Skip Navigation


Human Molecular Genetics Advance Access originally published online on February 12, 2004
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/7/693    most recent
ddh087v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (25)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ishikawa-Sakurai, M.
Right arrow Articles by Ozawa, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ishikawa-Sakurai, M.
Right arrow Articles by Ozawa, E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, 2004, Vol. 13, No. 7 693-702
DOI: 10.1093/hmg/ddh087

ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to ß-dystroglycan

Michiko Ishikawa-Sakurai1, Mikiharu Yoshida1, Michihiro Imamura1, Kay E. Davies2 and Eijiro Ozawa1,*

1Department of Cell Biology, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi-chou, Kodaira, Tokyo 187-8502, Japan and 2MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK

Received November 13, 2004; Accepted February 2, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An intracellular protein, dystrophin, plays an important role in keeping muscle fibers intact by binding at its N-terminal end to the subsarcolemmal cytoskeletal actin network and via its C-terminal end to the transmembraneous protein ß-dystroglycan. Duchenne muscular dystrophy is caused by the loss of dystrophin, which can result from the loss of this binding. The N-terminal part of the latter binding site of dystrophin has been well documented using overlay assay and X-ray diffraction assays. However, the binding site at the C-terminal region of dystrophin has not been examined in detail. In the present work, we report a detailed analysis of the C-terminal binding domain as follows. (1) The full binding activity corresponding to the effective binding in vivo is expressed by the dystrophin fragment spanning amino acids 3026–3345 containing the ZZ domain at the C-terminus. Determination of this binding range is important not only for understanding of the mechanism of dystrophy, but also useful for the design of truncated dystrophin constructs for gene therapy. (2) The ZZ domain binds to EF1 domain in the dystrophin fragment to reinforce the binding activity. (3) The cysteine 3340 in the ZZ domain is essential for the binding of dystrophin to ß-dystroglycan. A reported case of DMD due to missense mutation C3340Y may be caused by inability to fix dystrophin beneath the cell membrane. (4) The binding mode of utrophin is different from that of dystrophin. The difference is conspicuous concerning the cysteine residues present in the ZZ domain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dystrophin is a long slender protein with a molecular mass of 427 kDa and composed of four domains: the actin-binding, rod, cysteine-rich and C-terminal domains (1). Utrophin is the autosomal homolog of dystrophin having a molecular mass of 395 kDa and is similarly divided into four domains (2). Its cysteine-rich domain is extraordinarily highly homologous to the corresponding domain of dystrophin (3). Both dystrophin and utrophin are located beneath the sarcolemma. Dystrophin is distributed all over the cell membrane, whereas most utrophin is located in the junctional area. Dystrophin and the dystroglycan complex, which are collectively called the ‘dystrophin’ bolt (4,5), transmembranously connect the actin network and laminin, a major component of the basal lamina (6–9). The fixation of the basal lamina and the actin network by the dystrophin bolt is considered to be essentially important for the protection of the fragile muscle cell membrane composed of the lipid bilayer from damage due to repeated muscle contractions. It has long been known by gene analysis that out-of-frame deletions and nonsense mutations of dystrophin gene cause loss of C-terminal parts of dystrophin molecule. Today, nonsense-mediated decay of the mutant dystrophin mRNA is considered to be another cause of loss of dystrophin (10). Presumably, both mechanisms are working. The mdx mouse with a nonsense point mutation in its dystrophin gene, thus losing dystrophin from the sarcolemma, is widely used for the studies of the pathogenesis of dystrophin deficiency disorders (11).

In the early days of dystrophin study, we obtained a 31 kDa fragment of dystrophin keeping a firm binding to dystrophin-associated glycoproteins from rabbit normal skeletal muscle (7). Based on this finding, we considered this fragment includes the physiological binding site of dystrophin. Then, we showed using overlay-assay method a direct binding to ß-dystroglycan of the dystrophin fragment (amino acid acid numbers 3026–3442) that mainly spans the cysteine-rich domain and the first half of the C-terminal domain (12). We also found that the fragments (amino acids 3026–3264) obtained by a deletion of the C-terminal side of the above fragment showed a reduced binding. On these bases, details of the binding have been studied in various laboratories (13,14, reviewed in 15).

In the N-terminus of this region, the sequence (amino acids 3055–3092) applicable to the WW domain was found (16,17). The WW domain contains two highly conserved tryptophan (W) residues spaced 21 amino acids apart and binds to another protein having a consensus ligand sequence, PPXY. Since the sequence composed of 15 C-terminal residues of ß-dystroglycan was shown to be responsible for its binding to dystrophin (13) and PPPY, which corresponds to the consensus sequence, was found in the extreme C-terminal sequence of ß-dystroglycan, it was naturally assumed that the WW domain was responsible for the binding. On the other hand, it was indicated that the presence of only the WW domain was insufficient for the binding (18), and the long flanking polypeptide sequence tandem connected to the WW domain was necessary (13).

Recently, it has been indicated by the analysis of a predicted higher-order structure that the polypeptide sequence of the dystrophin WW domain was different from those of proteins that do not require additional polypeptides for their binding with partners (19). Moreover, the crystal structure of a protein complex composed of a dystrophin fragment (amino acids 3046–3306) and a ß-dystroglycan C-terminal polypeptide was analyzed by an X-ray diffraction technique, and the mechanism by which the domains following the WW domain participate in the binding of ß-dystroglycan was shown at the molecular level (20). These studies revealed the sophistication of the structure of the binding of dystrophin to ß-dystroglycan. However, there are issues that should be clarified.

Following our initial study of the dystroglycan binding site on dystrophin (7), it was found using in vitro overlay binding assay (12) that the binding activity increases stepwise when the C-terminal polypeptide of the binding region is elongated. More concretely, the dystrophin fragment composed of amino acids 3026–3442 showed an activity of binding to ß-dystroglycan nearly the same as that of the full-length C-terminal fragment (amino acids 3026–3685), whereas that of amino acids 3026–3264 showed a markedly reduced activity. The fragment that showed the full binding activity included the sequence amino acids 3307–3354 that was later identified to be the ZZ domain (21). In contrast, the fragment used for X-ray diffraction study (amino acids 3046–3306) did not contain the ZZ domain (20), although it contained the fragment that showed reduced binding activity. Our finding of the presence of two types of binding activity, which was reproduced in another laboratory (13), raised questions regarding the mechanism by which the binding reinforcement due to the elongation of the polypeptide is accomplished. In addition, one crucial problem emerged during these studies, namely, whether or not the reduced binding is enough to make an effective binding of dystrophin to ß-dystroglycan in vivo. This must be determined, because this is necessary not only for understanding DMD pathomechanisms but also for design of dystrophin cDNA construct for gene therapy of dystrophinopathy.

