Hislol Hislopalhol (1997) 12: 195-203 and 001: 10.14670/HH-12.195 Histopathology http://www.hh.um.es From Cell Biology to Tissue Engineering

Invited Review

The role of , a novel receptor of laminin and agrin, in cell differentiation

K. Matsumura, H. Yamada, F. Saito, Y. Sunada and T. Shimizu Department of Neurology and Neuroscience, Teikyo University School of Medicine, Tokyo, Japan

Summary. Dystroglycan was originally identified as the -associated (DAPs) (for overviews on extracellular and transmembrane constituents of a large the DGC and the DAPs see Tinsley et aI. , 1994; oligomeric complex of sarcolemmal proteins associated Campbell, 1995; Ozawa et aI., 1995; Worton, 1995). In with dystrophin , the product of the Duchenne DMD patients and mdx mice, the absence of dystrophin muscular dystrophy (DMD) gene. During the last few leads to a drastic reduction of all the DAPs in the years, dystroglycan has been demonstrated to be a novel . Extensive studies over the last several years receptor of not only laminin but also agrin, two major have indicated that the DAPs are classified into three proteins of the extracellular matrix having distinct groups: (1) the syntrophin complex; (2) the biological effects. The fact that the drastic reduction of complex; and (3) the dystroglycan complex (Fig. I). The dystroglycan in the sarcolemma, caused by the absence syntrophin complex is comprised of the members of the of dystrophin, leads to death in DMD syntrophin family, a-, 131- and 132-syntrophin, which are patients and mdx mice indicates that , as a laminin cytoplasmic proteins complexed with the carboxyl­ receptor, dys troglycan contributes to sarcolemmal terminal domain of dystrophin or its homologues. The stabilization during contraction and stretch of striated sarcoglycan complex is comprised of three trans­ muscle cells. Dystroglycan is also expressed in the membrane glycoproteins, a-, 13- and y-sarcoglycan. and non-muscle tissues such as Although the physiological functions of the syntrophin kidney, brain and peripheral nerve, and, as a receptor of and sarcoglycan complexes remain obscure at present, laminin/agrin , has been implicated in such diverse and recent studies have demonstrated that primary deficiency specific developmental processes as epithelial of a-, 13- and y-sarcoglycan causes three forms of morphogenesis, synaptogenesis and myelinogenesis. autosomal recessive muscular dystrophies, LGMD2D, These findings point to the fundamental role of LGMD2E and LGMD2C, respectively, suggesting that dystroglycan in the cellular differentiation process the sarcoglycan complex plays a crucial role in the shared by many different cell types. In this paper, we stabilization of the sarcolemma. review the recent publications on the biological In contrast to the syntrophin and sarcoglycan functions of dystroglycan and discuss its roles in cell complexes, the dystroglycan complex has been differentiation. characterized extensively during the last few years and this has led to the amazing discovery that it is a receptor Key words: Dystroglycan, Laminin, Agrin, Dystrophin, of the extracellular matrix (ECM) proteins laminin and Extracellular matrix agrin in various tissues. As a receptor of laminin/agrin, the dystroglycan complex is presumed to play roles in diverse and specific physiological processes such as Introduction epithelial morphogenesis, synaptogenesis and myelino­ genesis, as well as stabilization of the sarcolemma. In In , dystrophin, a large membrane­ this paper, we review the recent publications on the associated cytoskeletal protein encoded by the Duchenne biological functions of the dystroglycan complex and muscular dystrophy (DMD) gene, exists in a large discuss its roles in cell differentiation in various tissues. oligomeric complex (the dystrophin-glycoprotein complex or DGC) tightly associated with several The primary structure of dystroglycan sarcolemmal proteins, which are collectively called the Dystroglycan was originally identified as the 156 Offprint requests to: Dr. Kiichiro Matsumura, M.D. , Ph.D. , Department of kDa and 43 kDa constituents of the DGC in skeletal Neurology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi­ muscle (Ervasti et aI., 1990). Interestingly, cDNA ku. Tokyo 173. Japan cloning disclosed that both the 156 kDa and 43 kDa 196 Functions of dystrog/yean eomp/ex

