Review Increasing Complexity of the Dystrophin-Associated Protein Complex Jonathon M

Home , Dystrophin, Syntrophin, Utrophin

Proc. Nadl. Acad. Sci. USA Vol. 91, pp. 8307-8313, August 1994 Review Increasing complexity of the dystrophin-associated protein complex Jonathon M. Tinsley, Derek J. Blake, Richard A. Zuellig, and Kay E. Davies Molecular Genetics Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom ABSTRACT Duchenne muscular dys- Purkinje neurons. Alternatively spliced dystrophin-1 (Dp7l) and apo-dystro- trophy is a severe X chromosome-linked, isoforms originating from the carboxyl- phin-3 are regulated by a promoter situ- muscle-wasting disease caused by lack of terminal coding region ofdystrophin have ated between exons 62 and 63 of the the protein dystrophin. The exact function also been described. The significance of dystrophin gene and are expressed in of dystrophin rem to be determined. these isoforms at the RNA and protein nonmuscle tissues, including brain, lung, However, analysis of its interaction with a level has not been elucidated. liver, and kidney. Apo-dystrophin-1 tran- large oligomeric protein complex at the Dystrophin is a 427-kDa protein local- scripts are only detectable in fetal and sarcolemma and the identicaton of a ized to the cytoplasmic face of the sar- newborn muscle. Muscle samples taken structurally related protein, utrophin, is colemma, enriched at myotendinous after 15 days postnatally have no apo- leading to the characterization ofcandidate junctions and the postsynaptic mem- dystrophin-1 transcript as determined by genes for other neuromusular disorders. brane of the neuromuscular junction reverse transcription-PCR. In rat brain, (NMJ). Dystrophin colocalizes with apo-dystrophin-1 transcripts continue in- Duchenne muscular dystrophy (DMD) is f3-spectrin and vinculin in three distinct creasing until they reach a maximum the most common muscular dystrophy, domains at the sarcolemma (overlaying after -1 mo. Thus apo-dystrophin-1 ap- affecting 1 in 3,300 boys. It leads to both I bands and M lines), lying as an pears to be developmentally regulated in severe muscle wasting and eventual array ofthick bands localizing at the sites muscle and brain (4). Dpll6 (apo- death in the late teens or early twenties of attachment of the sarcomeres to the dystrophin-2) is regulated by a promoter due to cardiac or respiratory failure. muscle plasma membrane. The carboxyl- situated between exons 55 and 56 of the Becker muscular dystrophy (BMD) is an terminal region of dystrophin is bound to dystrophin gene and is expressed in pe- allelic disorder that is less common (1 in the protoplasmic half of the plasma- ripheral nerve. In contrast to dystrophin, 30,000 boys) and less severe, with a later lemma. Thus dystrophin forms an intri- neither apo-dystrophin-1 nor apo-dystro- onset and much longer survival rate. cate part of the muscle cytoskeleton and phin-2 is expressed before organogenesis Both disorders are caused by mutations may function to link the normal contrac- in the developing mouse embryo (5). within the dystrophin gene, resulting in a tile apparatus to the sarcolemma. In the Apo-dystrophin-1 is first detected at 11.5 lack of dystrophin (usually DMD) or the brain, dystrophin is also localized at the days in the ventral midline of the hind- expression ofmutant forms ofdystrophin postsynaptic regions of some neurons. brain. Later expression of apo-dystro- (usually BMD). Dystrophin is a member The amino terminus of dystrophin has phin-1 occurs in central nervous system of a growing family of related proteins, been shown in vitro to contain a functional glia, teeth primordia, and in the liver. including the autosomally encoded pro- actin-binding domain. Deletions within Apo-dystrophin-2 appears to be a rare tein utrophin. There have been several this region ofthe dystrophin gene result in transcript during development but is de- recent reviews covering the structure and a range of phenotypes from mild to se- tected at low levels in the region of the potential functions of dystrophin and vere, suggesting that the ability to bind caudate putamen in newborn mouse utrophin, and the