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At least 19 recordsLinked to original sources
PubMed1997

Tissue-specific heterogeneity in alpha-dystroglycan sialoglycosylation. Skeletal muscle alpha-dystroglycan is a latent receptor for Vicia villosa agglutinin b4 masked by sialic acid modification.

Because the polypeptide core of alpha-dystroglycan is encoded by a single gene, the difference in apparent molecular mass between alpha-dystroglycans expressed in various tissues is presumably due to differential glycosylation. However, little is presently known about the tissue-specific differences in alpha-dystroglycan glycosylation and whether these modifications may confer functional variability to alpha-dystroglycan. We recently observed that laminin-1 binding to skeletal muscle alpha-dystroglycan was dramatically inhibited by heparin, whereas the binding of commercial merosin to skeletal muscle alpha-dystroglycan was only marginally inhibited (Pall, E. A., Bolton, K. M., and Ervasti, J. M. (1996) J. Biol. Chem. 3817-3821). In contrast to 156-kDa skeletal muscle alpha-dystroglycan, both laminin-1 and merosin binding to 120-kDa brain alpha-dystroglycan were sensitive to heparin. We have now examined the laminin binding properties of 140-kDa alpha-dystroglycan purified from cardiac muscle and observed that like skeletal muscle alpha-dystroglycan, heparin inhibited cardiac alpha-dystroglycan binding to laminin-1, but not to merosin. On the other hand, cardiac and brain alpha-dystroglycans could be distinguished from skeletal muscle alpha-dystroglycan by their reactivity with the terminal GalNAc-specific lectin Vicia villosa agglutinin. Interestingly, skeletal muscle alpha-dystroglycan became reactive with V. villosa agglutinin upon digestion with sialidase from Clostridium perfringens, Arthrobacter neurofaciens, or Streptococcus, but not Vibrio cholerae or Newcastle disease virus sialidase. While none of the sialidase treatments affected the laminin binding properties of alpha-dystroglycan, the sum of our results suggests that skeletal muscle alpha-dystroglycan contains a novel sialic acid residue linked alpha2-6 to GalNAc. These properties are also consistent with the cellular characteristics of a GalNAc-terminated glycoconjugate recently implicated in neuromuscular synaptogenesis. Thus, variations in alpha-dystroglycan sialoglycosylation may prove as useful markers to further elucidate the role of alpha-dystroglycan glycoforms in different tissues and perhaps within a single cell type.

Animals↗
PubMed2001

Identification of the beta-dystroglycan binding epitope within the C-terminal region of alpha-dystroglycan.

Dystroglycan is a receptor for extracellular matrix proteins that plays a crucial role during embryogenesis in addition to adult tissue stabilization. A precursor product of a single gene is post-translationally cleaved to form two different subunits, alpha and beta. The extracellular alpha-dystroglycan is a membrane-associated, highly glycosylated protein that binds to various extracellular matrix molecules, whereas the transmembrane beta-dystroglycan binds, via its cytosolic domain, to dystrophin and many other proteins. alpha- and beta-Dystroglycan interact tightly but noncovalently. We have previously shown that the N-terminal region of beta-dystroglycan, beta-DG(654-750), binds to the C-terminal region of murine alpha-dystroglycan independently from glycosylation. Preparing a series of deleted recombinant fragments and using solid-phase binding assays, the C-terminal sequence of alpha-dystroglycan containing the binding epitope for beta-dystroglycan has been defined more precisely. We found that a region of 36 amino acids, from position 550-585, is required for binding the extracellular region, amino acids 654-750 of beta-dystroglycan. Recently, a dystroglycan-like gene was identified in Drosophila that showed a moderate degree of conservation with vertebrate dystroglycan (31% identity, 48% similarity). Surprisingly, the Drosophila sequence contains a region showing a higher degree of identity and conservation (45% and 66%) that coincides with the 550-585 sequence of vertebrate alpha-dystroglycan. We have expressed this Drosophila dystroglycan fragment and measured its binding to the extracellular region of vertebrate (murine) beta-dystroglycan (Kd = 6 +/- 1 microM). These data confirm the proper identification of the beta-dystroglycan binding epitope and stress the importance of this region during evolution. This finding might help the rational design of dystroglycan-specific binding drugs, that could have important biomedical applications.