To clarify these issues, detailed analyses were conducted of the binding of dystrophin to ß-dystroglycan using an overlay assay and the minimum size determined of an extra sequence necessary for the full binding activity. The results demonstrate that the ZZ domain, which is truncated from the original one by nine C-terminal amino acids (21), is essential for dystrophin to obtain the full binding activity and that its role is binding reinforcement by intramolecular interaction with the first EF hand-like structure termed EF1 (20). The comparison of results of our in vitro experiments with those of in vivo experiments by the use of transgenic mice (14) suggests that the full binding activity is necessary for the effective binding. Thus, these results also suggest that the reported crystal structure (20) does not span the whole region for the effective binding. The binding of utrophin to ß-dystroglycan was similarly examined. As expected, the ZZ domain of utrophin was found to also be essential for the binding, but in other respects its binding characteristics seem to be slightly different from those of dystrophin.

Because it has previously been shown that some cysteine residues in dystrophin are obligatory for the binding (12) and the ZZ domain contains many cysteine residues (21), the effect of replacement of each cysteine residues in the ZZ domain was analyzed using a substitution technique. This clarified that the reported case of a missense mutation present in the ZZ domain responsible for DMD (22) prevented the binding. This may be the first case where the pathomechanisms of DMD due to missense mutation were explained at the protein level.

In the present study, some differences were noticed between the modes of dystropin and utrophin fragments. Thus, it was also examined whether the binding of utrophin to ß-dystroglycan is the same as that of dystrophin in vitro.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Terminology and definition
The binding of recombinant dystrophin and utrophin fragments to ß-dystroglycan prepared from muscle cell membrane was analyzed by overlay binding assay. For this purpose, we constructed several dystrophin and utrophin fragments. The largest fragment spans the C-terminal short sequence of the rod domain, the cysteine-rich domain and the first half of the C-terminal domain of dystrophin, and another fragment spans the full-length C-terminal sequence of utrophin starting from the C-terminal short sequence of the rod domain. We described fragments using a string name composed of an initial letter ‘D’ for dystrophin or ‘U’ for utrophin, and the amino acid number corresponding to both N and C termini of each fragment. By this terminology, D3026–3442 means a dystrophin fragment spanning amino acid residues from 3026 to 3442.

In Figure 1A, is shown schematically functional subdomains of dystrophin (20), which start from the C-terminal short sequence of the rod domain to the following cysteine-rich domain. These terminologies of the subdomains are similar to those used by others (1), although defined differently. Koenig et al. (1) used the term ‘the first and second EF hand-like domains’ to indicate two small domains that have similar sequences to those of the authentic EF hand, whereas Huang et al. (20) called these domains the first EF hand-like domain (EF1), and its subsequent region the second EF hand-like domain (EF2), despite the dissimilarity in the sequence. Here, we followed the terminologies of Huang et al. for ease in the comparison of our results with theirs. In dystrophin, the WW domain spans amino acids 3046–3084, and EF1 and EF2 span amino acids 3125–3207 and amino acids 3208–3290, respectively (Table 1) (20). The ZZ domain of dystrophin was originally defined to span amino acids 3307–3354 (21). For our present purpose, however, we define the ZZ domain to span amino acids 3307–3345 (Table 1).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 1. Analysis of ß-dystroglycan-binding regions of dystrophin and utrophin. (A) Top: schematic representation of the domain structures of dystrophin and utrophin. The C-terminal region spanning from the last small sequence of the rod domain to the cysteine-rich domain (CR) contains four functional subdomains. WW, the WW domain; EF1 and EF2, the first and the second EF hand-like domains (20); ZZ, the ZZ domain. Bottom: the dystrophin constructs used in this study are presented as bold lines. The names of the polypeptide fragments are indicated on the right side of lines and the subsequent letters in parentheses F, R and N indicate the full, reduced and no binding activities of the corresponding fragments, respectively. (B) Binding of dystrophin fragment to ß-dystroglycan shown by in vitro overlay binding assay. Purified complex composed of dystrophin and its associated proteins (0.7 µg) is loaded to each lane. The names of the fragments are indicated on the top of lanes. An arrowhead on the left side of lanes indicates the position of ß-dystroglycan. (C) The utrophin constructs used in this study are presented as bold lines. In U2783–3069, but we depicted its activity with N*, since this protein only showed a trace activity. (D) Binding of utrophin fragment to ß-dystroglycan shown by in vitro overlay binding assay.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Alignment of amino acid sequences in subdomains of dystrophin and utrophin
 
Determination of the ß-dystroglycan binding region of dystrophin
All the fragments used in the present study did not bind to the dystrophin-associated proteins other than ß-dystroglycan, as reported previously (data not shown) (12). We previously reported that the binding activity of D3026–3442 (the full binding activity) decreased when its C terminus was deleted to form D3026–3264 (the reduced activity) (12). In the present study, we found that D3026–3345, prepared by a C-terminal 98-amino-acid deletion from D3026–3442, retained its full binding activity (Fig. 1B). On the other hand, D3026–3324, prepared by a C-terminal 22-amino-acid deletion from D3026–3345, showed a reduced binding activity that was the same as that of D3026–3264 (Fig. 1B). Therefore, D3026–3345 is the minimal fragment showing the full binding activity and the last small sequence of D3026–3345 seems to be critical for the full binding activity. D3026–3232 prepared from a C-terminal 33-amino-acid deletion from D3024–3264 did not show any binding activity (Fig. 1B). Therefore, D3026–3264 is the minimal fragment showing the reduced binding activity.

D3026–3105 spanning the WW domain and D3095–3345, which was D3026–3345 lacking the WW domain, showed no binding activity. The following fragments also showed no binding activities: D3026–3264{Delta}3121–3188, which was D3026–3264 with the deleted internal region amino acids 3121–3188 spanning most of EF1 (Fig. 1B); D3120–3312, spanning EF1 and EF2 but lacking both the WW and ZZ domains, and D3264–3345, which was the difference between two minimal fragments providing the full and reduced binding activities and thus tentatively termed D{Delta}F. The results are summarized in Figure 1A These results are compatible with those of previous studies (12,13,23).

In an in vitro experiment, it was shown that the second dystrophin-binding sequence exists at a juxtamembrane region of ß-dystroglycan, and that it binds to dystrophin at the region amino acids 3090–3306 including EF1 (23). A similar result was reported in the utrophin study (24). Under our experimental conditions, however, we could not confirm these reports (Fig. 1).

Determination of the ß-dystroglycan binding region of utrophin
U2783–3102 corresponding to D3026–3345 bound to ß-dystroglycan to the same extent as U2783–3434, spanning the entire region from the WW domain to the C terminus (Fig. 1D). This fragment was thus considered to have the full binding activity of utrophin. On the other hand, the following fragments had no binding activities: U2783–2862, spanning the WW domain; U3021–3102, spanning U{Delta} F, which corresponds to D{Delta} F, and U2852–3102, which is the U2783–3102 lacking the WW domain. These results are almost compatible with those of Tommasi di Vignani et al. (25) but not necessarily compatible with those of Chung et al. (24).