reader is referred to actin is an important, but not an absolute, brain (5). these for detailed references (1-3). This requirement for dystrophin function. The Utrophin is a 395-kDa protein encoded review focuses on the proteins with central rod domain consists ofa numberof by a large (1 Mb) multi-exonic gene lo- which dystrophin and utrophin interact. a-helical coiled-coil repeats that are sim- cated on chromosome 6q24(6). The amino ilar to spectrin and probably give the acid sequence shows that utrophin has a Dystrphin and Utrophin Genes: molecule a flexible rod-shaped structure. strong similarity to dystrophin. Utrophin A Brief Overview In-frame deletions within this domain usu- has an amino-terminal actin-binding do- ally give rise to intermediate or milder main (S. J. Winder, L. Hemmings, S. K. The dystrophin gene is the largest gene so BMD. The carboxyl-terminal domain has Maciver, S. J. Bolton, J.M.T., K.E.D., far identified in humans, covering >2.5 no homology with any other identified D. R. Critchley, and J. Kendrick-Jones, megabases and containing 79 exons. The sequences apart from the related protein personal communication), a repeated unusually high mutation rate at the DMD utrophin. Mutations in the cysteine-rich coiled-coil rod domain, and a cysteine- locus may, inpart, be accountedforby the domain and first half of the carboxyl- rich carboxyl-terminal domain. In normal large size of the gene. The corresponding terminal domain almost always result in adult muscle, utrophin is localized at the 14-kb dystrophin mRNA is expressed pre- severe DMD phenotypes, indicating their membrane of the NMJ. High-resolution dominantly in skeletal, cardiac, and importance for the normal function of analysis of rat NMJs suggests that utro- smooth muscle; lower levels appear in dystrophin. Loss of the last 200 amino phin is colocalized with the acetylcholine brain. Transcription of dystrophin in dif- acids ofdystrophin appears to produce an receptors (AChRs) at the crests of the ferent tissues is regulated from either the intermediate DMD/BMDphenotype. The junctional folds (7) and may play a role in brain promoter (predominantly active in potential functions of the dystrophin car- neuronal cells) or muscle promoter (dif- boxyl terminus are discussed later. Abbreviations: DMD, Duchenne muscular ferentiated myogenic cells and primary Three shorter transcripts, expressed dystrophy; BMD, Becker muscular dystro- glial cells) giving rise to different first from two different phy; NMJ, neuromuscular junction; DAP, promoters, encode dystrophin-associated protein; DAG, dystro- exons. A thirdpromoterbetweenthe mus- proteins that have a majority ofsequence phin-associated glycoprotein; AChR, acetyl- cle promoter and the second exon ofdys- identical to that of the cysteine-rich and choline receptor; SCARMD, severe childhood trophin regulates expression in cerebellar carboxyl terminus of dystrophin. Apo- autosomal-recessive muscular dystrophy. 8307 Downloaded by guest on September 28, 2021 8308 Review: Tinsley et al. Proc. Natl. Acad. Sci. USA 91 (1994)

the clustering of AChRs (8). In fetal mus- complex and other associated proteins cle (18). The protein consists of a short cle development, utrophin is expressed localized to the sarcolemma (described signal sequence, one transmembrane do- before dystrophin. Higher levels of utro- later). main, and two potential sites for N-linked phin mRNA have been detected in human The 43DAG (A3) and 156DAG are de- glycosylation. In contrast to dystrogly- and mouse fetal tissue compared with rived from a single 97-kDa precursor pro- can, expression of the SODAG is specific adult. In normal humanfetal muscle, utro- tein translated from a 3.8-kb transcript to skeletal, cardiac, and smooth muscle phin is localized to the sarcolemma and (15). The 43-156DAG transcript is ex- (18, 19). This protein has been renamed NMJs, showing maximum expression at pressed not only in skeletal, cardiac, and adhalin (from the Arabic word for mus- 17-18 weeks of gestation (9). When dys- smooth muscle but also in other tissues- cle, adhal). trophin is expressed, utrophin is gradually namely, brain, lung, liver, and