proteins are encoded by a single 5.8 kb mRNA and that between 120 kDa to 190 kDa among different tissues, post-translational processing of a 97 kDa precursor whereas the core protein has a molecular mass of only protein resulted in two mature proteins (lbraghimov­ about 70 kDa, suggesting that it may be a target of Be krovnzaya et aI. , 1992). The 156 kDa and 43 kDa extensive post-translational modification , such as proteins were found to correspond to the amino- and glycosylation. This is consistent with the facts that: (I) carboxyl-terminal halves of the dystroglycan precursor a-dystroglycan contains three potential N-glycosylation protein and were named a- and l3-dystroglycan, sites and numerous potential O-glycosylation sites respectively (Ibraghimov-Beskrovnaya et aI. , 1992). (Ibraghimov-Beskrovnaya et aI., 1992); and (2) a­ Dystroglycan has no significant homology with any dystroglycan i far more negatively charged than known proteins. There is a signal sequence in the amino­ predicted from the primary structure (Gee et aI., 1993; terminus and a single transmembrane domain of 24 Smalheiser and Kim, 1995). Indeed, the results of lectin amino acids near the carbox.yl-terminus. This suggests staining and digestion with deglycosylation enzymes or that a- and l3-dy s troglycan are extracellular and O-sialogJycoprotease indicate that a-dystroglycan is a transmembrane proteins, respectively (Ibraghimov­ mucin-type glycoprotein , which is defined as a Beskrovanaya et aI. , 1992). Tryptic peptide mapping of glycoprotein heavily glycosylated in the O-sites (Ervasti purified a- and l3-dystroglycan and amino-terminal and Campbell , 1991; Smalheiser and Kim , 1995; amino acid sequencing of purified l3-dystroglycan have Yamada et aI. , 1996a). The most prominent feature of identified serine 654 of the dystroglycan precursor as the mucin domains of glycoproteins is the high content of single cleavage site (Deyst et aI. , 1995; Smalheiser and proline residues in the vicinity of serine and threonine Kim, 1995). The complex comprised of a- and 13- O-glycosylation sites . Such a characteristic sequence can dystrogJycan is called the dystroglycan complex . be found in the middle portion of a-dystroglycan , However, the precise nature of the interaction between suggesting that this may be a mucin domain. This region these two proteins has not yet been characterized. The is expected to take the form of a rigid rod , since complex dystrogJycan gene, containing two exons, is localized on secondary and tertiary structures are hindered by heavy chromosome 3p21 and 9 in human and mouse , glycosylation (Fig. I). Consistent with this prediction, respectively (Ibraghimov-Beskrovnaya et aI., 1993; analysis by electron microscopy demonstrated that the G6recki et aI., 1994). Thus far, no disease-causing isoJated a-dystroglycan consisted of two mutations have been identified in the dystroglycan gene. globular domains connected by a rod-like segment (Brancaccio et aI., 1995). At present, there is no Glycosylation of a-dystroglycan direct evidence which indicates that a-dystroglycan contains glycosaminoglycan chains as originally The molecular mass of a-dystroglycan ranges proposed.

Glycocalyx

Sarcolemma

Cytoplasm

Fig. 1. The molecular organization of the DGC in skeletal muscle. a-DG : a-dystroglycan; B-DG: B­ dystroglycan; a-SG : a-sarcoglycan ; B-SG: B­ sarcoglycan ; y-SG : y- sarcoglycan; 25: the 25 kDa DAP ; SYN: syntrophin; nNOS: neuronal ; DYS: dystrophin; UTR : utrophin. 197 Functions of dystroglycan complex

The dystroglycan complex and signal transduction and immunoelectron microscopy (Ervasti et aI., 1990; Ervasti and Campbell, 1991; Ibraghimov-Beskrovnaya et The intracellular signal transduction pathways aI., 1992; Cullen et aI., 1994; Wakayama et aI., 1995). In activated by adhesion of cells to other cells or the ECM cardiac muscle, it is localized in the transverse tubules in play crucial roles in cell differentiation, migration and addition to the sarcolemma (Klietsch et aI., 1993). The proliferation. The prototypical cell adhesion molecules molecular organization of the skeletal muscle are the cell sUiface receptors for the ECM glycoproteins. dystroglycan complex has been investigated extensively. As a receptor of the ECM glycoproteins laminin and The DGC was isolated from the detergent extracts of the agrin (discussed below), the dystroglycan complex skeletal muscle membrane fractions using wheat germ fulfills this criterion, raising a possibility that it may be agglutinin (WGA) and DEAE chromatography followed involved in the signaling pathways. This is supported by by sucrose density gradient centrifugation (Ervasti et aI. , the fact that the intracellular carboxyl-terminal tail of B­ 1990; Ervasti and Campbell, 1991). Both a- and B­ dystroglycan contains a phosphotyrosine consensus dystroglycan were identified as major constituents of the sequence and several proline-rich regions which could isolated DGC, together with dystrophin and the associate with SH2 and SH3 domains of signaling components of the sarcoglycan and syntrophin proteins (Ibraghimov-Beskrovnaya et aI., 1992). complexes (Ervasti et aI. , 1990; Ervasti and Campbell, Recently, the carboxyl-terminal tai l of B-dystroglycan 1991). The dystroglycan complex was dissociated from was found to bind to Grb2, an adaptor protein in the dystrophin and the sarcoglycan and syntrophin signal ing pathways, via the two SH3 domains, complexes by n-octyl-B-D-glucoside (Yoshida et aI., suggesting that B-dystroglycan may indeed playa role in 1994). Alpha-dystroglycan, but not B-dystroglycan, was signal trasduction (Yang et aI. , 1995). Since B­ extracted from the membranes or dissociated from the dystroglycan binds to dystrophin through the last 15 DGC at pH 12 (Ervasti and Campbell, 1991). In amino acids of the carboxyl-terminus (lung et al., 1995), addition, a probe which labels the hydrophobic segments the interaction with GrB2 may also modulate the of proteins reacted with B-dystroglycan, but not with a­ interaction of B-dystroglycan with the submembranous dystroglycan (Ervasti and Campbell, 1991). Together dystrophin-cytoskeleton and affect the molecular with the primary structure and the localization in the organization of the dystroglycan complex in the cell sarcolemma, these findings indicate that a-dystroglycan membrane. is an extracellular peripheral membrane protein, while B­ dystroglycan is an integral membrane protein of the Biological functions of the dystroglycan complex sarcolemma. The most amazing discovery relevant to the In contras t to dys troph in, w hose ex press ion is physiological functions of a-dystroglycan is that it binds restricted to muscle and nervous tissues, dystroglycan is laminin, a major component of the ECM basal lamina, broadly expressed in a variety of tissues. Northern blot with high affinity (lbraghimov-Beskrovnaya et aI., 1992; analysis demonstrated the expression of a single 5.8 kb Ervasti and Campbell, 1993). Laminin is a heterotrimer dystroglycan mRNA in not only skeletal and cardiac made up of three chains of classes a, Band y , and exists muscles but also non-muscle tissues such as brain, lung, in numerous trimeric isoforms in different tissues (for liver, pancreas , placenta and kidney (Ibraghimov­ review see Burgeson et aI., 1994; Sanes, 1994). Laminin Beskrovnaya et aI. , 1992, 1993). Immunoblot analysis a chains have globular domain (G) repeats in the has demonstrated, on the other hand, that the molecular carboxyl-terminus, and laminin-1, comprising the a I, mass of a-dystroglycan differs between muscle and non­ B I , and yl chains, binds to a-dystroglycan via the last muscle tissues. In striated muscle, the molecular mass of two G repeats in the al chain (Gee et aI., 1993). Later a­ a-dystroglycan is 156 kDa or over, while it is 120 kDa dystroglycan was also shown to bind laminin-2, in lung, kidney, brain and peripheral nerve (lbraghimov­ comprising the a2, B I and y I chains, which is a striated Beskrovnaya et aI., 1992; Evarsti and Campbell, 1993; muscle isoform of laminin (Sunada et aI., 1994). The Gee et aI., 1993; Matsumura et aI., 1993; Yamada et aI. , binding of laminin-I and 2 to a-dystroglycan is Ca2+­ 1994; Smalheiser and Kim, 1995). This difference is dependent. Heparin inhibits the binding of laminin-l explained by different levels of glycosylation of a­ more effectively than laminin-2 (Pall et aI., 1996). On dystroglycan (Ervasti and Campbell, 1993; Ibraghimov­ the cytoplasmic side of the sarcolemma, the carboxyl­ Beskovnaya et aI. , 1993) . Thus far, the celluI.ar terminal tail of B-dystroglycan is anchored to the distribution , molecular organization and biological cysteine-rich/carboxyl-terminal domains of dystrophin functions of the dystroglycan complex have been (Suzuki et aI., 1994; lung et aI. , 1995). The carboxyl­ investigated in detail in striated muscle, kidney, brain terminal domain of dystrophin interacts with al­ and peripheral nerve. syntrophin which, in turn, binds to neuronal nitric oxide synthase, a signaling enzyme (Brenman et aI., 1996). On 1. Striated muscle (Fig. 1) the other hand, the amino-terminal domain of dystrophin interacts with F- (Winder et aI., 1995). These In skeletal muscle, the dystroglycan complex was findings indicate that the DGC spans the sarcolemma to localized in the sarcolemma by immunohistochemistry link the ECM basal lamina with the subsarcolemmal 198 Functions of dystroglycan complex