U2783–3021, in contrast with the corresponding dystrophin fragment D3026–3264 having a reduced binding activity, did not bind to ß-dystroglycan (Fig. 1D). Among the fragments extending to the C-terminus, U2783–3069 showed faint activity, whereas U2783–3081 showed nearly full activity. Thus, in utrophin, the region providing the reduced activity observed in dystrophin is not clearly defined. The results are summarized in Figure 1C.

Intermolecular interaction of EF1 with the subsequent domains of dystrophin and utrophin
The reduced binding activity of D3026–3264 was reinforced by the elongation of the polypeptide termed D{Delta} F. To understand this mechanism, we examined whether an intramolecular interaction between D{Delta} F and preceding regions may exist in dystrophin. For this purpose we constructed the fragments corresponding to D{Delta} F and the ZZ domain (D3310–3345 or D-ZZ) as probes, and various fragments spanning preceding domains as their putative receptors (Fig. 2A). These tested fragments were produced as GST-fusion proteins and the GST tag was then removed by thrombin cleavage so as to avoid misinterpretation of results due to a possible interaction between GST tags.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 2. Specific binding of ZZ domain-containing fragments to other regions of dystrophin and utrophin. (A) Top: schematic representation of the domain structures as in Figure 1A. Bottom: the dystrophin fragments containing the domains preceding the ZZ domain are presented as bold lines and the ZZ domain-containing fragments are presented as open bars. D{Delta}F, D3264–3345; D-ZZ, D3310–3345; D{Delta}F*, D3264–3345 with C3337Y mutation; D{Delta} F**, D3264–3345 with C3340Y mutation. (B) Binding of D{Delta}F (with or without mutations) and the ZZ domain fragment of dystrophin (D-ZZ) to various fragments tested for a putative receptor. The [32P]-labeled fragments D{Delta}F, D-ZZ, D{Delta} F* and D{Delta} F** were overlaid onto various dystrophin fragments. The names of fragments tested are indicated on the left side of lanes. CBB means the Coomassie brilliant blue-staining patterns of tested fragments. (C) The utrophin fragments containing the domains preceding the ZZ domain are presented as bold lines and the ZZ domain-containing fragments are presented as open bars. U{Delta} F, U3021–3102; U-ZZ, U3067–3102; U{Delta} F{dagger}, U3021–3102 with C3076Y mutation; U{Delta} F{dagger}{dagger}, U3021–3102 with C3097Y mutation. (D) Binding of U{Delta}F (with or without mutations) and the ZZ domain fragment of utrophin (U-ZZ) to various fragments tested. The figure is depicted similarly as (B).

 
We first examined [32P]-labeled D3264–3345 spanning D{Delta}F. This was overlaid onto various dystrophin fragments immobilized onto a membrane (Fig. 2B). EDTA was included in the reaction mixture to exclude the possible role of divalent cations such as Ca2+ or Zn2+. This probe bound to the EF1-containing fragments D3026–3264 and D3095–3183, but not to the fragments without EF1, namely, D3026–3105, D3227–3312 and D3026–3264{Delta}3121–3188. Consistent results were obtained using [32P]-labeled D-ZZ as a probe.

Similar experiments were also performed using utrophin fragments (Fig. 2C). [32P]-labeled U3021–3102 spanning U{Delta} F bound to the EF1-containing U2852–2940 fragment, but not to the EF1-free U2783–2862 fragment (Fig. 2D). Consistent results were obtained with [32P]-labeled U3067–3102 spanning the ZZ domain (U-ZZ) as a probe.

Effects of mutations in the ZZ domain of dystrophin on the full binding activity to ß-dystroglycan
To further examine the role of the ZZ domain, we prepared the D3026–3345 mutants, in which each of the cysteine or asparagine residues present in the ZZ domain was replaced with another amino acid by site-directed mutagenesis (Fig. 3A). These experiments were related to our previous report that N-ethylmaleimide, a specific modifier of cysteine residue, inhibits the binding (12), and also related to clinical reports on missense mutations (22,26). The D3026–3345 mutants having the C3340Y or C3340S mutation showed null or markedly diminished binding activity, respectively (Fig. 3C). In contrast, all of the D3026–3345 mutants with C3313Y, C3316Y, C3319Y, C3337Y or D3335H mutation exhibited the same binding activity as that of the wild-type.



View larger version (48K):
[in this window]
[in a new window]
  		Figure 3. ß-Dystroglycan-binding activities of the dystrophin and utrophin fragments with a single amino acid replacement. (A, B) Top: amino acid representation of the ZZ domain of dystrophin (A) and utrophin (B). Amino acids that are different between them are asterisked. Bottom: the site of amino acid replacement and the name of mutation. (C, D) Activities of D3026–3345 (C) and U2783–3102 (D) with and without a mutation, respectively, binding to ß-dystroglycan shown by in vitro overlay binding assay. Purified complex composed of dystrophin and its associated proteins (0.7 µg) is loaded to each lane. The name of mutation is on the top of each lane. The position of ß-dystroglycan is indicated by an arrowhead.

 
To examine the influence of the mutation on the binding between EF1 and D{Delta} F, the D3264–3345 mutants D{Delta} F* and D{Delta} F** were prepared that have C3337Y and C3340Y mutations, respectively (Fig. 2A). Both [32P]-labeled probes bound to D3095–3183 to the extent similar to the wild-type D{Delta} F (Fig. 2B).

Effects of mutations in the ZZ domain of utrophin on binding to ß-dystroglycan
We also prepared the U2783–3102 mutants, in which each of the five cysteine residues present in the ZZ domain was alternately replaced with tyrosine to examine the effect of mutation on binding activity (Fig. 3B). C3097Y mutation of utrophin corresponding to C3340Y mutation of dystrophin did not affect the binding, as previously reported (25). C3094Y mutation also had no influence on the binding. In contrast to the case of dystrophin, C3070Y, C3073Y and C3076Y mutations, corresponding to dystrophin C3313Y, C3316Y and C3319Y mutations, respectively, markedly inhibited the binding (Fig. 3D).

The U3021–3102 mutants U{Delta}F{dagger} and U{Delta}F{dagger}{dagger}, having C3076Y and C3097Y mutations, respectively, and the wild-type U{Delta}F showed a similar binding activity to U2852–2940 (Fig. 2D).