kidney. At present the cloning of the 35DAG depleted from the sarcolemma to be re- Cleavage and posttranslational modifica- (A4) and 25DAP (AS) has not been re- placed by dystrophin, leaving the NMJs tion of the carboxyl-terminal portion ported, so their potential functions have as the only site ofutrophinlocalization (9). gives rise to the 43DAG. The 43DAG has yet to be elucidated. However, 35DAG It is not known whether utrophin has a sites for glycosylation, a transmembrane expression appears restricted to striated functional role while located at the fetal domain, and a cytoplasmic tail (15). On muscle (19). A 94-kDa protein, AO, which sarcolemma-i.e., the fetal form of dys- acrylamide gels, the 43DAG (A3) is a also copurifies with the DAP complex, trophin-or is simply in the process of component of a doublet, A3a and A3b has been identified (12). Circumstantial iigrating with the developing AChR clus- (12, 16). The 43DAG is the same as A3a, evidence suggests that AO may be related ters beforeformation ofthe mature NMJs. as determined by peptide sequence anal- to the Torpedo 87-kDa postsynaptic pro- In contrast to dystrophin, utrophin is de- ysis of the purified gel band. A3b is tein (discussed later). tectable in most tissues-particularly the distinct from A3a, as it has a different Recently, the purified DAP complex lung, blood vessels, and nerves. Utrophin proteolytic cleavage pattern (14) and is purified from rabbit skeletal muscle has is enriched in the vascular regions of the unable to bind dystrophin (16). The 56- been divided into three subcomplexes brain-for instance, the choroid plexus kDa amino-terminal core protein once based upon detergent solubilization using and at the astrocytic foot processes ofthe glycosylated at many of the potential 1-octyl f-D-glucoside (14). The groups blood-brain barrier. O-glycosylation sites gives rise to the consisted of a- and f-dystroglycan (dys- fully processed 156DAG (15). Recently a troglycan complex), adhalin, 35DAG and The Dystrophin-Associated Protein protein of120 kDa called laminin-binding A3b (sarcoglycan complex), and a com- Complex in Muscle protein 120 (LBP120) has been isolated plex of dystrophin, syntrophin, and AO. from the brain and shown to have peptide The restricted expression patterns of ad- In skeletal muscle, previous analysis of sequences identical to 156DAG (17). The halin and 35DAG suggests that the sar- the functional domains ofdystrophin iden- size difference between the two proteins coglycan complex may be specific to tified a carboxyl-terminal domain that is probably due to different glycosyla- striated muscle. bound to a large oligomeric complex ofsix tion. Because of their posttransla- novel proteins localized to the sarco- tional modification and their interaction Emergence of the Syntrophin Family lemma. These sarcolemmal proteins were with dystrophin, these proteins have of Proteins identifiedalmostsimultaneouslybyCamp- now been renamed a-(156DAG) and bell et al (10, 11) and Yoshida and Ozawa (3-(43DAG) dystroglycan (15). In 1987, Froehner et al. (20) described a (12). Tight association of these proteins The 59-kDa membrane-bound protein 58-kDa protein that was associated with was demonstrated by copurification from named 59DAP or Al has been indepen- the cytoplasmic side of the AChRs in rabbit muscle membranes, cosedimenta- dently cloned from a number of species, Torpedo electroplaques. Analysis ofadult tion on sucrose density centrifugation, and recent evidence shows this is a family rat muscle using a monoclonal antibody immunoprecipitation, colocalization to of related genes, some of which bind to raised against this 58-kDa protein strongly the sarcolemma, and protein crosslinking dystrophin. They have been renamed syn- stained the NMJ but also, to a lesser experiments. Table 1 details the names of trophins (syntrophos from Greek, mean- extent, stained the sarcolemma (20). In the dystrophin-associated glycoproteins ing companion). These constitute an in- 1990, initial analysis of rabbit 59DAP/A1 (DAGs) and dystrophin-associated pro- creasingly complex family ofproteins and by one-dimensional PAGE demonstrated teins (DAPs), their