actin-cytoskeleton and a signaling pathway (Fig. I) . 2. Neuromuscular junction (Fig. 1) In DMD patients and mdx mice , absence of dystrophin causes a severe reduction in all of the DAPs, Immunohistochemical analysis demonstrated that all including the dystroglycan complex , and eventually the components of the DAPs were highly enriched in the leads to mu scle cell death. In the patients with neuromuscular junction (Matsumura et aI., 1992). In LGMD2C , LGMD2D or LGMD2E, the loss of the addition, utrophin, an autosomal homologue of whole sarcoglycan complex leads to muscle cell death. dystrophin, is selectively localized at the neuromuscular Furthermore, deficiency of the laminin a2 chain in the junction, and is associated with the DAPs, that is, the basal lamina leads to muscle cell death in congenital dystroglycan , sarcoglycan and syntrophin complexes muscular dystrophy (CMD) patients and dy mice. Thus, (Matsumura et aI., 1992). Recently a-dystroglycan was the disruption of the linkage anywhere between the shown to bind agrin, a major component of the basal subsarcolemmal cytoskeleton and the ECM via the DGC lamina of the neuromuscular junction, with high affinity leads to muscle cell death (Fig. 2) (for overviews see (Bowe et aI., 1994; Campanelli et aI., 1994; Gee et aI., Campbell, 1995; Ozawa et aI., 1995; Worton , 1995). All 1994; Sugiyama et aI., 1994). Agrin has the G repeats these findings indicate that the DGC contributes to the homologous to those of laminin a chains in the sarcolemmal stability during contraction and stretch of carboxyl-terminus and mediates the clustering of mature muscle cells. At present, the role of the acetylcholine receptors (AChRs) in the postsynaptic dystroglycan complex in muscle cell diffe rentiation membrane of the neuromuscular junction (for overviews remains unknown. see Fallon and Hall, 1994; Carbonetto and Lindenbaum,

Normal DMD

Sarcolemma x F-actin

LGMD2C,D,E CMD

Basal Lamina I x Sarcolemma

Fig. 2. Disruptions of the link between the basal lamina and the cytoskeleton via the DGC in muscular dystrophies. DG : the dystroglycan complex; SG : the sarcoglycan complex. 199 Functions of dystroglycan complex

