Dystroglycan: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Dystroglycan

Synonyms -

Cytological map position - 52D2--15

Function - receptor

Keywords - cell polarity, oogenesis

Symbol - Dg

FlyBase ID: FBgn0034072

Genetic map position - 2-

Classification - N-terminal mucin-like domain; transmembrane domain; C-terminal region with WW-, SH2- and SH3-binding domains

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | Entrez Gene | UniGene |
BIOLOGICAL OVERVIEW

The transmembrane protein Dystroglycan is a highly glycosylated central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin (see Drosophila Laminin A), agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Drosophila Dystroglycan (Dg) is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity (Deng, 2003).

Muscular dystrophies are genetic disorders that are characterized by progressive muscle degeneration. These diseases are caused by mutations in different members of the Dystrophin-associated glycoprotein complex (DGC), which is composed of multiple cytocortical, transmembrane and extracellular proteins (Burton, 2002; Henry, 1999; Winder, 2001). How these mutations cause the observed muscle defects is not fully understood and no cures for the diseases exist. In addition to muscle cells, the DGC is required in other cell types such as epithelial cells and neural cells (Durbeej, 1995; Durbeej, 1999; Williamson, 1997; Michele, 2002; Moore, 2002). Reduced expression of the DGC components is observed in breast and prostate cancers (Henry, 2001a). Dystroglycan (DG), a transmembrane protein, is a central player of the DGC. It acts as a receptor for the extracellular matrix (ECM) component Laminin (Ibraghimov-Beskrovnaya, 1992), and connects to the actin cytoskeleton through an actin-binding protein, Dystrophin (Deng, 2003).

The cellular function and regulation of the interactions of Dystroglycan remain elusive. Drosophila is an excellent model organism with which to study basic cellular functions of evolutionarily conserved genes, particularly human disease genes. The DG homolog in Drosophila has been analyzed and shown to be required for the establishment of cellular polarity (Deng, 2003).

Since Dg is highly expressed in the follicle cells, it was first asked whether Dg plays a role in establishing or maintaining epithelial morphology in this tissue. The follicle cell epithelium (FE) has a typical apical-basal polarity, with its apical side facing the germline cells. Since all follicle cells are derived from two to three somatic stem cells, mosaic analysis provides an excellent tool with which to study gene functions in epithelial development (Deng, 2003).

The FLP/FRT system was used to generate follicle cell clones of all three Dg alleles, and the Gal4/UAS-mediated RNAi technique was applied to silence DG expression in all follicle cells (tubP-Gal4/dsDG). Similar phenotypes are observed in these different Dg mutant backgrounds. Some mutant cells lost their epithelial appearance and formed multiple layers, a typical terminal phenotype for polarity defects in epithelial cells. Within the multi-layer groups, the mutant cells from the mosaic egg chambers were frequently excluded from the layer that contacts the germline cells. Discontinuity of the epithelium was also visible in egg chambers containing Dg follicle cell clones. These phenotypes are similar to loss-of-function phenotypes of crumbs (crb), dlt, dlg or lgl in follicle cells and suggest that DG is required for proper epithelial polarity. The mutant follicle cells eventually die off; clones 9-10 days after heat shock were rarely seen, while sister clones (twin spots) were readily observed (Deng, 2003).

To characterize the apicobasal polarity defect in more detail, the expression and distribution of molecular markers were examined in mutant cells that still maintained their columnar shape. In Dg follicle cell clones and tubP-Gal4/dsDG follicle cells, mislocalization of apical markers, Dlt and ß-Heavy-Spectrin (ßH-Spec) was observed. Instead of a strict apical localization, Dlt and ßH-Spec were present at both the apical and basal sides of the mutant epithelia. Dlg, a basolateral marker, exhibits a significant reduction of staining in the basolateral domain in Dg RNAi follicle cells. The function of Dg in apicobasal polarity formation is not restricted to the FE, since mislocalization of Dlt to the lateral and basal sides was also observed in the mutant epithelial cells in an antennal disc. Taken together, these results suggest that DG is required in different epithelial cells for proper formation or maintenance of apicobasal polarity (Deng, 2003).