molecular weights, are discussed later. that the protein separated as a triplet of and location within muscle. Fig. 1 depicts The 50-kDa glycoprotein (5ODAG/A2) bands thought to be a result of differing diagrammatically the location of the DAP has been cloned from rabbit skeletal mus- posttranslational modifications or possi- Table 1. Nomenclature of the dystrophin-associated proteins Yoshida and Ozawa Campbell et al. (10, 11) (12) Sarcolemmal Name Size,* kDa Name Size,* kDa Renamed localization Non-DAP binding 156DAG 156 a-Dystroglycan Extracellular Merosin, agrin, laminin 59DAP 59 59-1DAP a-Al 60 al-Syntrophin (-Al 64 (31-Syntrophin Cytoplasmic Dystrophin, apo-dystrophins, utrophin ,82-Syntrophin 50DAG 50 A2 52 Adhalin Transmembrane 43DAG 43 A3a 43 /3-Dystroglycan Transmembrane Dystrophin, utrophin, apo-dystrophins A3b 35DAG 35 A4 36 Transmembrane 25DAP 25 A5 24 Transmembrane AO 94 Cytoplasmic Dystrophin, utrophin,t apo-dystrophinst *Estimated size of the protein. tBinding not shown experimentally. Assumed because of sequence similarity to dystrophin carboxyl terminus. Downloaded by guest on September 28, 2021 .mc;view: Tinsley et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8309

CIA ...... ,,...... ,..,,,...... '...... p~ ~ f .(. Ad a i

Y~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Pdyrstroghlyn:! FIG.1.Schematicrepresentation of the dystrophin-associated protein complex embedded in the sarcolemma. This is based ofSzukieta.(1)ad th incrportio of he dstrolycn sucompexesi~e,on.the.modeldytrogycan sacoglcanand (14)Thedysrophnro doain s rpresntedby he rctagles thecyseinerichdominshdedline.thefirt.haf.oystophi/s...... /Ai rereseted y te thecarboxyltermnusis representd by the hatchedline, and the later half of the arboxyl terniinu is represented y the black line

bly different isoforms (12, 21). Recent phin species (22). Recently, using peptide sequence data bases using the human analysis of 59DAP/A1 by two-dimen- antibodies specific to mouse syntrophin-1 ,(Al cDNA sequence identified a skele- sional gel electrophoresis resolved the or mouse syntrophin-2, syntrophin-1 was tal muscle-expressed sequence tag proteins into two major groups-namely, localized to rat muscle sarcolemma and (EST25263) that had strong homology to the (3-Al group which was larger (64 kDa) AChRs, whereas syntrophin-2 was local- mouse syntrophin-2 (25). and more basic than the a-Al group (60 ized only at the AChRs (23). It has been agreed by the laboratories kDa) (16). In retrospect, the 58-kDa Tor- The cDNA coding for 59-lDAP has that have adopted the syntrophin nomen- pedo protein and the 59DAP/Al proteins been cloned from rabbit skeletal muscle clature to rename the syntrophins on the all originate from the same family of and shown to have 94% amino acid iden- basis of their amino acid similarity and genes, the to mouse their acidic (a, pI z6.7) or basic ((3, pI syntrophins. tity syntrophin-1, suggesting -9+) nature (23). Thus mouse syntro- Syntrophin genes of Torpedo, mouse, that this is the rabbit homolog of syntrophin-1, rabbit 59-lDAP, and Torpedo rabbit, and human have recently been phin-1 (24). The 2.4-kb transcript has an syntrophin are now named al-syntro- cloned. In the mouse, two related proteins almost identical expression pattern as phins. Mouse syntrophin-2 and the hu- were shown to be derived from different mouse syntrophin-1. An antibody to rab- man gene encoding the EST25263 frag- genes. These were named syntrophin-l bit syntrophin-1 detects only the lowest ment are ,32-syntrophins. Finally the hu- and syntrophin-2 (22). Expression of the band ofthe 59DAP/Al triplet ofproteins man (3-Al is a member of the (31- 2.2-kb mouse syntrophin-1 transcript was seen by one-dimensional PAGE (24). syntrophins. Table 2 shows the amino highest in striated muscle, lower levels Finally, a third syntrophin gene has acid similarity between the cloned syn- were found in brain, and expression was been identified from human tissue (25). trophin species. The overall amino acid barely detectable in the other tissues Purification and partial peptide sequenc- similarity of Torpedo syntrophin is in the tested. There are multiple