1995). These findings indicate that, in the neuromuscular agrin-induced clustering of AChRs (Martin and Sanes, junction, agrin is anchored to the submembranous 1995). Although skeletal muscle a -dystroglycan with a utrophin-cytoskeleton via the identical sets of membrane molecular mass of 156 kDa is distributed throughout the proteins as laminin-2 . In the Torpedo postsynaptic sarcolemma and was reported negative for VVAB4 membrane, on the other hand, dystrophin, utrophin or (Martin and Sanes, 1995), a possibility exists that the the components of the sarcoglycan and syntrophin fraction of a-dystroglycan localized and enriched in the complexes are not detected in the isolated dystroglycan neuromuscular junction may be glycosylated differently complex (Bowe et aI. , 1994). from the extrajunctional form and be positive for Similar to laminin-I and 2, the binding of agrin to a­ VVAB . These findings suggest that a-dystroglycan 2 4 dystroglycan is Ca +-dependent and inhibited by may be identical to the VVAB4-positive glycoconjugate heparin . Agrin has several isoforms generated by involved in the agrin-induced clustering of AChRs in the alternative mRNA splicing at two sites in the carboxyl­ neuromuscular junction. In any case, more studies are terminus (named A and B in chick and Y and Z in rat). needed to clarify the precise role of the dystroglycan Agrin isoforms are expressed among many different complex in AChR clustering. tissues, but the neuronal isoform, which has inserts of four and eight amino acids at the A (Y) and B (Z) sites 3. Kidney respectively, has the highest activity of AChR clustering. The neuronal isoform is secreted by nerve terminals and It has been known that the a6B I integrin in the cell accumulates in the junctional basal lamina. Since the surface binds to the E8 fragment of laminin-I and AChR clustering activity of agrin is Ca2+-dependent and antibody against the a6 subunit of integrin interfers with inhibited by heparin , it was proposed that the dystro­ kidney tubule development in vitro. On the other hand, it glycan complex might be the functional agrin receptor has also been known that antibodies to the E3 fragment itself (Bowe et aI., 1994; Gee et aI. , 1994; Campanelli et of laminin-I , which contains the a-dystroglycan-binding aI. , 1994). This was supported by the finding that a­ site, inhibit kidney tubule development, suggesting the dystroglycan was deficient in the mutant muscle cells existence of a cell surface laminin-l-receptor distinct which are defective in proteoglycan synthesis and from the a6B1 integrin. Recently, dystroglycan was response to agrin. The 43 kDa AChR associated protein localized to the basal side of epithelial cells in rapsyn was suggested to function as a linker between the developing kidney, and dystroglycan mRNA, together AChRs and the dystroglycan complex (Apel et aI., with that of laminin a I chain, was shown to increase 1995). during kidney epithelial differentiation (Durbeej et aI., This view is still disputed for several reasons (for 1995). Furthermore, monoclonal antibody IIH6 against more extensive reviews see Fallon and Hall, 1994; a-dystroglycan, which inhibits the binding of Carbonetto and Lindenbaum, 1995) . Firstly, the isoform laminin/agrin to a-dystroglycan, perturbed epithelial of agrin, which lacks amino acid inserts at the A (Y) and development in kidney organ culture (Durbeej et aI. , B (Z) sites and thus is inactive for AChR clustering, 1995). These findings indicate that, in concert with the binds to a-dystroglycan with higher affinity than the a6B I integrin, the dystroglycan complex acts as a active isoform (Sugiyama et aI. , 1994). Secondly, studies receptor of laminin-I during epithelial morphogenesis in looking at the effects of monoclonal antibody IlH6 kidney. against a-dystroglycan, which inhibits the binding of laminin/agrin to a-dystroglycan, on the agrin-induced 4. Brain clustering of AChRs gave somehow confusing results. In three studies, the c lu stering was affected, to varying In brain, biochemical studies had led to the extents, by IIH6 (Campanelli et aI., 1994; Gee et aI. , identification of a laminin-binding protein with a 1994; Cohen et aI. , 1995), whereas it was not affected in molecular mass of 120 kDa, called cranin or LBP 120 one study (Sugiyama et aI., 1994). Thirdly, the domains (Smalheiser and Schwartz, 1987; Gee et aI., 1993). of agrin necessary to induce AChR clustering and the Surprisingly, amino acid sequencing of the purified heparin-binding s ite of agrin were found to be cranin/LBP 120 demonstrated that it was identical to a­ overlapping, but not identical to the a-dystroglycan­ dy troglycan (Gee et aI., 1993; Smalheiser and Kim, binding site (Gesemann et aI., 1996; Hopf and Hoch, 1995). Similar to striated muscle, brain a-dystroglycan 1996). These findings suggest the existence and is complexed with B-dystroglycan. However, the other involvement of another and hitherto- unidentified components of the DGC are apparently not detected in functional agrin receptor for AChR clustering. the isolated brain dystroglycan complex (Gee et aI., It should be noted, on the other hand , that the 120 1993; Smalheiser and Kim, 1995), indicating differences kDa brain and peripheral nerve a-dystroglycan are both in the composition of the proteins associated with the positive for VVAB4 , a lectin which binds GaI/NAc­ dystroglycan complex from skeletal muscle. The cellular terminated saccharides (Smalheiser and Kim, 1995; distribution of the dystroglycan complex in brain is not Yamada et aI., 1996a). Interestingly, VVAB4-positive fu lly established. In situ hybridization has demonstrated glycoconjugates, selectively localized at the neuro­ the expression of dystroglycan mRNA in the muscular junction, were reported to playa role in the hippocampal formation (Anmon's horn and the dentate 200 Functions of dystrog/ycan camp/ex