To ask whether Dg, when overexpressed, is sufficient to interfere with the epithelial cell polarity two UAS constructs were used, the full-length DG-construct (UAS-DG) and the short construct with cytoplasmic and transmembrane domains (UAS-DGcyto). These constructs were expressed in the follicular epithelium (FE) and in the embryonic salivary glands. Both constructs expressed proteins of the expected sizes and were induced by the following Gal4 driver lines: daughterlessGal4 (daGal4), for maternal expression; elavGal4, for the salivary gland expression, and the flip-out Gal4 system for the FE expression. Similar defects in epithelial polarity were observed with all three drivers (Deng, 2003).

In wild-type salivary glands, Crb is localized to the apical membrane of the epithelium, facing the lumen of the gland, while Dg expression is undetectable. Embryos that overexpress Dg show strong ectopic Dg staining on both the apical and basolateral membranes of the salivary gland. In about 75% of these salivary glands, the expression of Crb was strongly reduced. Whereas Crb localization is disrupted by overexpression of full-length Dg (UAS-DG), it is unaffected by overexpression of the form of DG lacking the extracellular domain (UAS-DGcyto). These results suggest that the mislocalization of Crb was not due to nonspecific interference with the secretory apparatus but due to a defect in cell polarity. The lateral membrane domain was unaffected as assayed by the localization of Neurotactin, a lateral marker. As seen in the salivary glands, follicle cells that overexpressed Dg lose the apical markers, ßH-Spec and Bazooka (Baz), while normal apical localization of these proteins is observed in neighboring wild-type cells. Again, overexpression of the DGcyto-form does not cause any obvious defects in the follicle epithelial polarity (Deng, 2003).

Since Laminin A is required in the posterior follicle cells for proper oocyte polarity at stages 7-10, attempts were made to ask whether Dg functions in the germline cells to receive the polarity signal from the Laminin ECM by clonal analysis. Unfortunately, egg chambers bearing germline clones of all deletion alleles are arrested at previtellogenic stages, prior to the stage at which signaling could be detected between the posterior follicle cells and the oocyte. Therefore, the establishment of oocyte polarity in earlier stages, a process that is marked by a posterior movement of the MTOC, was examined. During these stages, a low-level expression of Dg is detected at the oocyte membrane (Deng, 2003).

To detect whether the early oocyte polarity is properly established in Dg germline clones, the localization was examined of two MTOC markers, Nod-ß-Galactosidase (Nod-ß-Gal) and ORB, which (in the wild type) are localized at the anterior of the oocyte at stage 1 and move to the posterior in later stages. Mislocalization of both markers was observed in the mutant germline clones [Nod-ß-Gal mislocalization: 60%; ORB mislocalization: 76% in Dg323; 60% Dg248]. In half of the mislocalization cases, the markers either remained in the anterior of the oocyte or surrounded the nuclei after stage 3. The remaining egg chambers exhibited diffuse staining. Compared with wild type, the staining was significantly reduced. Furthermore, no accumulation of alpha-tubulin was observed in the mutant oocytes, while normal posterior accumulation was detected in the control oocytes between stages 2 and 6. In conclusion, these data suggest that Dg is required in the early oocyte for the maintenance or translocation of the MTOC from the anterior to the posterior of the oocyte. This step is crucial in establishing AP polarity in the oocyte and the future embryo (Deng, 2003).

Although links between DG and MT cytoskeleton have been suggested (Lumeng, 1999), the linkage between DG and the actin cytoskeleton via dystrophin/utrophin is far more evident. Therefore the actin distribution was examined in the developing oocyte in the wild-type and Dg germline clones. Actin is known to be enriched at the cortex of early wild-type oocytes. Interestingly, this actin enrichment is disrupted in the Dg germline clones. In addition, 'spreading' of the ring canals normally observed in stage 1-2 oocyte is not detected in egg chambers that lack germline Dg (Deng, 2003).