syntrophin-2 ing of the human a- and 3-Al proteins order of 50%o when compared with either transcripts (10 kb, 5.0 kb, and 2.2 kb) (separated by two-dimensional PAGE) the al-syntrophins or the (-syntrophins. expressed in all tissues tested with lowest confirmed that a-Al is the human ho- It will be ofinterest to see if Torpedo has levels in skeletal muscle. However, the molog of mouse syntrophin-1. A cDNA only one syntrophin, suggesting that the relative expression of each of these tran- containing the (3Al peptide sequences other syntrophin genes have evolved scripts varies from tissue to tissue. For was isolated encoding a protein of 58.7 from a single ancestor. example, the 2.2-kb transcript was pre- kDa, which is related to but distinct from dominant in testes, the 5.0-kb transcript mouse syntrophin-l and -2. There appear Association of Other Proteins with was predominant in lung, and the 10.0-kb to be multiple transcripts (7.8, 7.2, 6.0, Muscle DAPs transcript was predominant in brain (22). 3.8, and 2.4 kb) expressed from the (3-Al An antibody against Torpedo syntrophin gene, which are detected in various As the name DAP suggests, dystrophin (20) reacts with both ofthe mouse syntro- amounts in all tissues tested. A search of was the first protein shown to interact Downloaded by guest on September 28, 2021 8310 Review: Tinsley et al. Proc. Natl. Acad. Sci. USA 91 (1994) Table 2. Similarity of syntrophins al-Syntrophin (3-Syntrophin Rabbit Mouse Torpedo Human (.82)* Mouse (,12) Human (131) (506 aa) (504 aa) (489 aa) (? aa) (440 aa) (538 aa) al-Syntrophin Rabbit +++ Mouse 94 +++ Torpedo 59 59 +++ 3-Syntrophin Human ,(2 31 31 25 +++ Mouse 132 53 52 54 82 +++ Human p1 52 52 51 48 59 +++ Numbers show the overall percentage amino acid identity between each syntrophin gene product. Strong amino acid identities are in boldface type. Numbers in parentheses indicate the size of the protein in amino acids. *Amino acid similarity only compared with the coding potential of the 210-bp expressed sequence tag 25263 fragment (25). with this complex of sarcolemmal pro- Utrophin localization to muscle DAPs trix components such as fibronectin, col- teins. Purification ofthe complex always has been demonstrated in vivo. Antibody lagen I, collagen IV, entactin, or heparin resulted in the copurification of dystro- to a-dystroglycan stains both the AChRs, sulfate proteoglycan (33). The major effect phin, demonstrating their tight associa- where utrophin is located, and beyond ofbinding laminin to a-dystroglycan would tion. Suzuki et al. (26) demonstrated in the AChR by :0.3 ,um, where dystrophin be to link the sarcolemma to the extracel- vitro that the cysteine-rich domain and is located (30). In rat skeletal muscle, lular matrix, potentially providing an addi- the first half of the carboxyl terminus of antibody specific to 32-syntrophin local- tional strengthening mechanism to allow dystrophin bound to the DAG complex. izes only to the NMJs, suggesting a spe- the muscle fibers to withstand the enor- More recent evidence showed that the cific interaction with utrophin (23). In mous stresses of contraction and stretch. f3-dystroglycan binds to this region of dystrophin-deficient mdx mouse skeletal Laminin-Sepharose also bound a-dystro- dystrophin but also demonstrated that muscle, a-dystroglycan and syntrophin glycan from nonmuscle tissues. However, syntrophin and another protein of87 kDa strongly stain only the NMJ (23, 29, 31). the other members of the muscle DAP (AO) probably bind to the latter halfofthe Utrophin association with the DAP com- complex were lacking, suggesting they do carboxyl terminus (13). In vitro studies plex has also been shown in vitro by not exist in nonmuscle tissue, that they are have shown that f-dystroglycan is bound copurification of a DAP/utrophin com- antigenically dissimilar, or that they simply directly to a purified dystrophin fusion plex from mdx skeletal muscle (31). The do not bind together as tightly as the mus- protein consisting of only the cysteine- binding ofutrophin to components of the cle complex (33). rich domain of dystrophin (residues DAP complex is not surprising since the Agrin