Relative binding activities of dystrophin and utrophin to ß-dystroglycan
We compared the binding activities of D3026–3345 and U2783–3102 to ß-dystroglycan by the competitive binding assay. [32P]-labeled D3026–3345 was allowed to react with ß-dystroglycan blotted on the membrane in the presence of the unlabeled fragment of D3026–3345 or U2783–3102 as a competitor (Fig. 4). The concentrations causing a half-maximal binding of the labeled fragment were estimated and compared. From three independent experiments, the binding activity of D3026–3345 was evaluated to be 2.7, 2.0 and 1.9 times stronger than that of U2783–3102.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Relative ß-dystroglycan-binding activity of dystrophin and utrophin. Typical results are shown. [32P]-labeled D3026–3345 was allowed to react with ß-dystroglycan blotted on the membrane in the presence of the unlabeled fragment of D3026–3345 (•) or U2783–3102 ({circ}) as a competitor at increasing concentration. The concentration of the competitor causing a half-maximal binding of the labeled fragment was estimated. The ratio of the concentration of dystrophin to that of utrophin was calculated (described in the text).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Dystrophin has to be fixed beneath the sarcolemma by binding to both actin filament and ß-dystroglycan to prevent muscle fibers from dystrophic degeneration. Therefore, both binding domains of dystrophin are physiologically important. In the present work, we focused on the ß-dystroglycan binding.

In 1992, we obtained a 31 kDa dystrophin fragment keeping a firm binding to dystrophin-associated glycoproteins by the calpain digestion of the complex composed of dystrophin and its associated proteins prepared from the rabbit skeletal muscle. This was the first demonstration that the glycoprotein-binding site physiologically working is present in this fragment that spans mainly the cysteine-rich domain and the first half of the C-terminal domain (7). In 1994, we showed by overlay assay using truncated recombinant proteins of dystrophin that the D3026–3442 corresponding to the 31 kDa fragment directly binds to ß-dystroglycan. We further showed that the fragments D3026–3264 with a deletion of the C-terminal side of D3026–3442 showed reduced binding (12). On these bases, many reports from other laboratories have been made including the WW domain present at the N-terminal part of the binding site and details of this part by overlay assay and X ray analysis (12,13,20). Jung et al. (13) confirmed the results of our overlay assay and also showed that the binding site of ß-dystroglycan exists its C-terminal domain 15 amino acid residues, which includes PPPY, the binding counterpart consensus domain of WW domain. However, the border of the C-terminal side of dystrophin used for the effective binding has not been precisely determined and the nature of the C-terminal region has not been detailed. Accordingly, we examined the binding mode of dystrophin to ß-dystroglycan focusing our attention on the C-terminus of the binding site of dystrophin in this report. We confirmed and extended our previous findings that the dystrophin fragments D3026–3232 showed no binding activity despite the presence of the WW domain and its following sequences with 190 amino acid residues. The longer fragments with C-terminally extending sequences D3026–3264 and D3026–3324 showed the reduced activities and D3026–3345 the full activity (Fig. 1).

Our first question was whether or not the reduced binding is enough to make an effective binding of dystrophin to ß-dystroglycan in vivo. To answer this question is important not only to understand molecular pathogenesis of DMD but also to design cDNA constructs for gene therapy. For this purpose, we at first determined the minimum size of the fragments that show full binding activity. For the following discussion, we further define the difference (D{Delta}F) between two fragments showing the reduced and full activities, namely the difference of D3026–3264 and D3026–3345. This D{Delta}F includes most of the originally defined ZZ domain whose nature has not been clarified (21) at its C terminus. Originally, the ZZ domain was described to span amino acids 3307–3354, but here we call the sequence amino acids 3307–3345 the ZZ domain. We thus conclude that D{Delta}F or the ZZ domain is absolutely required for the full activity of binding to ß-dystroglycan.

Our in vitro studies provide the answer to this question by comparison with in vivo studies. Chamberlain's group prepared several transgene/mdx mice expressing various dystrophin cDNA constructs and showed that the dystrophic change of mdx muscle was rescued in some case but not in others (14,27). Because the binding of dystrophin to ß-dystroglycan is required to prevent the expression of the dystrophic phenotype, deletion models can be considered to exhibit effective and physiological binding activity in these mice. They showed that the full-length cDNA with deletion of exons 68–70 (Fig. 5B) failed to rescue the phenotype (14). Since this model is considered to be comparable to D3026–3264 with reduced binding activity, the pathology shown by these mice demonstrates that the reduced binding activity is not sufficient for the effective binding in vivo. Another model with deletion of exons 64–67 (Fig. 5B) also failed to rescue the phenotype. This model, which includes the WW domain encoded by exons 62–63 but lacks the subsequent EF1 and EF2, is compatible with previous reports that the WW domain alone is not sufficient for the effective binding to ß-dystroglycan (13,18). On the other hand, the model with deletion of exons 71–78 (Fig. 5B) could rescue the phenotype, suggesting that the C-terminal region encoded by these exons is not essential for the binding (27). All these result are compatible with our in vitro result that the C-terminal region spanning amino acids 3346 is dispensable for the binding.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. DGB domain of dystrophin. (A) Top: schematic representation of the domain structure of ß-dystroglycan-binding region of dystrophin as in Figure 1A. Bottom: the important regions discussed in the present study are drawn in bold lines. R-min, a minimal region showing a reduced binding activity; R-max, a maximal region showing a reduced binding activity; F-min, a minimal region showing a full binding activity; D{Delta}F, the difference between the minimal regions showing the full and the reduced binding activities; D-ZZ, the ZZ domain of dystrophin. ‘X-ray’ is the target region for X-ray analysis (20). (B) Comparison of the dystrophin fragments used in the present study with the deletion loci in the dystrophin cDNA mutants used in the transgene/mdx mice studies (14,27). (C) Proposed molecular model of the DGB domain composed of the WW, EF1, EF2 and ZZ domains. The linker sequence present between EF2 and the ZZ domain is tentatively termed FZ (see text). It should be noted that the ZZ domain non-covalently interacts with EF1.

 
On the basis of our present results for dystrophin and utrophin together with the report that the fragment of dystrophin homolog DRP2, which spans the region from the WW domain to the ZZ domain, binds to ß-dystroglycan (28), and also with the results of Chamberlain's group described above, we consider that members of these dystrophin families require the region having the same domain structures for their binding to ß-dystroglycan. Therefore, we consider that the entire region from the WW domain to the ZZ domain forms one functional unit and is required for the binding in vivo. The WW domain spans a region from the terminal part of the rod domain to the initial part of the cysteine-rich domain, and the ZZ domain resides in the terminal part of the cysteine-rich domain. Thus, this does not coincide with any single domain, originally defined by Kunkel's group. Since no terminology has yet been coined for this functional unit, we propose to call this region the dystroglycan-binding (DGB) domain. Using this proposed terminology, we can avoid the discordance between the standard classification of domains and this functional unit.