At the basal side of the FE, actin filaments have a planar polarity that is perpendicular to the long axis, the AP axis, of the egg chamber. Integrins and receptor tyrosine phosphatase Lar are involved non-cell-autonomously in organizing this basal actin orientation. In an analysis of the ßH-Spec staining in follicle cells that express dsDG, it was noticed that ßH-Spec is mislocalized to the basal side of the FE to bind the basal actin fibers. Noticeably, the fibers decorated with ßH-Spec in different follicle cells appear to be oriented in a random fashion. To test whether this defect reflects problems in basal actin orientation, planar polarity of the actin arrays was analyzed in control egg chambers and in the mutant Dg follicle cell clones. Instead of normal perpendicular orientation to the AP axis, random misorientation was observed in the Dg mutant egg chambers. Moreover, the basal actin fibers in follicle cells adjacent to the mutant clones were also misoriented, revealing a non-cell autonomous requirement for DG function. Although the actin filaments are not organized perpendicular to the AP axis in the mutant cells, they align with the neighboring cells, suggesting that some communication of the orientation from one cell to the other still exists. These results suggest that Dg has a non-cell-autonomous role in organizing the actin cytoskeleton in the follicle cells, similar to other receptors such as Integrin and Lar. Losing any one of these receptors still allows some orientation transfer but the global direction is defective suggesting that multiple receptor-ECM interactions are required for precise orientation (Deng, 2003).

Thus, Dg is required cell-autonomously for polarizing two different cell types: epithelial cells and the oocyte. In Dg mutant epithelial cells, apicobasal polarity is disrupted, while in oocytes, anteroposterior polarity is abnormal. Loss of Dg function in follicle and disc epithelia results in expansion of apical markers to the basal side of the cells and overexpression results in a seemingly opposite phenotype, reduced localization of apical markers. Dg mutations in the germline, however, disturb the enrichment of the oocyte cortical actin and the movement of the MTOC to the posterior oocyte: a process that is the prerequisite for the establishment of all polarity within the egg chamber and embryo. In addition, Dg has a non-cell-autonomous effect on the planar polarity of basal F-actin in follicle cells. The non-cell-autonomous phenotype probably results from a lack of instructive interaction between the actin cytoskeleton and the ECM, since Dg is required for and sufficient in organizing Laminin in follicle cells (Deng, 2003).

Drosophila Dg contains most of the hallmarks of vertebrate DG, but is significantly longer than its vertebrate orthologs, due to a ~250 amino acid duplication in the extracellular domain. The N-terminal half of fly Dg harbors a mucin-like domain, similar to vertebrate DG, but is otherwise only weakly conserved. Since the mucin-like sugars have been implicated in Laminin binding it is interesting to note that splicing variants of Dg that lack exon 8, also lack most of this domain. In addition, altered glycosylation of DG is related to two forms of congenital muscular dystrophy (Brockington, 2001; Hayashi, 2001; Michele, 2002; Moore, 2002, and reduced expression of DG is observed in a mouse model (Ervasti, 1993) of Duchene's muscular dystrophy (Deng, 2003).

The C-terminal half of Drosophila DG is conserved with 31% identity (46% similarity). Especially well conserved are the protein-protein interaction sites in the cytoplasmic domain of DG, including the binding site for Dystrophin. Seven of the eight amino residues that are crucial for Dystrophin binding (Huang, 2000) are conserved in Drosophila. Recent studies demonstrate that phosphorylation of the tyrosine residue within the dystrophin/utrophin binding motif can interfere with binding to utrophin, leading to recruitment of SH2 domain proteins (Sotgia, 2001; Ilsley, 2002). The putative SH2-binding motif involved in this interaction is conserved in Drosophila. The third protein-protein interaction described for vertebrate DG is the binding of the SH2-SH3 adaptor GRB2. GRB2 helps initiate the Ras-MAP kinase signal transduction cascade and is involved in controlling cytoskeletal organization (Yang, 1995). However, the SH3-binding motif, thought to mediate GRB2 binding, is not fully conserved in Drosophila (Deng, 2003).