Amino Acid Sequence↗
PubMed2001

Processing of beta-dystroglycan by matrix metalloproteinase disrupts the link between the extracellular matrix and cell membrane via the dystroglycan complex.

The dystroglycan complex is a membrane-spanning complex composed of two subunits, alpha- and beta-dystroglycan. alpha-dystroglycan is a cell surface peripheral membrane protein which binds to the extracellular matrix (ECM), whereas beta-dystroglycan is an integral membrane protein which anchors alpha-dystroglycan to the cell membrane. The dystroglycan complex provides a tight link between the ECM and cell membrane. Dysfunction of the dystroglycan complex has commonly been implicated in the molecular pathogenesis of severe forms of hereditary neuromuscular diseases, including Duchenne muscular dystrophy, Fukuyama-type congenital muscular dystrophy and sarcoglycanopathy (LGMD2C, -D, -E and -F). To begin to clarify the pathway by which the dysfunction of the dystroglycan complex could lead to muscle cell degeneration, we investigated the proteolytic processing of the dystroglycan complex in this study. We demonstrate that (i) a 30 kDa fragment of beta-dystroglycan is expressed in peripheral nerve, kidney, lung and smooth muscle, but not skeletal muscle, cardiac muscle or brain, and (ii) this fragment is the product of proteolytic processing of the extracellular domain of beta-dystroglycan by the membrane-associated matrix metalloproteinase (MMP) activity. Importantly, furthermore, we demonstrate that this processing disintegrates the dystroglycan complex. Our results indicate that the processing of beta-dystroglycan by MMP causes the disruption of the link between the ECM and cell membrane via the dystroglycan complex, which could have profound effects on cell viability. Based on these and previously reported findings, we propose a hypothesis that this processing may play a crucial role in the molecular pathogenesis of sarcoglycanopathy.

Animals↗
PubMed1995

The alpha-dystroglycan-beta-dystroglycan complex. Membrane organization and relationship to an agrin receptor.

Aberrant expression of the dystrophin-associated protein complex is thought to underlie the pathogenesis of Duchenne dystrophy, Becker muscular dystrophy, and severe childhood autosomal recessive muscular dystrophy. Recently, our laboratory identified an agrin receptor from Torpedo electric organ postsynaptic membranes. It is a heteromer of 190- and 50-kDa subunits with similarity to two components of the dystrophin-associated protein complex of alpha- and beta-dystroglycan. We now confirm the relationship between the Torpedo agrin receptor and mammalian dystroglycans and provide further information about the structure of the alpha-dystroglycan-beta-dystroglycan complex. The sequences of three peptides from each Torpedo subunit were 69% identical to mammalian dystroglycans. An antiserum to mammalian beta-dystroglycan recognizes the Torpedo 50-kDa polypeptide. Additionally, like alpha-dystroglycan, the 190-kDa agrin receptor subunit binds laminin. Previous studies have indicated that alpha- and beta-dystroglycan arise by cleavage of a precursor protein. Tryptic peptide mapping of both subunits and amino-terminal sequencing of Torpedo beta-dystroglycan indicate a single cleavage site, corresponding to serine 654 of the mammalian dystroglycan precursor. Gel electrophoresis analysis indicates there is at least one intrachain disulfide bond in beta-dystroglycan. These results provide precise primary structures for alpha- and beta-dystroglycan.

Agrin↗
PubMed1996

Agrin binding to alpha-dystroglycan. Domains of agrin necessary to induce acetylcholine receptor clustering are overlapping but not identical to the alpha-dystroglycan-binding region.

The synaptic basal membrane protein agrin initiates the aggregation of acetylcholine receptors at the postsynaptic membrane of the developing neuromuscular junction. Recently, alpha-dystroglycan was found to be a major agrin-binding protein on the muscle cell surface and was therefore considered a candidate agrin receptor. Employing different truncation fragments of agrin, we determined regions of the protein involved in binding to alpha-dystroglycan and to heparin, an inhibitor of alpha-dystroglycan binding. Deletion of a 15-kDa fragment from the C terminus of agrin had no effect on its binding to alpha-dystroglycan from rabbit muscle membranes, even though this deletion completely abolishes its acetylcholine receptor aggregating activity. Conversely, deletion of a central region does not affect agrin's clustering activity, but reduced its affinity for alpha-dystroglycan. Combination of these two deletions resulted in a fragment of approximately 35 kDa that weakly bound to alpha-dystroglycan, but displayed no clustering activity. All of these fragments bound to heparin with high affinity. Thus, alpha-dystroglycan does not show the binding specificity expected for an agrin receptor. Our data suggest the existence of an additional component on the muscle cell surface that generates the observed ligand specificity.