A crystal structure of the complex composed of the C-terminal polypeptide of ß-dystroglycan (K881–Y895) and the dystrophin fragment composed of the WW domain and the subsequent EF1 and EF2 were elegantly described, and various interesting inter- and intramolecular interactions were shown (20). The interaction by which the hydrogen bond is formed via a water molecule between R3246 on EF2 and Y886 on the dystroglycan molecule may be very important, because arginine is present in the critical region the removal of which from D3026–3264 leads to a change from a reduced to null activity. Unfortunately, however, the fragment used in the X-ray analysis was amino acids 3046–3306. It is positioned between D3026–3264 and D3026–3324, both of which have a reduced binding activity and did not contain the whole DGB domain. Thus, further structural analysis is needed for elucidating the structure of DGB domain.

The second question was what the role is of the region that was not examined by X-ray analysis (20). We found that extending the region resulted in the full binding activity, suggesting that the ZZ domain is required for reinforcing the activity. We found by probe overlay assay that D{Delta}F and the ZZ domain bind to EF1. Based on these results, we slightly modified the three-dimentional structural model proposed based on X-ray analysis as schematically shown in Figure 5B. In this model, the region from the WW domain to most of EF2 forms the tertiary structure as observed by X-ray analysis, but the rest of EF2 and some flanking polypeptides before the ZZ domain (FZ in Fig. 5) turns back enabling the ZZ domain to bind to EF1. We are of the opinion that a conformational change induced by the interaction between EF1 and the ZZ domains is necessary to acquire the full binding activity. Further structural analysis of the dystrophin fragment spanning the region from the WW domain to the ZZ domain may clarify the binding mechanism.

We previously reported that N-ethylmaleimide, a specific modifier of the cysteine residue, greatly reduced the binding activity of dystrophin (12). This suggests that at least one of the 15 cysteine residues present in the cysteine-rich domain of dystrophin has some role in its binding to ß-dystroglycan. Our third question was which cysteine residue present in the ZZ domain is essential for the binding? Further, is there any relation of the cysteine mutation in the ZZ domain to missence mutation causing DMD? The present mutation analysis of the five cysteine residues in the ZZ domain indicated the importance of C3340, because the replacement of this residue with tyrosine or serine residue blocked the binding of dystrophin to ß-dystroglycan.

It has been reported that a missense mutation of G10227A, that gives rise to C3340Y mutant dystrophin, caused DMD, mental retardation and absence of ERG b-wave (22). This patient showed calf enlargement and Gowards' sign with high serum creatine kinase activity. He became wheelchair bound at the age of 9 years old, indicating severity of the phenotype. Although residual amount of 427 kDa dystrophin was detected in both immunoblot and immunohistochemical analysis, the muscle showed degenerative changes. This suggests dystrophin does not work in the patient's muscle. Based on our present results, it is most likely that the C3340Y mutation resulted in dissociation of dystrophin and ß-dystroglycan, which gives rise to DMD phenotype. A residual amount of dystrohin is sometimes observed in DMD. Even in complete absence of distal parts of dystrophin, including the C-terminal region of rod, cysteine-rich and C-terminal domains, the presence of normal amount of dystrophin has been observed in some cases (29–31). Since dystrophin binds to actin-filaments at its actin-binding domain and probably also at the actin-binding site present on rod (32,33), some dystrophin molecules may be fixed beneath the sarcolemma detectable by immunohistochemistry, although it is not functional for preventing muscular dystrophy. Another missense mutation D3335H in the ZZ domain of dystrophin was reported to cause DMD (26), but under our experimental conditions the activity of binding to ß-dystroglycan was not disrupted by the introduction of the same mutation to the dystrophin construct. A replacement of cysteine at amino acids 3313 with phenylalanine by a missence mutation in DMD patient was recently reported (34). As far as we observed by replacement of this cysteine with tyrosine, the importance of the cysteine residue for the binding was not detected. Since the mutation did not affect the intramolecular interaction between EF1 and the ZZ domain, some unknown role of the ZZ domain other that one described above may work in these cases.

The fourth question is whether the binding mode of dystrophin and that of utrophin is completely the same or not, because their cysteine-rich domains are extremely homologous (3). There were some differences between dystrophin and utrophin in the binding mode of the ZZ domain. U2783–3102 spanning the entire region from the WW domain to the ZZ domain of utrophin showed the full activity of binding to ß-dystroglycan. In contrast to dystrophin, further truncation of this utrophin fragment from the C terminus inactivated the binding rather steeply and the phase at which reduced binding was observed in dystrophin was not clearly observed. Whereas the intramolecular interaction between EF1 and the ZZ domain was also detected in utrophin, observed effects of mutations in the ZZ domain on binding to ß-dystroglycan were different between dystrophin and utrophin. These results may seem unusual, because the sequence similarity between D{Delta}F and the corresponding region of utrophin U{Delta}F is as high as 94% (homology 88%) and particularly in the ZZ domain, only two valine residues were replaced with isoleucine (3) (Fig. 3A and B). However, we do not necessarily consider these results unusual for the following reasons. One is that both the EF1 of dystrophin and utrophin, the binding partners of the ZZ domain, are somewhat different, that is, they have lower similarity (77% similar, 65% homology) than their surrounding regions. The other reason is that valine and isoleucine are similar in their electrical properties, but the former is less bulky than the latter. The tertiary structure may be changed, if small, by replacing two valine residues separated by 13 intervening amino acids with isoleucine, because of the difference in the degree of rotational freedom in the backbone structure, giving rise to difference in the binding mode.

In the present study, it was demonstrated that utrophin binds to ß-dystroglycan more weakly than dystrophin by about 2-fold. Although it is known that forced upregulation of utrophin in the skeletal muscle can prevent muscular dystrophy (35,36), our result will not contradict these previous reports if the number of molecules expressed is taken into consideration. It was previously found that utrophin is up-regulated on sarcolemma in mdx and DMD muscles (37,38) and that ß-dystroglycan is greatly reduced but distinctly present in DMD muscles (39). These findings lead us to assume that up-regulated utrophin in DMD may serve to replace dystrophin, keeping the cell membrane stable to some extent but the number of utrophin molecules that fix to sarcolemma is much lower than that of dystrophin in normal muscle. It was then assumed that, in DMD muscle, a reduced number of connections is present between the subsarcolemmal actin filaments and laminin molecules in the basal lamina via dystroglycan complex, and that decrease of the connections may be the reason why DMD muscles finally succumb to the disease, in spite of the presence of utrophin underneath the sarcolemma (for review see 4). The idea that utrophin serves to functionally replace dystrophin was further supported by the findings that the utrophin–dystrophin double-deficient mice show more severe phenotypes than mdx mice, although utrophin-deficient mice scarcely showed pathological phenotypes (40,41). Our present results further suggested that the connection is inferior not only in its number but also in strength in DMD muscles.