Reduced expression of DG is often associated with tumor formation, suggesting that DG can act as a tumor suppressor (Henry, 2001a). It is likely that loss of DG function in some cancers leads to abnormal cell-ECM interactions and thus contributes to progression to a metastatic state. Defects in epithelial interactions normally result in cell death, when associated with abnormal cell growth and division. However, such defects could induce metastasis. The analysis in Drosophila supports this hypothesis: lack of Dg function results in tumor-like structures and abnormal cell movement because of the lack of epithelial integrity and cellular polarity (Deng, 2003).

Reduction of Dg function expands the apical domain and overexpression of Dg reduces this domain in epithelial cells. In Dg loss-of-function follicle cell clones, a component of the Lgl-complex, Dlg, is mislocalized. This mislocalization could explain the expansion of apical markers in the clones, since Dlg and Scrib are each required for the lateral localization of each other and their function is essential to restrict the apical markers Crb and Dlt to the apical surface. Further experiments need to be carried out to distinguish whether mislocalization of Dlg is caused directly by lack of physical interaction with Dg or indirectly by lack of proper cytoskeletal arrangements (Deng, 2003).

Drosophila oocyte polarity is essential for morphogen localization and therefore for the formation of the major body axes. The establishment of oocyte polarity is a gradual process that involves multiple steps. Key events in the process are cytoskeletal rearrangements. Initially, the MTOC is present in the anterior region of an early oocyte. By stage 3, the first rearrangement has occurred and the MTOC is positioned in the posterior portion of the developing oocyte. By the end of stage 6, a signal from the posterior follicle cells has initiated a new MT rearrangement, the posterior MTOC disappears and a new anterior MTOC forms. Although this signaling pathway remains a mystery, several molecules including Laminin A have been shown to be involved (Riechmann, 2001; Deng, 2000). As for the first rearrangement, genes encoding the Drosophila Par3/Par6/aPKC-complex, Par-1, and Maelstrom are required. However, the mechanism for the MTOC movement or anchoring is not clear. Dg, similar to the Par proteins, is required in the germline for this first rearrangement step. Since Dg germline clones also exhibit a defect in cortical actin enrichment in the oocyte, it is possible that the cortical actin plays an important role in MTOC movement and/or anchoring. Alternatively, since DGC contains proteins that can interact with either actin or microtubular cytoskeletons, it could play a role in coordinating actin and microtubule functions in this process (Deng, 2003).

The fact that Dg is required for both epithelial and oocyte polarity re-iterates the idea that common strategies may exist for polarizing these two very different cell types. In addition to Dg, Par proteins also act in polarity formation in both cell types, suggesting that the Par proteins and Dg complex have functional similarities. Interestingly, Dg can affect localization of the Par complex, since one of the members, Baz (Par3), is mislocalized when Dg is overexpressed. In addition, both Par-proteins and the Dg-complex interact with molecules that can associate with either actin or microtubular cytoskeletons. Par-1 associates with Myosin II heavy chain and also phosphorylates a MT-associated protein. Dg can interact with actin through Dystrophin-like proteins. Furthermore, the mammalian Dystrophin-associated protein, Syntrophin, interacts with MT-associated proteins via a two-hybrid assay. It is possible that both Par proteins and the DG complex facilitate interactions between actin and microtubules and that these interactions between the two cytoskeletal systems are key regulators for establishment of polarity in both cell types (Deng, 2003).