is an extracellular matrix protein 3080-3265). However, binding to afusion sequence comparison ofutrophin reveals that appears to be able to direct the accu- protein also containing the first halfofthe an 88% amino acid similarity to the mulation of AChRs at developing and re- carboxyl terminus (residues 3266-3442) j-dystroglycan-binding domain of dys- generating NMJs. Independent evidence contributes to a more stable interaction. trophin (32). Utrophin also has the same from three different groups suggests that Interestingly, a protein consisting ofonly leucine zipper motif in the latter half of a-dystroglycan is an agrin receptor (34- the carboxyl terminus of dystrophin was the carboxyl terminus as dystrophin and 36). Bowe et al. (34) purified a complex unable to bind f-dystroglycan, suggest- the 87-kDa Torpedo protein (1). from Torpedo electric organ postsynaptic ing that this region is unable to bind It is not known what DAP complex is membranes capable of binding agrin and directly but may be important for main- present at the fetal muscle sarcolemma showed that the complex consisted oftwo taining the correct conformational bind- before dystrophin expression. Expression proteins (190 kDa and 50 kDa). Partial ing structure (13). Immunogold labeling and formation of the DAP complex may peptide analysis of the two proteins dem- ofmuscle localized P-dystroglycan to the simply mimic the expression pattern of onstrated that they had very strong homol- plasma membrane at the same site as the dystrophin. However, given the overall ogy to a- and (B-dystroglycan. Campanelli dystrophin carboxyl terminus (27). domain similarity between dystrophin and et al. (35) and Gee et al. (36) demonstrated The latter halfofthe carboxyl terminus utrophin, itwouldbe reasonable to hypoth- that a-dystroglycan could bind agrin in of dystrophin has a leucine-zipper motif esize that utrophin may be the fetal form of vitro by different methods and that the (1), which may play arole in the binding of dystrophin and function similarly. Dystro- binding was calcium and heparin depen- syntrophin and/or the 87-kDa protein phin may be more specialized and adapted dent (35). Analysis ofmouse C2 myotubes (13). One interpretation of the literature to dealing with the much greater stresses of by immunofluorescence demonstrated that (although not formerly proven) is that this skeletal and cardiac muscle development agrin, utrophin, and a-dystroglycan were 87-kDa protein may be the same as that after birth. However, until this time, utro- concentrated at the AChRclusters (35, 36). labeled AO (12, 13), which in turn may be phin could give some form of structural Thus, in muscle the DAP complex ap- the same as the 87-kDa cytoplasmic pe- rigidity to the developing myotubes. pears to have two very important roles. (i) ripheral membrane protein identified in Laminins are a family of basement- The complex (possibly containing al- Torpedo (28). This is a good candidate membrane glycoproteins. The a-dystro- syntrophin) links the sarcolemma to the because in Torpedo, 87-kDa protein ex- glycan binds to laminin and merosin (M- extracellular matrix via merosin and the pression is restricted to the electric organ, laminin) situated in the extracellular matrix internal cytoskeleton via dystrophin in a brain, and skeletal muscle (28) and, most (33). Laminin-Sepharose, as well as puri- regular array along the length of the myo- importantly, is copurified as a complex fying a-dystroglycan, also copurified all tubes to give the myotubes strength (Fig. containing dystrophin and syntrophin the other members of the DAP complex 1). (ii) The DAP complex (containing (2- (29). Sequence analysis of the 87-kDa (33). The binding of a-dystroglycan to syntrophin) is involved in the formation/ protein reveals homology to the carboxyl- laniinin is specific-probably at one ofthe stabilization ofAChR clusters linking agrin terminal region of dystrophin, including heparin-binding domains, with no demon- in the extracellular matrix and utrophin the putative leucine zipper (1, 29). strable binding of other extracellular ma- within the cell to establish the junctional Downloaded by