gyrus), cerebellum, olfactory bulb, thalamus and the dystroglycan-binding site of dystrophin, these proteins choroid plexus (Gorecki et aI. , 1994). However, the are the candidates that anchor the dystroglycan complex localization of a-dystroglycan has been confirmed only in the Schwalm cell outer membrane to the underlying in the regions of synaptic contact upon the Purkinje cell cytoskeleton. dendrites by immunohistochemical analysis (Smalheiser In peripheral nerve, laminin-2 is expressed in the and Kim , 1995). endoneuria I basal lamina (Leivo and Engvall, 1988; Isolated brain a-dystroglycan binds laminin in a Sanes et a!., 1990), and is a ligand of the Schwann cell Ca2+-dependent manner in vitro (Ibraghimov­ dystroglycan complex (Yamada et aI., 1994, 1996a). Beskrovnaya et aI. , 1992; Ervasti and Campbell, 1993; G lycosylation of a-dystroglycan (sialylation in Gee et aI., 1993; Smalheiser and Kim, 1995). However, particular) was implicated in the interaction with laminin basal lamina does not exist surrounding neuronal cells in 2 (Yamada et aI. , 1996a). Similar to brain a-dystro­ mature brain and the native ligands for the neuronal a­ glycan, Schwal1l1 cell a-dystroglycan is HNK- I positive, dystroglycan remain elusive. Neurexins, a large family raising the possibi li ty that the HNK-I carbohydrate of receptors found on nerve terminals, were proposed as epitope may also be involved in the interaction with the candidates, based on the fact that they have the G repeats ECM ligands (Yamada et aI., 1996a). In addition , homologous to those of laminin a chains and agrin in laminin-2, an isoform of agrin, which lacks inserts at the carboxyl-terminus (Carbonetto and Lindenbaum, both A (Y) and B (Z) sites and thus is inactive for AChR 1995). Sugar moieties of brain a-dystrogJycan have clustering, is also expressed in the endoneurial basal been characterized by lectin and antibody staining lamina of peripheral nerve, and is a ligand of the (Smalheiser and Kim, 1995). One interesting feature is Schwann cell dystroglycan complex (Ruegg et a!. , 1992; that brain a-dystroglycan is HNK- I positive, which Yamada et a!., 1996b). The interaction of laminin-2 and indicates that it has 3-sulphated glucuronic acid. Since agrin with Schwann cell a-dystroglycan is Ca2+­ the HNK-I carbohydrate epitope, contained in a number dependent (Yamada et a!., 1994, 1996b). These findings of glycoproteins of nervous system, has been implicated indicate that the dystroglycan complex is a dual receptor in cell adhesion, to laminin in particular (Schachner and for laminin-2 and agrin in the Schwann cell outer Martini, 1995), it would be interesting to see if the membrane. HNK-l carbohydrate epitope is involved in the binding The results of in vitro experiments had shown that of brain a -dystroglycan with the ligands. the deposition of laminin in the basal lamina was Immunohistochemical analysis revealed that a- and essential for ensheathment and myelination by Schwann l3-dystroglycan were expressed in the outer plexiform cells (Eldridge et aI. , 1989; Anton et a!., 1994; Obremski layer of retina, where both dystrophin and utrophin, but and Bunge, 1995). Furthermore, peripheral myelination not laminin, were present (Montanaro et aI. , J 995). In is known to be greatly disturbed in CMD patients and dy situ hybridization identified two neuronal populations, mice deficient in laminin a2 chain (Arahata et aI., 1993; photo receptors and retinal ganglion cells, that expressed Sunada et a!. , 1994; Xu et a!., 1994; Tome et a!., 1994; dystroglycan mRNA (Montanaro et a!., 1995). Helbling-Leclerc et aI. , 1995; ShoreI' et aI., 1995) . These findings, all together, point to the role for the interaction 5. Peripheral nerve of the Schwann cell dystroglycan complex with laminin- 2 in peripheral myelinogenesis. So far, the activity of In peripheral nerve, utrophin, a- and l3-dystroglycan, agri n has been di scussed only in terms of AChR but not dystrophin and a-sarcoglycan, are expressed, clustering, and the biological functions of the agrin indicating differences in the composition of the proteins isoform lacking amino acid inserts at both A (Y) and B associated with the dystroglycan complex from skeletal (Z) sites remain to be elucidated. It would be of interest muscle (Matsumura et aI., 1993). Indeed, the to see if, in addition to laminin-2 , the agrin isoform, components of the sarcoglycan complex were not inactive for AChR clustering, is also involved in detected in the peripheral nerve dystroglycan complex peripheral myelinogenesis. isolated from the detergent extracts of the peripheral nerve membrane fractions using WGA and laminin­ How does the dystroglycan complex mediate diverse affinity chromatography (Yamada et aI. , 1996a). Alpha­ cell differentiation processes? (Fig. 3) and l3-dystroglycan are localized to the Schwalm cell membrane (Yamada et a!. , 1994, 1996a). It is noteworthy At present, the mechanism by which the dystro­ that, in contrast to tbe·other well known myelin proteins, g lycan complex mediates such diverse and specific their expression is restricted to the Schwann cell outer biological processes like sarcolemmaI stabilization, membrane that abuts on the endoneurial basal lamina , epithelial morphogenesis, synaptogenesis and but not the compact myelin or the inner membrane myeli nogenesis is unclear. We may envisage several (Yamada et aI. , 1996a). On the other hand, utrophin and hypothetical scenarios . Firstly, the interaction of Dp116, a 116 kDa protein product of the DMD gene different ECM ligands with the dystroglycan complex (Byers et a!., 1993), are localized in the Schwann cell may transmit downstream intracellularly distinct signals cytoplasm (Yamada et aI. , 1996a). Since both utrophin for each of these processes (Fig . 3a). Differential and Dp 116 are cytoskeletal proteins sharing the 13- glycosylation of a-dystroglycan among different tissues 201 Functions of dystroglycan complex