Agrin↗
PubMed1997

Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of alpha-dystroglycan with laminin.

alpha-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein beta-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve alpha-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the alpha-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the alpha-dystroglycan with laminin.

Animals↗
PubMed2001

A synthetic peptide corresponding to the 550-585 region of alpha-dystroglycan binds beta-dystroglycan as revealed by NMR spectroscopy.

We have probed the binding of a synthetic peptide corresponding to the region 550-585 of the alpha subunit of dystroglycan with a recombinant protein fragment corresponding to the N-terminal extracellular region of beta-dystroglycan (654-750), using NMR in solution. In a 30:1 molar ratio, the peptide binds to the recombinant protein fragment in the fast/intermediate exchange regime. By monitoring the peptide intra-residue HN-Halpha peak volumes of the 2D TOCSY NMR spectra, both in the absence and in the presence of the recombinant fragment, we determined the differential binding affinities of each amino acid. We found that the residues in the region 550-565 (SWVQFNSNSQLMYGLP) are more influenced by the presence of the protein, whereas the C-terminal portion is marginally involved. These NMR results have been confirmed by solid-phase binding assays.

Amino Acid Motifs↗
PubMed1997

A role of dystroglycan in schwannoma cell adhesion to laminin.

Dystroglycan is encoded by a single gene and cleaved into two proteins alpha- and beta-dystroglycan by posttranslational processing. Recently, alpha-dystroglycan was demonstrated to be an extracellular laminin-binding protein anchored to the cell membrane by a transmembrane protein beta-dystroglycan in striated muscle and Schwann cells. However, the biological functions of the dystroglycan-laminin interaction remain obscure, and in particular, it is still unclear if dystroglycan plays a role in cell adhesion. In the present study, we characterized the role of dystroglycan in the adhesion of schwannoma cells to laminin-1. Immunochemical analysis demonstrated that the dystroglycan complex, comprised of alpha- and beta-dystroglycan, was a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. It also demonstrated the presence of alpha-dystroglycan, but not beta-dystroglycan, in the culture medium, suggesting secretion of alpha-dystroglycan by RT4 cells. RT4 cells cultured on dishes coated with laminin-1 became spindle in shape and adhered to the bottom surface tightly. Monoclonal antibody IIH6 against alpha-dystroglycan was shown previously to inhibit the binding of laminin-1 to alpha-dystroglycan. In the presence of IIH6, but not several other control antibodies in the culture medium, RT4 cells remained round in shape and did not adhere to the bottom surface. The adhesion of RT4 cells to dishes coated with fibronectin was not affected by IIH6. The known inhibitors of the interaction of alpha-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, also perturbed the adhesion of RT4 cells to laminin-1, whereas the reagents which do not inhibit the interaction, including dextran, chondroitin sulfate, dermatan sulfate, and GlcNAc, did not. Altogether, these results support a role for dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells.

Animals↗
PubMed2003

Inhibition of dystroglycan cleavage causes muscular dystrophy in transgenic mice.

Dystroglycan (DG) is an essential component of the dystrophin-glycoprotein complex, a molecular scaffold that links the extracellular matrix to the actin cytoskeleton. Dystroglycan protein is post-translationally cleaved into alpha dystroglycan, a highly glycosylated peripheral membrane protein, and beta dystroglycan, a transmembrane protein. Despite clear evidence of the importance of dystroglycan and its associated proteins in muscular dystrophy, the purpose of dystroglycan proteolysis is unclear. By introducing a point mutation at the normal site of proteolysis (serine 654 to alanine, DGS654A), we have created a dystroglycan protein that is severely inhibited in its cleavage. Transgenic expression of DGS654A in mouse skeletal muscles inhibited the expression of endogenously cleaved dystroglycan, while overexpression of wild type dystroglycan by similar amounts did not. DGS654A animals had increased serum creatine kinase activity and most muscles had increased numbers of central nuclei. Overexpression of wild type dystroglycan, by contrast, caused no dystrophy by these measures. Dystrophy in DGS654A muscles correlated with reduced binding of antibodies that recognize glycosylated forms of alpha dystroglycan. Lastly, neuromuscular junctions in DGS654A muscles were aberrant in structure. These data show that aberrant processing of the dystroglycan polypeptide causes muscular dystrophy and suggest that dystroglycan processing is important for the proper glycosylation of alpha dystroglycan.

