biorad) was used at a dilution of 1 : 3000. Blots 1608 K. Grisoni et al. (Eur. J. Biochem. 269) Ó FEBS 2002 were revealed using the ECL+ kit (Amersham) as recommended by the supplier. Functional assay in C. elegans First, a dyb-1 functional construct was obtained by m odi- fying a previously built dyb-1:gfp construct [19]. The dyb-1:gfp construct, which has been previously described, was shortened on the 5¢ end to leave 2.9 kb of upstream sequence, and various restriction e nzyme s ites were removed and a dded by performing synonymous point mutations to yield the construct dyb-1:gfp VII, which h as single Age Iand MluI sites at codons 390 and 543. This co nstruct encod es a functional Dyb-1 gene as it can rescue dyb-1 muta tions ( data not shown). Secondly, Dyb-1 construct AN450 and the deletion derivatives described above were transferred from pGEX into dyb-1:gfp VII using PCR-amplifying procedures with primers carrying AgeIandMluI sites, and cloned into the single AgeIandMluI sites of dyb-1:gfp VII (Fig. 5). Positives c lones were c hecked by determining their seq- uence. dyb-1:gfp V II and constructs carrying e ither A N450 or the deletions were injected at a concentration of 1ngÆlL )1 along with the transformation marker KP13 [22] using standard procedures [23] into worms carrying the putative null allele dyb-1(cx36) [17]. Transgenic strains were grown at 23 °C. RESULTS Mapping of the dystrophin-binding site on Dyb-1 The results of a previous study suggested that the Dys-1- binding region on Dyb-1 w as located in the second helix of the predicted coiled-coil domain [18]. We refined this analysis by creating additional deletions by random muta- genesis and testing their affinity for Dys-1. C lones 2¢5, 5¢1, 5¢2B, a nd 5¢5B were obtained by inducing exonuclease digestion of the referen ce clone AN450, which encodes t he amino acids 390–543 of Dyb-1 fused to the GST protein [18]. These four clones contain various breakpoints within the second helix of the predicted coiled-coil domain (H2) (Fig. 1). The d eleted amino acids were 489–499 (clone 2¢5), 487–513 (clone 5¢1), 489–528 (clone 5¢2B) and 471–517 (clone 5¢5B). Clone 2¢1, lacking amino acids 478–543, was used as a negative control [18]. Clone 6¢4, lacking amino acids 391–450, was used as a second positive control [18]. The constructs were used to produce Dyb-1–GST chimeric proteins in E. coli, which were affinity purified on gluthati- one–Sepharose beads and subjected to in vitro binding with 35 S-labelled Dys-1. Clones 2¢5and5¢2B bound to Dys-1 at levels that were not significantly different from those of the positive controls AN450 (Fig. 2) and 6¢4 (gel not shown). In contrast, the binding activity of clones 5¢1and5¢5B was weaker (Fig. 2). The difference between clones 2¢5, 5¢1and Fig. 1. Deletions used in this s tudy. The ‘WT’ line represents the amino- acid sequence of the wild-type Dyb-1 protein in the predicted coiled- coil domain region. The predicted helices forming the domain are shown by hatched boxes. Numbers above the wild-type sequence indicate the amino-acid coordinates of t he helices. Deletions are s hown below the wild-type se quenc e. Numbers i ndicate the coordinates o f the breakpoints. D eletions we re ge ne rated b y e xonuclease digestion . No te that the 6¢4 de letion extends on the left side further tha n sh own on the drawing. For in vitro binding experiments, the corresponding DNAs were cloned into the pGEX vector to produce Dyb-1–GST fusion proteins [18]. T he righ t column g ives t he binding affinity of the v arious constructs to 35 S-labelled Dys-1 in arbitrary units (mean ± SD). One unit is defined as t he autoradiogram intensity obtained with the neg- ative control GST. Asterisks indicate values significantly different from wild-type. Constructs 5¢1, 5¢5B and 2¢1 have significantly reduced affinity to Dys-1. Fig. 2. In vitro binding of Dyb-1 (dystrobrevin) to Dys-1 (dystrophin). Representative example of in vitro binding experiments. The same gel is shown in Coomassie staining (top) and autoradiography (bottom). The gel was loaded with various GST–Dyb-1 fusion proteins (and GST alone) after inc ubation with equ al amounts of in vitro translated 35 S-labelled DYS-1. The signal intensity of the autoradiogram was quantitated with a radiographic analyser (Biorad). MW, Molecular mass markers. T, aliquot of the in vi tro translation product. Ó FEBS 2002 Dystrophin–dystrobrevin interactions in C. elegans (Eur. J. Biochem. 