may also be in volved in this mechani sm. As discussed to the d ys troglycan comple x may e nable the more a bove, however, thi s scena ri o is not nece saril y specific and functional cell surface receptors to interact supported by the recent findings on the agrin-induced w ith these ligands ( Fig . 3c ) . Witho ut the initia l AChR clu stering in the neuromuscul ar junction. inte racti o n w ith the d ystrog lycan comple x , it may The other three scenarios propose the existence and require very hi gh concentrations of these ligands to in vol vement of more specific and functional cell surface inter act with the func tional ce ll s urface receptors. receptors fo r each of the cell di fferentiati on processes Fourthly, the dystroglycan complex may function purely describe d a bove . In the second scena ri o , we may as a structural prote in in the maturational stages of envisage that the interaction of different ECM li gands development in a broad range of cell types; the binding w ith the d ystroglycan complex may transmit intra­ of the ECM ligands to the cell surface dystroglycan cellular signals, which, in turn , induce the more specific complex m ay trigger the re organi zation o f the a nd func ti o na l ce ll s urface recepto rs, s uc h as the s u bme m brano us dystro ph i n/ u troph in-cytoske leton , members of the integrin family for instance, to interact potentiall y via Grb2 and other signaling/adaptor proteins with these ligands (Fig . 3b). Thirdly, the dystroglycan (Fig . 3d ) . This process c ould be c ritical for the complex may function as a helper protein or co-receptor; maturation of the cytoskeletal network that supports the the initial and hi gh-affinity binding of the ECM ligands cell me mbrane . C learl y, m any mor e future s tudies (a) (b) o

.'m.....~.."..J1L"'r..°...... D.,. tL ...... "", .. .. ,.." . ')')"", .., ....¥o'.., . '''''"...... , ...... , ".... ¥ ,,~"'~...--e>...... ¥...0...... ,,".... . t t \ .. 1 t t t t + (c) (d)

t Signal

VNon-Dystroglycan ,eM Ug"d, I Cell Surface Receptor °D [J\' Fig . 3. Schematic model of the biological func tions of the dyslrog lycan complex in cell differentiation. 202

Functions of dystrog/ycan complex

are needed before we wi ll know if any of these Cohen M.W., Jacobson C. , Godfrey E.R ., Campbell K.P. and hypotheses, alone or in various combinations, hold true . Carbonetto S. (1995) . Distribution of a -dystroglycan during embryonic nerve-muscle synaptogenesis. J. Cell BioI. 129, 1093- Acknowledgements. The authors' group was supported by the grants 1101 . from Ciba-Geigy Foundation (Japan) for the Promotion of Science, Nitoh Cullen M.J. , Walsh J. and Nicholson L.V.B. (1994) . Immunogold Foundation, Uehara Memorial Foundation, Kato Memorial Bioscience localization of the 43-kDa dystroglycan at the plasma membrane in Foundation and the Cell Science Research Foundation, the Science control and dystrophic human muscle. Acta Neuropathol. 87, 349- Research Promotion Fund from Japan Private School Promotion 354. Foundation, Research Grant (5A-2) for Nervous and Mental Disorders Deyst K.A. , Bowe M.A., Leszyk J.D. and Fallon J.R. (1995). The a­ from the Ministry of Health and Welfare, and Grants-in-Aid for Scientific dystroglycan-B-dystroglycan complex : membrane organization and Research on Priority Areas, for Scientific Research (07264239, relationship to an agrin receptor. J. BioI. Chem. 270, 25956-25959. 06454280, 08457195 and 06770463) and for Scientific Research on Durbeej M. , Larsson E. , Ibraghimov-Beskrovnaya 0 ., Roberds S.L., Developmental areas (05557037) from the Ministry of Education, Campbell K.P. and Ekblom P. (1995). Non-muscle a-dystroglycan is Science, Sports and Culture. involved in epithelial development. J. Cell BioI. 130, 79-91 . Eldridge C.F., Bunge M.B. and Bunge R.P. (1989). Differentiation of axon-related Schwann cells in vitro: II. Control of myelin formation by References basal lamina. J. Neurosci. 