Surprisingly, Dg mutant follicle cells generate actin defects in neighboring cells; the basal actin is misoriented in adjacent follicle cells. How would a defective Dg in one cell alter the dynamics of actin organization in the neighboring cell? It is proposed that the interaction between ECM and Dg is bi-directional: in one case, DG organizes the Laminin ECM architecture (Henry, 2001b; Deng, 2003), suggesting that a defect in Dg will be transmitted to a defect in ECM organization; in the other case, a defective Laminin lattice will extend to the surface of the neighboring cell and there this architectural information could be transmitted to the cellular actin cytoskeleton by Dg in the neighboring cell (Colognato, 1999). Three pieces of evidence support this hypothesis: (1) Drosophila Dg is capable of organizing the Laminin lattice; (2) the Laminin lattice in the basal side of follicle cells is oriented in the same orientation as the underlying basal actin lattice; (3) Laminin, similar to Dg, could also be involved in basal actin organization. Interestingly, two other Laminin receptors, Integrin and Lar, are also required for basal actin planar polarity in follicle cells. It is possible that one connector alone would not give enough rigidity or allow enough flexibility in relaying information between the ECM and the basal actin (Deng, 2003).

In summary, Drosophila Dg has two separate functions in cell polarity: cell autonomous in apical-basal and anteroposterior polarity, and non-cell-autonomous in planar polarity. Future research aims to take advantage of Drosophila as a model organism to genetically dissect the partners of DG in these two functions (Deng, 2003).


GENE STRUCTURE

Drosophila Dystroglycan gene was identified by EP screen and independently by homology to mouse DG using the Protein BLAST program. LD04782, which maps to the genomic region identified by this search contains the 3' half of the gene. Several cDNAs were isolated from the 5' region by PCR using an embryonic cDNA library. The 5' end of Dg was mapped near the insertion site of EP(2)2241 and approximatelyh 4 kb downstream of Rho 1. Several EST 5' sequences overlap the 5' UTR of Dg. The following ESTs were analyzed: LD11619, GH09323 and SD06707. The intron-exon structure was found to be different from the Gadfly prediction in two incidents (Gadfly annotation has been corrected). At least two exons [exon 8 (265 amino acids) and 9 (83 amino acids)] are subjected to alternative splicing. Five different cDNAs were tested for the presence of exon 8 and 9. Two lacked exon 9, one of which was LD11619 (1179 amino acids). Two cDNAs lacked both exon 8 and exon 9, one of which was SD06707 (914 amino acids). One lacked exon 8 (GH09323, 997 amino acids). None of the cDNAs tested contained both exon 8 and exon 9 (Deng, 2003).


PROTEIN STRUCTURE

Amino Acids - isoforms listed in various sources: 997, 983, 1179, 1248, 1262

Structural Domains

To identify genes that affect the polarity of the Drosophila oocyte, the EP/Gal4 system was used to screen for genes that, when overexpressed in follicle cells, cause a polarity defect in the underlying oocyte. From the over 2000 EP insertions screened, two components of the mammalian DGC were identified: Laminin A and Drosophila DG [EP(2)2241]. An independent homology search with mouse DG protein verified the identification of CG18250 as the Drosophila homolog of the mammalian Dystroglycan gene (Deng, 2003).

Conceptual translation of the longest cDNA (LD11619) reveals an open reading frame of 1179 amino acids. This deduced Drosophila DG protein contains all the hallmarks of vertebrate DG: a mucin-like domain, a transmembrane domain and a C-terminal region with WW-, SH2- and SH3-binding domains. The best conserved region between human and Drosophila is the C-terminal half of the protein showing 31% identity. The last 12 amino acids of the C terminus include the WW domain-binding motif (PPxY), which is the Dystrophin binding site. Of 12 amino acids within the C terminus, 10 are perfectly conserved in Drosophila. Vertebrate DG contains a second PPxY motif in its cytoplasmic domain, which is also conserved in Drosophila. In addition, two of the six putative SH3 binding sites and all three SH2-binding sites in the cytoplasmic domain of vertebrate DG can be found in Drosophila. The putative C. elegans homolog DGN-1 (T21B6.1) shows 20% identity to Drosophila in the C-terminal half. However, T21B6.1 contains no mucin-like domain, Dystrophin-binding site or second PPxY motif (Deng, 2003).


Dystroglycan: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 April 2003

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