Animals↗
PubMed2000

Expression of dystroglycan and laminin-2 in peripheral nerve under axonal degeneration and regeneration.

In Schwann cells, the transmembrane glycoprotein beta-dystroglycan composes the dystroglycan complex, together with the extracellular glycoprotein alpha-dystroglycan which binds laminin-2, a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerve, the expression of beta-dystroglycan and laminin-alpha2 chain was studied in rat sciatic nerves undergoing axonal degeneration and regeneration as well as in normal condition. In normal sciatic nerve, immunoreactivity for the cytoplasmic domain of beta-dystroglycan was consistently and selectively localized in the Schwann cell cytoplasm underlying the outer (abaxonal) membrane apposing the basal lamina. While beta-dystroglycan expression was gradually down-regulated in Schwann cells losing contact with axons during axonal degeneration, it was progressively up-regulated as the regenerating process of ensheathment and myelination proceeded during regeneration. Interestingly, beta-dystroglycan expression, when detectable, was always restricted to the Schwann cell cytoplasm beneath the outer membrane apposing the basal lamina during both axonal degeneration and regeneration. Furthermore, laminin-alpha2 immunoreactivity roughly paralleled that of beta-dystroglycan during both axonal degeneration and regeneration, indicating that the expression of beta-dystroglycan and laminin-alpha2 is induced and maintained by the Schwann cell contact with axons. Our results indicate that the dystroglycan complex is involved in the adhesion of the Schwann cell outer membrane with the basal lamina and suggest that the dystroglycan complex may play a role in the process of Schwann cell ensheathment and myelination through the interaction with laminin-2.

Animals↗
PubMed2001

Dystroglycan is present in rat thyroid and rat thyroid cells and responds to thyrotropin.

Dystroglycan is a high affinity laminin-binding glycoprotein originally described as a member of the dystrophin-associated glycoprotein complex in muscle. We have demonstrated the presence of dystroglycan in the thyroid using immunocytochemistry, immunoblots, ligand binding assays, and relative quantitative RT-PCR. In intact rat thyroid glands, antibodies against the alpha (extracellular, laminin-binding subunit) and beta (cytoplasmic/membrane bound) portions of the dystroglycan protein reacted at basolateral membranes where they colocalized with laminin. Western-blotted protein from the Fischer rat thyroid cell line FRTL-5 reacted with both the alpha- and beta-dystroglycan antibodies. The alpha-dystroglycan-reactive band colocalized with laminin-binding activity, and the protein and binding activity were decreased by TSH. In contrast, in the culture medium of these cells, alpha-dystroglycan was increased by TSH. The beta-dystroglycan antibody recognized the full-length 43-kDa band and an approximately 30-kDa truncated form. The truncated form was reduced in cells cultured with TSH, whereas the full-length form was not significantly diminished by TSH. Immunofluorescence of FRTL-5 cells in the absence of TSH showed a colocalization of dystroglycan and laminin. This was disrupted by the addition of TSH and was correlated to morphological changes. PCR amplification of complementary DNA with primer pairs from alpha- and beta-dystroglycan produced appropriately sized bands, whose sequence had identical protein-coding sequences and more than 96% nucleotide homology to mouse dystroglycan sequences. Relative quantitative RT-PCR of beta-dystroglycan messenger RNA showed reduced expression in cells cultured with TSH. We conclude that dystroglycan is present in rat thyroid and in FRTL5 rat thyroid cells and that TSH reduces its expression.

Animals↗
PubMed1997

Subcellular concentration of beta-dystroglycan in photoreceptors and glial cells of the chick retina.