guest on September 28, 2021 Rmveview: Tinsley et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8311 regions between the motor neuron and Further analysis of A3b and 35DAG is levels (15). Patients with mutations in the muscle (Fig. 2). required to answer these questions. carboxyl terminus of dystrophin always Possibly the DAP complex in nonmus- result in a severe phenotype with a re- Association of Other Proteins with cle cell types could consist of only the a- duction in the DAPs (40). In some cases Nonmuscle DAPs and (3-dystroglycan, P2-syntrophin, and these patients express a truncated form possibly AO. From the proposed DAP of dystrophin that appears to localize It is very probable that utrophin also binding complex ofErvasti and Campbell correctly to the sarcolemma-presum- binds to a related complex in nondystro- (21) and Suzuki et al. (13), this would be ably due to an intact amino-terminal ac- phin-expressing cell types. The presence the minimum complex required to link tin-binding domain (41). Dystrophin mu- of a-dystroglycan expression in nonmus- laminin (in the extracellular matrix) to tations originating within the actin- cle tissues (15) and the identification of a actin via utrophin. As actin and laminin binding domain giving rise to an in-frame smaller (120 kDa) brain a-dystroglycan fusion protein result in variable pheno- isoforms are found in all cells, this could types, suggesting that the presence of a isoform (17) suggests that this protein provide a scaffold to link stably these functional dystrophin carboxyl terminus may have other binding partners. In intra- and extracellular proteins and thus is capable of stabilizing a proportion of mouse, f32-syntrophin expression occurs be a mechanism to give rigidity to all the DAP complexes (42). in all tissues examined (22, 24), mimick- cells. In BMD there is a general mild reduc- ing utrophin expression and suggesting a Syntrophin preparations from various tion in the DAPs-probably because higher specificity for binding utrophin. rat tissues copurify a protein of 71 kDa, most mutations involve in-frame dele- Specific antibodies only detect 82- which reacted with an antibody raised tions ofthe rod domain, leaving the actin- syntrophin in rat kidney and not in al- against the unique amino terminus of apo- binding and DAP-binding regions intact syntrophin (23). In peripheral nerves dystrophin-1 (Dp71) (38). (43). These truncated forms of dystro- found among muscle fibers ofnormal and phin must be partially effective in both DMD muscle, utrophin and f-dystrogly- Potential for Disease Caused by Defects organizing the DAP complex into the can are clearly detected, but no 50DAG is in the DAPs or Associated Proteins correct conformation and linking with the detected (37). The peripheral nerves cytoskeletal actin. Interestingly, symp- must contain a DAP complex deficient in In DMD the levels of all the DAPs are tomatic DMD carriers have significantly 50DAG. It is possible that in nonmuscle reduced (11, 37, 39), although the dystro- reduced DAPs in dystrophin-deficient re- cells there is no sarcoglycan complex or glycan mRNA (and presumably the other generating fibers, whereas they were ex- there is a related sarcoglycan complex. DAP mRNAs) is expressed at normal pressed at normal levels in the normal dystrophin-positive fibers (44, 45). Motor Axon All of these results have led to the notion that dystrophin localizes or stabi- lizes the DAP complex. Without dystrophin the complex is unable to organize properly, and consequently the linkage between the extracellular matrix and the subsarcolemma is disrupted; this eventu- ally leads either to the gross muscle necrosis seen in DMD or to the less severe phenotype in BMD, where expression of a partially functional dystrophin results in a partial retention of the DAPs. Further analysis of autosomal muscular dystrophies has strengthened this hy- pothesis. Deficiency in only adhalin (50DAG) is observed in patients suffering from severe childhood autosomal-recessive muscular dystrophy (SCARMD) (46-48). SCARMD has been mapped to 13q12 by linkage analysis of 16 Tunisian pedigrees (49, 50). However analysis of three Brazilian SCARMD families was unable to demonstrate linkage to 13q, yet affected individuals were deficient in ad-