9, 625-638. Ervasti J.M. and Campbell K.P. (1991) . Membrane organization of the Anton E.S. , Sandrock A.W.J. and Mathew W.D. (1994). Merosin dystrophin-glycoprotein complex . Cell 66, 1121-1131. promotes neurite growth and Schwann cell migration in vitro and Ervasti J.M. and Campbell K.P. (1993) . A role for the dystrophin­ nerve regeneration in vivo: evidence using an antibody to merosin , glycoprotein complex as a transmembrane linker between laminin ARM-1. Dev. BioI. 164, 133-145. and actin. J. Cell BioI. 122, 809-823. Apel E.D., Roberds S.L. , Campbell K.P. and Merlie J.P. (1995) . Rapsyn Ervasti J.M., Ohlendieck K., Kahl S.D ., Gaver M.G. and Campbell K.P. may function as a linker between the acetylcholine receptor and the (1990). Deficiency of a glycoprotein component of the dystrophin agrin-binding dystrophin-associated glycoprotein complex. Neuron complex in dystrophiC muscle. Nature 345, 315-319. 15,115-126. Fallon J. R. and Hall Z. W. (1994) . Building synapses: agrin and Arahata K. , Hayashi Y.K., Koga R. , Goto K. , Lee J.H., Miyagoe Y. , Ishii dystroglycan stick together. Trends Neurosci. 17, 469-473. H., Tsukahara T. , Takeda S. , Woo M., Nonaka I. , Matsuzaki T. and Gee S.H. , Blacher R.w., Douville P.J., Provost P.R. , Yurchenco P.D. Sugita H. (1993). Laminin in animal models for muscular dystrophy: and Carbonetto S. (1993). Laminin-binding protein 120 from brain is defect of laminin M in skeletal and cardiac muscles and peripheral closely related to the dystrophin-associated glycoprotein, nerve of the homozygous dystrophic dy/dy mice. Proc. Jpn. Acad . B dystroglycan, and binds with high affinity to the major heparin 69, 259-264. binding domain of laminin. J. BioI. Chem. 268, 14972-14980. Bowe M.A., Deyst K.A. , Leszyk J .D. and Fallon J.R. (1994) . Gee S.H., Montanaro F. , Lindenbaum M.H. and Carbonetto S. (1994). Identification and purification of an agrin receptor from Torpedo Dystroglycan-a, a dystrophin-associated glycoprotein, is a functional postsynaptic membranes: a heteromeric complex related to the agrin receptor. Cell 77, 675-686. . Neuron 12, 1173-1180. Gesemann M., Cavalli V., Denzer A.J., Brancaccio A., Schumacher B. Brancaccio A., Schulthess T. , Gesemann M. and Engel J. (1995). and Ruegg M.A. (1996) . Alternative splicing of agrin alters its Electron microscopiC evidence for a mucin-like region in chick binding to heparin, dystroglycan, and the putative agrin receptor. muscle a-dystroglycan. FEBS Lett. 368,139-142. Neuron 16, 755-767. Brenman J.E., Chao D.S., Gee S.H ., McGee A.W., Craven S.E. , Gorecki D.C ., Derry J.M.J. and Barnard E.A. (1994). Dystroglycan: brain Santillano D.R. , Wu Z., Huang F. , Xia H., Peters M.F., Froehner S.C. localization and chromosome mapping in the mouse. Hum. Mol. and Bredt D.S. (1996) . Interaction of nitric oxide synthase with the Genet. 3, 1589-1597. postsynaptic density protein PSD-95 and a1-syntrophin mediated by Helbling-Leclerc A. , Zhang X., Topaloglu H., Cruaud C., Tesson F., PDZ domains. Cell 84, 757-767. Weissenbach J., Tome F.M.S., Schwartz K., Fardeau M., Burgeson R.E., Chiquet M., Deutzmann R. , Ekblom P., Engel J., Tryggvason K. and Guicheney P. (1995) . Mutations in the laminin Kleinman H., Martin G.R. , Meneguzzi G., Paulsson M. , Sanes J. , a 2-chain gene (LAMA2) cause merosin-deficient congenital Timpl R., Tryggvason K., Yamada Y. and Yurchenco p.o. (1994) . A muscular dystrophy. Nature Genet. 11 , 216-218. new nomenclature for the laminins. Matrix BioI. 14, 209-211 . Hopf C. and Hoch W. (1996) . Agrin binding to a-dystroglycan : domains Byers T.J., Lidov H.G .W. and Kunkel L.M. (1993). An alternative of agrin necessary to induce acetylcholine receptor clustering are dystrophin transcript specific to peripheral nerve. Nature Genet. 4, overlapping but not identical to the a-dystroglycan-binding region. J. 77-81 . BioI. Chem . 271, 5231-5236. Campanelli J.T., Roberds S.L. , Campbell K.P. and Scheller R.H. (1994). Ibraghimov-Beskrovnaya 0., Ervasti J.M., Leveille C.J., Slaugther CA, A role for dystrophin-associated glycoproteins and utrophin in agrin­ Sernett S.W. and Campbell K.P . (1992) . Primary structure of induced AChR clustering. Cell 77, 663-674. dystrophin-associated glycoproteins linking dystrophin to the Campbell K.P. (1995) . Three muscular dystrophies: loss of cyto­ extracellular matrix. Nature 355, 696-702. skeleton-extracellular matrix linkage. Cell 80, 675-679. Ibraghimov-Beskrovnaya 0., Milatovich A., Ozcelik T., Yang B. , Carbonetto S. and Lindenbaum M. (1995). The basement membrane at Koepnick K. , Francke U. and Campbell K.P. (1993). Human the neuromuscular junction : a synaptic mediatrix. Curro Opin. dystroglycan: skeletal muscle cDNA, genomic structure, origin of Neurobiol. 5, 596-605. tissue specific isoforms and chromosomal localization. Hum . Mol. 203 Functions of dystroglycan complex