Mutations in the dystrophin-glycoprotein complex cause muscle degeneration and dysfunctions in the central nervous system, including an impaired synaptic transmission in the outer plexiform layer (OPL) of the retina. To investigate the basis for this ocular phenotype, we analyzed the distribution of beta-dystroglycan, a central member of the dystrophin-glycoprotein complex, in the chick retina by using the 43DAG/8D5 monoclonal antibody. This antibody reacted specifically with chick beta-dystroglycan, as indicated by its staining of the neuromuscular junction, and its reactivity with a single 43-kilodalton band in Western blots. In the retina, beta-dystroglycan was highly concentrated in the OPL and at the vitreal border of the retina, around the inner limiting membrane. Mechanically isolated and flat-mounted inner limiting membranes were stained by the anti-beta-dystroglycan antibody, and this immunoreactivity could be extracted by detergent, indicating that beta-dystroglycan is associated with membranous structures bound to the basal lamina. Consistently, electron microscopy showed a concentration of beta-dystroglycan in the endfeet of Müller glial cells exclusively in the region of direct contact to the inner limiting membrane. In the OPL, beta-dystroglycan immunoreactivity was concentrated in the distal extensions of rod and cone terminals protruding into the outer plexiform layer. There, beta-dystroglycan codistributed with the alpha1beta subunit of the N-type voltage-gated calcium channel. By contrast to previous reports, we did not detect beta-dystroglycan directly associated with the synaptic regions of conventional or ribbon synapses of the retina. These results show that in the retina beta-dystroglycan is exclusively expressed by photoreceptors and glial cells and that beta-dystroglycan is highly concentrated in subcellular regions of glial cell endfeet and photoreceptor terminals. Moreover, the colocalization of beta-dystroglycan with N-type calcium channels in the outer plexiform layer indicates that both proteins might be part of a macromolecular complex.

Agrin↗
PubMed2000

Dystroglycan overexpression in vivo alters acetylcholine receptor aggregation at the neuromuscular junction.

Dystroglycan is a member of the transmembrane dystrophin glycoprotein complex in muscle that binds to the synapse-organizing molecule agrin. Dystroglycan binding and AChR aggregation are mediated by two separate domains of agrin. To test whether dystroglycan plays a role in receptor aggregation at the neuromuscular junction, we overexpressed it by injecting rabbit dystroglycan RNA into one- or two-celled Xenopus embryos. We measured AChR aggregation in myotomes by labeling them with rhodamine-alpha-bungarotoxin followed by confocal microscopy and image analysis. Dystroglycan overexpression decreased AChR aggregation at the neuromuscular junction. This result is consistent with dystroglycan competition for agrin without signaling AChR aggregation. It also supports the hypothesis that dystroglycan is not the myotube-associated specificity component, (MASC) a putative coreceptor needed for agrin to activate muscle-specific kinase (MuSK) and signal AChR aggregation. Dystroglycan was distributed along the surface of muscle membranes, but was concentrated at the ends of myotomes, where AChRs normally aggregate at synapses. Overexpressed dystroglycan altered AChR aggregation in a rostral-caudal gradient, consistent with the sequential development of neuromuscular synapses along the embryo. Increasing concentrations of dystroglycan RNA did not further decrease AChR aggregation, but decreased embryo survival. Development often stopped during gastrulation, suggesting an essential, nonsynaptic role of dystroglycan during this early period of development.

Animals↗
PubMed2003

Localization of phospho-beta-dystroglycan (pY892) to an intracellular vesicular compartment in cultured cells and skeletal muscle fibers in vivo.

beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. Tyrosine 892 is now thought to be the principal site for recognition by the c-Src tyrosine kinase; however, little is known about the regulation of this phosphorylation event in vivo. Here, we generated a novel monoclonal antibody probe that recognizes only tyrosine 892 phosphorylated beta-dystroglycan (pY892). We show that upon tyrosine phosphorylation, beta-dystroglycan undergoes a profound change in its sub-cellular localization (e.g., from the plasma membrane to an internal membrane compartment). One possibility is that the net negative charge at position 892 causes the redistribution of beta-dystroglycan to this intracellular vesicular location. In support of this notion, mutation of tyrosine 892 to glutamate (Y892E) is sufficient to drive this intracellular localization, while other point mutants (Y892F and Y892A) remain at the plasma membrane. Interestingly, our colocalization studies with endosomal markers (EEA1, transferrin, and transferrin receptor) suggest that these phospho-beta-dystroglycan containing internal vesicles represent a subset of recycling endosomes. At the level of these internal vesicular structures, we find that tyrosine phosphorylated beta-dystroglycan is colocalized with c-Src. In addition, we demonstrate that known ligands for alpha-dystroglycan, namely, agrin and laminin, are able to induce the tyrosine phosphorylation of beta-dystroglycan. Finally, we show that tyrosine phosphorylated beta-dystroglycan is also detectable in skeletal muscle tissue lysates and is localized to an internal vesicular membrane compartment in skeletal muscle fibers in vivo. The generation of a phospho-specific beta-dystroglycan (pY892) mAb probe provides a new powerful tool for dissecting the role of dystroglycan phosphorylation in normal cellular functioning and in the pathogenesis of muscular dystrophies.

Amino Acid Sequence↗
PubMed2001

Dystroglycan binding to laminin alpha1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro.

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.

Animals↗
PubMed1999

Biochemical characterization of the epithelial dystroglycan complex.

Dystroglycan is a widely expressed extracellular matrix receptor that plays a critical role in basement membrane formation, epithelial development, and synaptogenesis. Dystroglycan was originally characterized in skeletal muscle as an integral component of the dystrophin glycoprotein complex, which is critical for muscle cell viability. Although the dystroglycan complex has been well characterized in skeletal muscle, there is little information on the structural composition of the dystroglycan complex outside skeletal muscle. Here we have biochemically characterized the dystroglycan complex in lung and kidney. We demonstrate that the presence of sarcoglycans and sarcospan in lung reflects association with dystroglycan in the smooth muscle. The smooth muscle dystroglycan complex in lung, composed of dystroglycan, dystrophin/utrophin, beta-, delta-, epsilon-sarcoglycan, and sarcospan, can be biochemically separated from epithelial dystroglycan, which is not associated with any of the known sarcoglycans or sarcospan. Similarly, dystroglycan in kidney epithelial cells is not associated with any of the sarcoglycans or sarcospan. Thus, our data demonstrate that there are distinct dystroglycan complexes in non-skeletal muscle organs as follows: one from smooth muscle, which is associated with sarcoglycans forming a similar complex as in skeletal muscle, and one from epithelial cells.

Animals↗
PubMed1995

Non-muscle alpha-dystroglycan is involved in epithelial development.

The dystroglycan complex is a transmembrane linkage between the cytoskeleton and the basement membrane in muscle. One of the components of the complex, alpha-dystroglycan binds both laminin of muscle (laminin-2) and agrin of muscle basement membranes. Dystroglycan has been detected in nonmuscle tissues as well, but the physiological role in nonmuscle tissues has remained unknown. Here we show that dystroglycan during mouse development in nonmuscle tissues is expressed in epithelium. In situ hybridization revealed strong expression of dystroglycan mRNA in all studied epithelial sheets, but not in endothelium or mesenchyme. Conversion of mesenchyme to epithelium occurs during kidney development, and the embryonic kidney was used to study the role of alpha-dystroglycan for epithelial differentiation. During in vitro culture of the metanephric mesenchyme, the first morphological signs of epithelial differentiation can be seen on day two. Northern blots revealed a clear increase in dystroglycan mRNA on day two of in vitro development. A similar increase of expression on day two was previously shown for laminin alpha 1 chain. Immunofluorescence showed that dystroglycan is strictly located on the basal side of developing kidney epithelial cells. Monoclonal antibodies known to block binding of alpha-dystroglycan to laminin-1 perturbed development of epithelium in kidney organ culture, whereas control antibodies did not do so. We suggest that the dystroglycan complex acts as a receptor for basement membrane components during epithelial morphogenesis. It is likely that this involves binding of alpha-dystroglycan to E3 fragment of laminin-1.

Animals↗
PubMed2001

A stoichiometric complex of neurexins and dystroglycan in brain.

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. We have now identified alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. Our data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells.

Alternative Splicing↗