Furthermore, transgenic forced expression of utrophin corrects pathological changes of mdx mice (35,36), including strong mechanical coupling between subsarcolemmal actin-association and the sarcolemma (42). It is noteworthy that utrophin is overexpressed in these transgene/mdx mice as expected in transgene mice. In these transgene muscles, the number of utrophin molecules expressed is fairly large and thus a large number of molecular connections between actin filament and ß-dystroglycan are formed. Presumably, this increase in the connection may be large enough to prevent the dystrophic process in the muscle fiber. Therefore, the decreased affinity of utrophin to ß-dystroglycan is not incompatible with the previous results.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Expression and purification of dystrophin and utrophin constructs as GST-fusion proteins
Human dystrophin (GenBank accession number M18533) cDNA constructs were generated by polymerase chain reaction (PCR) using the pGEX-3X/DCT685 expression vector (12) as a template. Human utrophin (GenBank accession number X69086) cDNA constructs were also amplified by PCR using pGEX-2TK/UCT434 as a template. Amplified PCR fragments were ligated into pGEX-2TK expression vectors (Amersham Biosciences, Tokyo, Japan) to express recombinant proteins as glutathione S-transferase (GST) fusion proteins with an artificially tagged phosphorylation site. The cDNA constructs are shown in Figure 1 schematically. The site-directed mutagenesis of a cDNA construct was performed using a Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The sequences of oligonucleotide primers containing the desired mutation (underlined) are as follows: for dystrophin mutation, C3313Y (5'-CTC TTT GCA GAT GTT ATA TTT GGC CTG ATG CTT G-3'), C3316Y (5'-GAT TGG ACA CTC TTT GTA GAT GTT ACA TTT GGC C-3'), C3319Y (5'-CCT GAA TCC AAT GAT TGG ATA CTC TTT GCA GAT G-3'), C3337Y (5'-AAA AAA GCA GCT TTG GTA GAT GTC ATA ATT AAA GTG-3'), C3340Y (5'-TCG ACC AGA AAA AAA GTA GCT TTG GCA GAT-3'), C3340S (5'-TCG ACC AGA AAA AAA GGA GCT TTG GCA GAT-3'), and D3335H (5'-TCG ACC AGA AAA AAA GCA GCT TTG GCA GAT GTG ATA ATT-3'); for utrophin mutation, C3070Y (5'-C TTT ACA GAT GTT GTA TTT GGC CTG ATG TTT TGC AGT CTC-3'), C3073Y (5'-CC GAC AAT TGG ACA TTC TTT ATA GAT GTT GCA TTT GGC CTG-3'), C3076Y (5'-CCC GAC AAT TGG ATA TTC TTT ACA GAT GTT GC-3'), C3094Y (5'-AAA GAA ACA ACT CTG GTA GAC ATC ATA GTT A-3'), and C3097Y (5'-TCG ACC CGA AAA GAA ATA ACT CTG GCA GAC-3'). Each of the nucleotide sequences of dystrophin and utrophin cDNA constructs was confirmed by dideoxynucleotide chain-termination method.

GST-fusion proteins were expressed in protease-lacking Escherichia coli strain BL21, and chromatographically purified at 4°C using the glutathione–Sepharose 4B and Superose 12 HR10/30 columns (Amersham Biosciences), as described previously (12,43). When necessary, the GST tag was removed from fusion proteins by thrombin cleavage in accordance with the manufacturer's instructions (Amersham Biosciences). All of these fusion proteins were identified electrophoretically based on their molecular weights. Fusion proteins were enzymatically labeled with [{gamma}-32P]ATP in accordance with the manufacturer's instructions (Amersham Biosciences). The concentrations of proteins were determined using a Coomassie plus protein assay reagent (Pierce, Rockford, IL, USA) with bovine serum albumin as a standard.

In vitro overlay binding assay
The overlay binding assay was performed according to previous studies(12,43)with a slight modification. The complex composed of dystrophin and its associated proteins, which was purified from rabbit skeletal muscle (44), was electrophoretically separated on SDS–polyacrylamide gel and the separated products were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). These membranes were blocked with 0.1% each of casein and gelatin for 30 min at room temperature and overlaid with a GST-fusion protein at 30 µg/ml in a reconstitution buffer [20 mM HEPES (pH 7.5), 0.15 M NaCl, 2 mM MgCl2, 1 mM dithiothreitol, 2 µg/ml leupeptin, 0.5 µg/ml aprotinin, 1 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride] for 2 h at room temperature. After the membranes were washed thoroughly with a washing buffer (phosphate-buffered saline containing 0.1% Tween 20), bound fusion proteins were detected with anti-GST antiserum, followed by the horseradish peroxidase-conjugated anti-rabbit IgG antibody (Bethyl Laboratories, Montgomery, TX, USA). These blots were detected with an enhanced chemiluminescence system (Amersham Biosciences).

In some studies, [32P]-labeled fusion proteins were used for the probe. In these cases, the membranes were overlaid with [32P]-labeled fusion proteins in the buffer [10 mM HEPES (pH 7.5), 0.15 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 2 µg/ml leupeptin, 0.5 µg/ml aprotinin, 1 mM benzamidine and 0.1 mM phenylmethylsulfonyl fluoride] at 1 µg/ml for 2 h, then thoroughly washed with the washing buffer. After the membranes were rinsed with milli-Q water and completely dried, radioactivity of a bound fusion protein was detected using a BAS5000 bioimaging analyzer (Fuji Photo Film, Tokyo, Japan). For the competitive binding assay, the membranes were overlaid with [32P]-labeled D3026–3345 at 1 µg/ml in the presence of unlabeled D3026–3345 or U2783–3102 as a competitor at increasing concentration. The concentration of unlabeled competitor causing 50% inhibition of the probe was estimated and the ratio of concentration of U2783–3102 to that of D3026–3345 was calculated.

Antibody
In accordance with the method previously described (45), a polyclonal anti-GST antibody was raised in rabbits against the synthetic polypeptide corresponding to the C-terminal sequence (SCQGW QATFG GGDHP PK) of Schistosoma japonicum GST (46) coupled to keyhole-limpet hemocyanin at the internal cysteine.


   ACKNOWLEDGEMENTS
 
This work is supported by the Research Grants (14B-4) for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, Japan.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +81 423412712 ext. 5266; Fax: +81 423461741; Email: ozawa{at}ncnp.go.jp


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Koenig, M., Monaco, A.P. and Kunkel, L.M. (1988) The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell, 53, 219–226.[CrossRef][Web of Science][Medline]

  2. Tinsley, J.M., Blake, D.J., Zuelling, R.A. and Davies, K.E. (1994) Increasing complexity of the dystrophin-associated protein complex. Proc. Natl Acad. Sci. USA, 91, 8307–8313.[Abstract/Free Full Text]

  3. Tinsley, J.M., Blake, D.J., Roche, A., Fairbrother, U., Riss, J., Byth, B.C., Knight, A.E., Kendrick-Jones, J., Suthers, G.K., Love, D.R., Edwards, Y.H. and Davies, K.E. (1992) Primary structure of dystrophin-related protein. Nature, 360, 591–592.[CrossRef][Medline]