Genet. 2, 1651 -1657. 15425-15433. Jung D., Yang B., Meyer J., Camberlain J.S. and Campbell K.P. (1995). Smalheiser N.R. and Schwartz N.B. (1987). Cranin, a laminin-binding Identification and characterization of the dystrophin anchoring site protein of cell membranes. Proc. Natl. Acad. Sci . USA 84 , 6457- on B-dystroglycan . J. BioI. Chem . 270, 27305-27310. 6461 . Klietsch R., Ervasti J.M., Arnold W., Campbell K.P. and Jorgensen A.O. Sugiyama J., Bowen D.C. and Hall ZW. (1994). Dystroglycan binds (1993). Dystrophin-glycoprotein complex and laminin colocalize to nerve and muscle agrin. Neuron 13, 103-115. the sarcolemma and transverse tubules of cardiac muscle. Circ. Sunada Y. , Bernier S.M ., Kozak CA, Yamada Y. and Campbell K.P . Res . 72 , 349-360. (1994). Deficiency of merosin in dystrophic dy mice and genetic Leivo I. and Engvall E. (1988). Merosin , a protein specific for basement linkage of laminin M chain gene to dy locus. J. BioI. Chem . 269, membranes of Schwann cells, striated muscle, and trophoblast, is 13729-13732. expressed late in nerve and muscle development. Proc. Natl. Acad . Suzuki A., Yoshida M., Hayashi K., Mizuno Y., Hagiwara Y. and Ozawa Sci. USA 85, 1544-1548. E. (1994). Molecular organization at the glycoprotein-complex­ Martin P.T. and Sanes J.R. (1995). Role for a synapse-specific binding site of dystrophin. Three dystrophin-associated proteins bind carbohydrate in agrin-induced clustering of acetylcholine receptors. directly to the carboxy-terminal portion of dystrophin. Eur. J. Neuron 14, 743-754. Biochem . 220, 283-292. Matsumura K., Ervasti J.M., Ohlendieck K., Kahl S.D. and Campbell Tinsley J.M., Blake D.J., Zuellig A.A. and Davies K.E. (1994). Increasing K. P. (1992). Association of dystrophin-related protein with complexity of the dystrophin-associated protein complex. Proc. Nail. dystrophin-associated proteins in mdx mouse muscle. Nature 360, Acad. Sci. USA 91 , 8307-8313. 588-591 . Tome F.M.S. , Evangelista T., Leclerc A., Sunada Y., Manole E., Matsumura K., Yamada H., Sh imizu T. and Campbell K.P. (1993). Estounert B., Barois A., Campbell K.P. and Fardeau M. (1994). Differential expression of dystrophin, utrophin and dystrophin­ Congenital muscular dystrophy with merosin deficiency. C.R. Acad . associated proteins in peripheral nerve. FEBS Lett. 334, 281-285. Sci. (III ). 317, 351 -357. Montanaro F., Carbonetto S. , Campbell K.P. and Lindenbaum M. Wakayama Y., Shibuya S., Takeda A., Jimi T., Nakamura Y. and Oniki (1995). Dystroglycan expression in the wild type and mdx mouse H. (1995). Ultrastructural localization of the C-terminus of the 43-kd neural retina : synaptic colocalization with dystrophin, dystrophin­ dystrophin-associated glycoprotein and its relation to dystrophin in related protein but not laminin . J. Neurosci. Res . 42, 528-538. normal murine skeletal myofiber. Am . J. Pathol. 146, 189-196. Obremski V.J. and Bunge M.B. (1995). Addition of purified basal-lamina Winder S.J., Hemmings L., Maciver S.K., Bolton S.J., Tinsley J.M., molecules enables Schwann cell ensheathment of sympathetic Davies K.E ., Critchley D.A. and Kendrick-Jones J. (1995) . Utrophin neurites in culture. Dev. BioI. 168, 124-137. actin binding domain: analysis of actin-binding and cellular targeting . Ozawa E., Yosh ida M., Suzuki A., Mizuno Y., Hagiwara Y. and Noguchi J. Cell Sci. 108,63-71 . S. (1995). Dystrophin-associated proteins in muscular dystrophy. Worton A. (1995). Muscular dystrophies: diseases of the dystrophin­ Hum . Mol. Genet. 4, 1711 -1716. glycoprotein complex. Science 270, 755-756. Pall EA, Bolton K.M. and Ervasti J.M. (1996). Differential heparin Xu H., Christmas P. , Wu X.R., Wewer U.M. and Engvall E. (1994). inhibition of skeletal muscle a-dystroglycan binding to laminins. J. Defective muscle basement membrane and lack of M-Iaminin in the BioI. Chem. 271 , 3817-3821. dystrophic dy/dy mouse. Proc. Nail. Acad . Sci. USA 91 , 5572-5576. Ruegg M.A., Tsim KW.K., Horton S.E., Kroger S., Escher G., Gensch Yamada H., Shimizu T., Tanaka T., Campbell K.P. and Matsumura K. E.M. and McMahan U.J. (1992). The agrin gene codes for a family (1994). Dystroglycan is a binding protein of laminin and merosin in of ba sal lamina proteins that differ in function and distribution. peripheral nerve. FEBS Lett. 352, 49-53. Neuron 8, 691 -699 . Yamada H., Chiba A., Endo T. , Kobata A. , Anderson L.V.B., Hori H. , Sanes J.R. (1994). The extracellular matrix. In: Myology. Engel A.G. and Fukuta-Ohi H., Kanazawa I. , Campbell K.P., Shimizu T. and Franzini-Armstrong C. (eds). McGraw-HilI. New York. pp 242-260. Matsumura K. (1996a). Characterization of dystroglycan-Iaminin Sanes J.R., Engvall E., Butskowski R. and Hunter D.O. (1990). interaction in peripheral nerve. J. Neurochem. 66, 1518-1524. Molecular heterogeneity of basal laminae. Isoforms of laminin and Yamada H., Denzer A.J., Hori H., Tanaka T., Anderson L.V.B., Fujita S., collagen IV at the neuromuscular junction and elsewhere. J. Cell Fukuta-Ohi H., Shimizu T., Ruegg M. and Matsumura K. (1996b) . BioI. 111 , 1685-1 699. Dystroglycan is a dual receptor for agrin and laminin-2 in Schwann Schachner M. and Martini A. (1995). Glycans and the modulation of cell membrane. J. BioI. Chem. 271 , 23418-23423. neural-recognition molecule function. Trends Neurosci. 18, 183-191 . Yang B., Jung D., Motto D., Meyer J., Koretzky G. and Campbell K.P. Shorer Z., Philpot J., Muntoni F., Sewry C. and Dubowitz V. (1995). (1995). SH3 domain-mediated interaction of dystroglycan and Grb2. Peripheral nerve involvement in congenital muscular dystrophy. J. J. BioI. Chem . 270, 11711 -11714. Child Neurol. 10, 472-475. Yoshida M., Suzuki A., Yamamoto H., Noguchi S., Mizuno M. and Smalheiser N.R. and Kim E. (1995). Purification of cranin , a laminin Ozawa E. (1994). Dissociation of the complex of dystrophin and its bindin'g membrane protein : identity with dystroglycan and associated proteins into several unique groups by n-octyl B-D ­ reassessment of its carbohydrate moieties. J. BioI. Chem. 270, glucoside. Eur. J. Biochem . 222, 1055-1061 .