269) 1609 5¢2B is of interest because these clones have left breakpoints differing b y only two amino a cids. Although clones 2¢5and 5¢2B (cutting at position 489) display a wild-t ype p attern of binding behaviour, clone 5¢1 (cutting at position 487) does not. Therefore, the third heptad repeat of the second helix of Dyb-1 (amino acids 484–490) seems to be critical for proper Dys-1 binding to occur in vitro. Dys-1/Dyb)1 interactions in the yeast two hybrid assay Next, we t ested the ab ilit y of t he various forms of Dyb-1 t o interact with Dys-1 in a two-hybrid assay. The Dyb-1 control and mutant clones w ere fused to the activation domain of the Gal4 yeast transcription factor and were tested against the entire C-terminal end of Dys-1 (amino acids 2857–3674) fused t o the DNA-binding domain of Gal4. The expression of wild-type and truncated proteins was checked using the Western blotting procedure. This confirmed that all the fusion proteins were correctly expressed and that the experiment was not biased by any differences in the protein expression levels (Fig. 3 ). Among the six constructs tested , only the wild-type Dyb-1 fragment and t he 6 ¢4 fragment resulted i n t he gr owth of yeasts on His - plates, which can occur only if Dyb-1 binds to Dys-1 (Fig. 4). Similar r esults were obtained when a shorter Dys-1 fragment (amino acids 3402–3674) encompassing the syn- trophin-binding domain and the coiled-coil domain (cor- responding to BB810 in [18]) was used (not shown). These results indicate that all the deletions affecting the second helix of Dyb-1, including the shortest deletion (clone 2¢5), greatly reduce t he interactions between Dys-1 and D yb-1 in the yeast system. Functional complementation of Dyb-1 deletions in C. elegans The only functional assay available for dystrobrevin resides in functional complementation. To test whether the dele- tions of various parts of the coiled-coil domain had an effect on the in vivo function of Dyb-1, we created transgenes carrying the s ame deletions as those tested in v itro andinthe yeast system. We transferred the deletions into the vector dyb-1:gfp V II, w hich is a functional transgene consisting of genomic Dyb-1 sequences (Fig. 5). Because the deletions are derivatives of clone AN450, a cDNA fragment that encompasses several exons, it was necessary first to check whether removing introns 7 and 8 had any effect on the rescuing activity of dyb-1:gfp VII. When the 1.2-kb AgeI– MluI genomic fragment of dyb-1:gfp VII was replaced by the 450 -bp A N450 cDNA fragment encoding the same amino acids (Fig. 5), rescue of dyb-1(cx36) animals still occurred in t wo out of thre e lines transgenic lines (Table 1), which i ndicates that r emoving i ntrons 7 and 8 did not impair the rescuing capacit y of dyb -1:gfp VII. The n we teste d the various deletions. T hree out of the four lines obtained w ith deletion 6¢4 showed consistent, a lthough only p artial, r escue (Table 1). In these lines, the dyb-1 behavioural phenotype (head bending and hyperlocomotion) was intermediate between mutant and wild-type. This indicates that, although deleting the syntrophin binding region (SBR) and the first helix reduces the activity of the protein, it remains partly functional. Five lines were obtained with deletion 2¢5(the shortest deletion affecting the second helix); only one out of the five lines tested show ed a w eak rescuing e ffect, which was far less c onspicuous than that observed with construct 6¢4 (Table 1). Worms carrying the remaining constructs (5¢1, 5¢2B and 5 ¢5B) did not display any visible rescue. All in all, these data indicate that deletions affecting t he second helix of the Dyb-1 coiled-coil domain strongly decrease or abolish the functional p roperties of D yb-1. DISCUSSION The aim of this study was twofold: first, to confirm and refine th e localization of D ys-1 binding sites o n Dyb-1, and secondly to test whether there may exist a correlation Fig. 3. Western blot of D yb-1 deletions produced i n yeast. Western blots were prepared with protein extracts of yeast carrying DN A-BD-Dys-1 plasmids and empty pACT2 (lane 1), AD-AN450 (lane 2), AD-2¢5 (lane 3), AD-5¢1(lane4),AD-5¢2B (lane 5), AD-5¢5B (lan e 6) a nd AD-6¢4 (lane 7). Th e blot was probed w ith mouse monoclonal antibodies directed against the HA epitope situated between the C-terminal end of the activation domain of the Gal4 protein and the various Dyb-1 proteins. Molecular mass standards are shown on the right. Fig. 4. Yeast two-hybrid assay. A plate containing SD media minus leucin, t ryp tophan and histidine was seeded with yeast c arrying DNA- BD-Dys-1 and various AD-Dyb-1 p lasmids or empty pACT2 (as a negative control), an d incubated at 30 °C for 3 days. Gro wth on m edia devoid of histidine can occur only if D ys-1 and Dyb-1 interact and the HIS3 reporter gene is transactivated. Only t he wild-type construct A D- AN450 and the AD-6¢4 construct promoted growth in this assay. The other constructs, all carrying deletions in the second h e lix of the Dyb-1 coiled-coil domain (H2), were unable t o p romote gr owth in this assay, which indicates that the H2 helix is a critical prerequisite for Dys-1/ Dyb-1 interactions to b e possible. 