  4. Ozawa, E., Yoshida, M., Suzuki, A., Mizuno, Y., Hagiwara, Y. and Noguchi, S. (1995) Dystrophin-associated proteins in muscular dystrophy. Hum. Mol. Genet., 4, 1711–1716.[Abstract]

  5. Ozawa, E., Nishino, I. and Nonaka, I. (2001) Sarcolemmopathy: muscular dystrophies with cell membrane defects. Brain Pathol., 11, 218–230.[Web of Science][Medline]

  6. Way, M., Pope, B., Cross, R.A., Kendrick-Jones, J. and Weeds, A.G. (1992) Expression of the N-terminal domain of dystrophin in E. coli and demonstration of binding to F-actin. FEBS Lett., 301, 243–245.[CrossRef][Web of Science][Medline]

  7. Suzuki, A., Yoshida, M., Yamamoto, H. and Ozawa, E. (1992) Glycoprotein-binding site of dystrophin is confined to the cysteine-rich domain and the first half of the carboxy-terminal domain. FEBS Lett., 308, 154–160.[CrossRef][Web of Science][Medline]

  8. Yoshida, M., Suzuki, A., Yamamoto, H., Noguchi, S., Mizuno, Y. and Ozawa, E. (1994) Dissociation of the complex of dystrophin and its associated proteins into several unique groups by n-octyl ß-D-glucoside. Eur. J. Biochem., 222, 1055–1061.[Web of Science][Medline]

  9. Ibraghimov-Beskrovnaya, O., Ervasti, J.M., Leveille, C.J., Slaughter, C.A., Sernett, S.W. and Campbell, K.P. (1992) Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature, 355, 696–702.[CrossRef][Medline]

  10. Kerr, T.P., Sewry, C.A., Robb, S.A. and Roberts, R.G. (2001) Long mutant dystrophins and variable phenotypes: evasion of nonsense-mediated decay? Hum. Genet., 109, 402–407.[CrossRef][Web of Science][Medline]

  11. Sicinski, P., Geng, Y., Ryder-Cook, A.S., Barnard, E.A., Darlison, M.G. and Barnard, P.J. (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science, 244, 1578–1580.[Abstract/Free Full Text]

  12. Suzuki, A., Yoshida, M., Hayashi, K., Mizuno, Y., Hagiwara, Y. and Ozawa, E. (1994) Molecular organization at the glycoprotein-complex-binding site of dystrophin. Three dystrophin-associated proteins bind directly to the carboxy-terminal portion of dystrophin. Eur. J. Biochem., 220, 283–292.[Web of Science][Medline]

  13. Jung, D., Yang, B., Meyer, J., Chamberlain, J.S. and Campbell, K.P. (1995) Identification and characterization of the dystrophin anchoring site on ß-dystroglycan. J. Biol. Chem., 270, 27305–27310.[Abstract/Free Full Text]

  14. Rafael, J.A., Cox, G.A., Corrado, K., Jung, D., Campbell, K.P. and Chamberlain, J.S. (1996) Forced expression of dystrophin deletion constructs reveals structure-function correlations. J. Cell Biol., 134, 93–102.[Abstract/Free Full Text]

  15. Winder, S.J., Gibson, T.J. and Kendrick-Jones, J. (1995) Dystrophin and utrophin: the missing links! FEBS Lett., 369, 27–33.[CrossRef][Web of Science][Medline]

  16. Andre, B. and Springel, J.Y. (1994) WWP, a new amino acid motif present in single or multiple copies in various proteins including dystrophin and the SH3-binding Yes-associated protein YAP65. Biochem. Biophys. Res. Commun., 205, 1201–1205.[CrossRef][Web of Science][Medline]

  17. Bork, P. and Sudol, M. (1994) The WW domain: a signalling site in dystrophin? Trends Biochem. Sci., 19, 531–533.[CrossRef][Web of Science][Medline]

  18. Chen, H.I. and Sudol, M. (1995) The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. Proc. Natl Acad. Sci. USA, 92, 7819–7823.[Abstract/Free Full Text]

  19. Zarrinpar, A. and Lim, W.A. (2000) Converging on proline: the mechanism of WW domain peptide recognition. Nat. Struct. Biol., 7, 611–613.[CrossRef][Web of Science][Medline]

  20. Huang, X., Poy, F., Zhang, R., Joachimiak, A., Sudol, M. and Eck, M.J. (2000) Structure of a WW domain containing fragment of dystrophin in complex with beta-dystroglycan. Nat. Struct. Biol., 7, 634–638.[CrossRef][Web of Science][Medline]

  21. Ponting, C.P., Blake, D.J., Davies, K.E., Kendrick-Jones, J. and Winder, S.J. (1996) ZZ and TAZ: new putative zinc fingers in dystrophin and other proteins. Trends Biochem. Sci., 21, 11–13.[CrossRef][Web of Science][Medline]

  22. Lenk, U., Oexle, K., Voit, T., Ancker, U., Hellner, K.A., Speer, A. and Hubner, C. (1996) A cysteine 3340 substitution in the dystroglycan-binding domain of dystrophin associated with Duchenne muscular dystrophy, mental retardation and absence of the ERG b-wave. Hum. Mol. Genet., 5, 973–975.[Abstract/Free Full Text]

  23. Rentschler, S., Linn, H., Deininger, K., Bedford, M.T., Espanel, X. and Sudol, M. (1999) The WW domain of dystrophin requires EF-hands region to interact with beta-dystroglycan. Biol. Chem., 380, 431–442.[CrossRef][Web of Science][Medline]

  24. Chung, W. and Campanelli, J.T. (1999) WW and EF hand domains of dystrophin-family proteins mediate dystroglycan binding. Mol. Cell. Biol. Res. Commun., 2, 162–171.[CrossRef][Medline]

  25. Tommasi di Vignano, A., Di Zenzo, G., Sudol, M., Cesareni, G. and Dente, L. (2000) Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex. FEBS Lett., 471, 229–234.[CrossRef][Web of Science][Medline]

  26. Goldberg, L.R., Hausmanowa-Petrusewicz, I., Fidzianska, A., Duggan, D.J., Steinberg, L.S. and Hoffman, E.P. (1998) A dystrophin missense mutation showing persistence of dystrophin and dystrophin-associated proteins yet a severe phenotype. Ann. Neurol., 44, 971–976.[CrossRef][Web of Science][Medline]

  27. Crawford, G.E., Faulkner, J.A., Crosbie, R.H., Campbell, K.P., Froehner, S.C. and Chamberlain, J.S. (2000) Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain. J. Cell Biol., 150, 1399–1410.[Abstract/Free Full Text]

  28. Sherman, D.L., Fabrizi, C., Gillespie, C.S. and Brophy, P.J. (2001) Specific disruption of a schwann cell dystrophin-related protein complex in a demyelinating neuropathy. Neuron, 30, 677–687.[CrossRef][Web of Science][Medline]