/- halin (51). Until the chromosomal localization ofthe adhalin gene is determined, it is impossible to suggest which, if any,

.__ ._2., b___c ofthe autosomal-recessive muscular dystrophies are caused by adhalin mutations. The autosomally recessive, dystrophic hamster (NSJ-my/my) may turn out to be a model for SCARMD. Analysis of the DAPs shows a specific decrease in the levels of adhalin and 35DAG and, to a lesser extent, 3-dystroglycan (52, 53). FIG. 2. Schematic representation ofa muscle nerve terminal. The lower part ofthe diagram In these animals there is widespread mus- represents the proteins associated with an AChR at the crests ofthejunctional folds, linked by cle fiber necrosis and active regenera- utrophin to the actin cytoskeleton. a-DG, a-dystroglycan; P-DG, 8-dystroglycan; SC, sarco- tion. In Fukuyama-type congenital mus- glycan complex; S, (32-syntrophin; 43K, 43-kDa AChR-associatedHe protein. cular dystrophy abnormal staining ofthe Downloaded by guest on September 28, 2021 8312 Review: Tinsley et al. Proc. Natl. Acad. Sci. USA 91 (1994) Table 3. DAP gene location and potential disease association H., Noguchi, S., Mizuno, Y. & Ozawa, Chromosome E. (1994) Eur. J. Biochem. 222, 1055- location 1061. Human Mouse 15. Ibraghimov, B. O., Ervasti, J. M., Lev- Gene Human Mouse Disease Locus Mutant Locus eille, C. J., Slaughter, C. A., Sernett, S. W. & Campbell, K. P. (1992) Nature Dystroglycan 3p21 9 du/du, tip/tip 9 (London) 355, 696-702. Adhalin SCARMD 13q12 NSJ-my/my (hamster) 16. Yamamoto, H., Hagiwara, Y., Mizuno, P2-syntrophin 16* Y., Yoshida, M. & Ozawa, E. (1993) J. ,81-syntrophin 8q23 Biochem. (Tokyo) 114, 132-139. Merosin 6q22-23 10 CMD 6q2 dy/dy 10 17. Gee, S. H., Blacher, R. W., Douville, Dystrophin Xp2l X DMD/BMD Xp2l mdx X P. J., Provost, P. R., Yurchenco, P. D. Utrophin 6q24 10 & Carbonetto, S. (1993) J. Biol. Chem. FCMD 9q31-33 268, 14972-14980. 18. Roberds, S. L., Anderson, R. D., FCMD, Fukuyama-type muscular dystrophy; CMD, congenital muscular dystrophy. Ibraghimov-Beskrovnaya, 0. & Camp- *Tentative gene localization. bell, K. P. (1993) J. Biol. Chem. 268, 23739-23742. DAPs is seen, particularly of (dystro- role as candidate genes has not yet been 19. Yamamoto, H., Mizuno, Y., Hayashi, glycan (43DAG), in some muscle fibers fully explored. Given the wide diversity K., Nonaka, I., Yoshida, M. & Ozawa, (54, 55). However abnormal ,B-dystrogly- of cell types in which members of the E. (1994) J. Biochem. (Tokyo) 115, 162- can staining can only be a secondary DAP complex are expressed, it is also 167. effect, as the genetic defect for Fuku- possible that other nonmuscle disorders 20. Froehner, S. C., Murnane, A. A., To- yama-type muscular dystrophy has been will be caused by aberrant expression of bler, M., Peng, H. B. & Sealock, R. mapped to 9q31-33, whereas the dystro- this complex family of interacting mem- (1987) J. Cell Biol. 104, 1633-1646. glycan gene is located at 3p21 (56) (Table brane proteins. 21. Ervasti, J. M. & Campbell, K. P. (1991) 3). In both these human diseases, dystro- Cell 66, 1121-1131. is We thank Prof. S. C. Froehner, Prof. M. 22. Adams, M. E., Butler, M. H., Dwyer, phin expression normal, but symptoms T. M., Peters, M. F., Murnane, A. A. & are akin to severe BMD. Fardeau, and Prof. E. Ozawa for providing copies of press. Froehner, S. C. (1993) Neuron 11, 531- Decreased laniinin expression has been manuscripts in We thank the 540. demonstrated in a number of muscular Muscular Dystrophy Group of Great Britain and Northern Ireland, the Muscular Dystro- 23. Peters, M. F., Kramarcy, N. R., Seal- dystrophies (57). Recently, analysis ofthe phy Association ock, R. & Froehner, S. C. (1994) Neu- U.S.A., and the Medical Re- in severely dystrophic, autosomally reces- search Council for generous support. roReport, press. sive dy/dy mouse demonstrated a com- 24. Yang, B., Ibraghimov-Beskrovnaya, O., plete lack of merosin and vastly reduced 1. Blake, D. J., Tinsley, J. M. & Davies, Moomaw, C. R., Slaughter, C. 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