1610 K. Grisoni et al. (Eur. J. Biochem. 269) Ó FEBS 2002 between th e binding of dystrobrevin to dystrophin and functional activity of dystrobrevin. The C. elegans mode l organism was particularly well suited for the latter part because transgenic a nimals can be quickly obtained in this species. As long as the catalytic, enzymatic, or other functional activity of the dystrobrevin protein will remain unknown, the only way of making functional investi- gations will continue to be through complementation of mutations. A preliminary in vitro study on Dys-1–Dyb-1 interactions pointed to the second helix (H2) of the predicted coiled-coil domain of Dyb-1 [18]. Here, we refined this analysis by studying additional d eletions that s ubdivide the H2 domain. Our results confirm the previously published data and show that H2 is involved in the interaction with Dys-1 in vitro. Within this domain, the first half seems to be particularly critical as deletions infringing on th is side lead to a d ecrease in binding. Constructs 2¢5, 5¢2B and 5¢1 are of particular interest; t he first two constructs break at amino acid 489 and retain binding to Dys-1, whereas the third one breaks at position 487 and its binding affinity is reduced two-fold. Alternatively, this discrepancy might also be attributable to differences in the three dimensional structure of the helix imposed by amino a cids on the C -terminal side of the breakpoint. The predicted three-dimensional structure of the various constructs has not been investigated so far. The two deletions of the H2 region that retained some in vitro binding activity (clones 2¢5and5¢2B) were unable to promote Dys-1–Dyb-1 interaction in the yeast two-hybrid system, which indicates that the yeast assay i s more selective than the in v itro assay. A possible explanation may r eside in the d ifferences in protein concentrations. In the in vitro pull down experiment, the GST–Dys-1 moiety is in great excess to Dyb-1, whereas Dys-1 and D yb-1 are thought to be in the same range of concentration in the yeast assay. In agree- ment with the yeast results, these two constructs (2¢5and Fig. 5. Drawing o f the c onstructs used for in vivo complementation t es ts. To p, map of the dyb-1:gfp VII construct. dyb-1:gfp V II is a 8-kb genomic fragment of the dyb-1 gene cloned i nto a pGEM backbone, in which the gfp coding sequence has been added termin- ally to t he dyb-1 coding sequence. Bottom: map of t he constructs used for t h e in vivo experiments. The various de letions are deri- vatives of the A N450 construct, a fragment of dyb-1 cDNA cloned into the GST-containing vector pGEX. Deleted regions are indica ted in dark. T he deletions were transfered into dyb-1:gfp VII by PCR using the unique Age I and MluI restriction sites o f dyb-1:gfp VII. As a result, introns 7 an d 8 of dyb-1:gfp VII we re removed in t he se constructs. These construct were injected in dyb-1(cx36) mutants to assay their ability to re sc ue the mutant phenotype. Table 1. Rescuing activity o f Dyb-1 deletions. Constructs were injec ted in dyb-1(cx36) animals a long with the t ransformation marker KP13 [22]. dyb-1(cx36) animals display a behavioral phenotype consisting of hyperactivity, exaggerated be nding of the head w h en moving forward , and a tendency to hyp ercontract when m oving backwards. + + +, t ransgenic animals n ot distinguishable from wild-type animals; + +, transgenic animals resemble w ild-type but remain slightly hyperactive and bend their h ead more than wild-type; +, transgenic animals remain hyperactive and bend their h ead, but can be distingu ished from nontransgenic siblings in blind tests; ±, some t ransgenic animals show a slightly improved behavior but transgenics cannot be reco gnized with certainty in b lind tests; –, no m odification of the phenotype c ou ld be observed. Construct Number of transgenic lines Number of rescuing lines Rescue in best line(s) dyb-1:gfp VII 3 2 + + + AN450 in dyb-1:gfp VII 3 2 + + 2¢5 in dyb-1:gfp VII 5 1 ± 5¢1 in dyb-1:gfp VII 6 0 – 5¢2B in dyb-1:gfp VII 4 0 – 5¢5B in dyb-1:gfp VII 8 0 – 6¢4 in dyb-1:gfp VII 4 3 + Ó FEBS 2002 Dystrophin–dystrobrevin interactions in C. elegans (Eur. J. Biochem. 269) 1611 5¢2B) had no or very limited functional a ctivity in t he worm. Howev er, the 6¢4 construct, which removes the syntroph in- binding re gion and most of t he fi rst helix, continued to s how full binding activity. The results obtained in vivo by transgenesis in C. elegans are in agreement with the yeast results. Although t he 6¢4 deletion was still partly fu nctional, only very w eak activity could b e observed with d eletion 2¢5. In conclusion, the results presented in this paper show that the s econd helix of the coiled-coil domain of Dyb-1 is necessary for binding to Dys-1 both in vitro and in vivo. 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