  29. Hoffman, E.P., Garcia, C.A., Chamberlain, J.S., Angelini, C., Lupski, J.R. and Fenwick, R. (1991) Is the carboxyl-terminus of dystrophin require for membrane-associatedion? A novel, severe case of Duchenne muscular dystrophy. Ann. Neurol., 30, 605–610.[CrossRef][Web of Science][Medline]

  30. Recan, D., Chafey, P., Leturcq, F., Hugnot, J.P., Vincent, N., Tome, F., Collin, H., Simon, D., Czernichow, P., Nicholson, L.V., Fardeau, M. and Kaplan, J.-C. (1992) Are cysteine-rich and COOH-terminal domains of dystrophin critical for sarcolemmal localization? J. Clin. Invest., 89, 712–716.[Web of Science][Medline]

  31. Helliwell, T.R., Ellis, J.M., Mountford, R.C., Appleton, R.E. and Morris, G.E. (1992) A truncated dystrophin lacking the C-terminal domains is localized at the muscle membrane. Am. J. Hum. Genet., 50, 508–514.[Web of Science][Medline]

  32. Renley, B.A., Rybakova, I.N., Amann, K.J. and Ervasti, J.M. (1998) Dystrophin binding to nonmuscle actin. Cell Motil. Cytoskeleton, 41, 264–270.[CrossRef][Web of Science][Medline]

  33. Warner, L.E, DelloRusso, C., Crawford, R.W., Rybakova, I.N., Patel, J.R., Ervasti, J.M. and Chamberlain, J.S. (2002) Expression of Dp260 in muscle tethers the actin cytoskeleton to the dystrophin-glycoprotein complex and partially prevents dystrophy. Hum. Mol. Genet., 11, 1095–1105.[Abstract/Free Full Text]

  34. Flanigan, K.M., von Niederhausern, A., Dunn, D.M., Alder, J., Mendell, J.R. and Weiss, R.B. (2003) Rapid direct sequence analysis of the dystrophin gene. Am. J. Hum. Genet., 72, 931–939.[CrossRef][Web of Science][Medline]

  35. Tinsley, J.M., Potter, A.C., Phelps, S.R., Fisher, R., Trickett, J.I. and Davies, K.E. (1996) Amelioration of the dystrophic phenotype of mdx mice using a truncated utrophin transgene. Nature, 384, 349–353.[CrossRef][Medline]

  36. Rafael, J.A., Tinsley, J.M., Potter, A.C., Deconinck, A.E. and Davies, K.E. (1998) Skeletal muscle-specific expression of a utrophin transgene rescues utrophin-dystrophin deficient mice. Nat. Genet., 19, 79–82.[CrossRef][Web of Science][Medline]

  37. Tanaka, H., Shimizu, T. and Ozawa, E. (1989) Expression of a dystrophin-like protein on the surface membrane of muscle cells in mdx mice. Proc. Japan Acad., 65b, 238–241.

  38. Tanaka, H., Ishiguro, T., Eguchi, C., Saito, K. and Ozawa, E. (1991) Expression of a dystrophin-related protein associated with the skeletal muscle cell membrane. Histochem., 96, 1–5.

  39. Mizuno, Y., Yoshida, M., Nonaka, I., Hirai, S. and Ozawa, E. (1994) Expression of utrophin (dystrophin-related protein) and dystrophin-associated glycoproteins in muscles from patients with Duchenne muscular dystrophy. Muscle Nerve, 17, 206–216.[CrossRef][Web of Science][Medline]

  40. Deconinck, A.E., Rafael, J.A., Skinner, J.A., Brown, S.C., Potter, A.C., Metzinger, L., Watt, D.J., Dickson, J.G., Tinsley, J.M. and Davies, K.E. (1997) Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell, 90, 717–727.[CrossRef][Web of Science][Medline]

  41. Grady, R.M., Teng, H., Nichol, M.C., Cunningham, J.C., Wilkinson, R.S. and Sanes, J.R. (1997) Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell, 90, 729–738.[CrossRef][Web of Science][Medline]

  42. Rybakova, I.N., Patel, J.R., Davies, K.E., Yurchenco, P.D. and Ervasti, J.M. (2002) Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle. Mol. Biol. Cell, 13, 1512–1521.[Abstract/Free Full Text]

  43. Suzuki, A., Yoshida, M. and Ozawa, E. (1995) Mammalian {alpha}1- and ß1-syntrophin bind to the alternative splice-prone region of the dystrophin COOH terminus. J. Cell Biol., 128, 373–381.[Abstract/Free Full Text]

  44. Yoshida, M. and Ozawa, E. (1990) Glycoprotein complex anchoring dystrophin to sarcolemma. J. Biochem. (Tokyo), 108, 748–752.[Abstract/Free Full Text]

  45. Yoshida, M., Mizuno, Y., Nonaka, I. and Ozawa, E. (1993) A dystrophin-associated glycoprotein, A3a (one of 43DAG doublets), is retained in Duchenne muscular dystrophy muscle. J. Biochem. (Tokyo), 114, 634–639.[Abstract/Free Full Text]

  46. Smith, D.B. and Johnson, K.S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 67, 31–40.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
K. Hnia, J. Gayraud, G. Hugon, M. Ramonatxo, S. De La Porte, S. Matecki, and D. Mornet
L-Arginine Decreases Inflammation and Modulates the Nuclear Factor-{kappa}B/Matrix Metalloproteinase Cascade in Mdx Muscle Fibers
Am. J. Pathol., June 1, 2008; 172(6): 1509 - 1519.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
A. Driss, L. Charrier, Y. Yan, V. Nduati, S. Sitaraman, and D. Merlin
Dystroglycan receptor is involved in integrin activation in intestinal epithelia
Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1228 - G1242.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
S. Li, E. Kimura, R. Ng, B. M. Fall, L. Meuse, M. Reyes, J. A. Faulkner, and J. S. Chamberlain
A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy
Hum. Mol. Genet., May 15, 2006; 15(10): 1610 - 1622.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
L. M. Judge, M. Haraguchiln, and J. S. Chamberlain
Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex
J. Cell Sci., April 15, 2006; 119(8): 1537 - 1546.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I. N. Rybakova, J. L. Humston, K. J. Sonnemann, and J. M. Ervasti
Dystrophin and Utrophin Bind Actin through Distinct Modes of Contact
J. Biol. Chem., April 14, 2006; 281(15): 9996 - 10001.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
R. Barresi and K. P. Campbell
Dystroglycan: from biosynthesis to pathogenesis of human disease
J. Cell Sci., January 15, 2006; 119(2): 199 - 207.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/7/693    most recent
ddh087v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (25)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ishikawa-Sakurai, M.
Right arrow Articles by Ozawa, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ishikawa-Sakurai, M.
Right arrow Articles by Ozawa, E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?