coracle

Protein Interactions

Neurexin is localized apicolaterally, adjacent to Crumbs, which delimits the zonula adherens. These two proteins are not coexpressed, placing NRX apicolaterally. Both Fasciclin3 and NRX colocalize at salivary gland synaptic junctions. NRX precisely colocalized with Coracle, the Drosophila homolog of mammalian protein 4.1, except in the PNS and CNS where Coracle is only expressed in a few cells. No defects in the localization of Discs large protein is detected in Nrx mutants. However, Coracle is not restricted to septate junctions in Nrx mutants. These results suggest that the short cytoplasmic portion of NRX that shows homology to glycophorin C is required to localize Coracle to septate junctions, creating a parallel with red blood cell cytoskeletal anchoring proteins (Baumgartner, 1996).

The protein 4.1 superfamily comprises a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homolog, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, it has been demonstrated that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. The localization of both Coracle and the transmembrane protein Neurexin to the septate junction displays an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and the cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members (Ward, 1998).

The interdependence between Coracle and NRX for proper localization suggests that at least one other protein in the presumptive septate junction serves as the initial target for both proteins to be properly localized. Based on the protein 4.1 paradigm of a ternary complex consisting of protein 4.1, glycophorin C, and p55 , it is predicted that a PDZ repeat-containing protein is a part of the complex containing Coracle and NRX. The most likely candidate for this additional protein is DLG, based on its extensive sequence similarity to p55. DLG is expressed maternally, and initially is uniformly distributed along the lateral membrane (and to a lesser extent throughout the cytoplasm). Coincident with the expression of Coracle and NRX, this subcellular localization is refined to the presumptive septate junction. This expression pattern might be expected of a protein that serves to "prepattern" the septate junction. However, attempts to detect any interaction between Coracle and DLG by immunoprecipitation have failed; no genetic interaction between coracle and dlg mutant alleles have been detected. The embryonic defects associated with dlg mutants are different from those of coracle and Nrx. These results suggest that DLG is not involved in a ternary complex together with Coracle and NRX, despite its structural similarity with p55. The question of whether there is another PDZ repeat-containing protein that functions to stabilize Coracle-NRX interactions remains to be answered, although the structural similarities between the respective Drosophila and human proteins strongly suggest that one exists. The recent identification of EBP50 as a PDZ repeat-containing protein that associates with ERM proteins suggests that an interaction with a PDZ repeat-containing protein may be a ubiquitous feature of protein 4.1 members. Regardless, the results described here strongly suggest that at least one other component is involved in Coracle/NRX localization and function (Ward. 1998).

Drosophila Discs large 1 interacts with protein 4.1 homologs. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. Synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila Discs-large tumor suppressor protein. Thus protein 4.1, known to interact with the cytoskeleton, also interacts with Discs large family members (Marfatia, 1995).

Distribution of two family 4.1 proteins, Expanded and Coracle, are disrupted in dlg mutants. Loss of Discs large also affects the distribution of Fasciclin III and neuroglian, two transmembrane proteins thought to be involved in cell adhesion (see in particular Discs large: Biological overview). These results suggest that Dlg serves as a binding protein linking cell surface receptors with the cytoskeleton via family 4.1 proteins (Woods, 1996).

One essential function of epithelia is to form a barrier between the apical and basolateral surfaces of the epithelium. In vertebrate epithelia, the tight junction is the primary barrier to paracellular flow across epithelia, whereas in invertebrate epithelia, the septate junction (SJ) provides this function. New proteins have been identified that are required for a functional paracellular barrier in Drosophila. In addition to the previously known components Coracle (Cora) and Neurexin (Nrx), four other proteins [Gliotactin, Neuroglian (Nrg), and both the alpha and ß subunits of the Na+/K+ ATPase] are required for formation of the paracellular barrier. In contrast to previous reports, it is demonstrated that the Na pump is not localized basolaterally in epithelial cells, but instead is concentrated at the SJ. Data from immunoprecipitation and somatic mosaic studies suggest that Cora, Nrx, Nrg, and the Na+/K+ ATPase form an interdependent complex. Furthermore, the observation that Nrg, a Drosophila homolog of vertebrate neurofascin, is an SJ component and is consistent with the notion that the invertebrate SJ is homologous to the vertebrate paranodal SJ. These findings have implications not only for invertebrate epithelia and barrier functions, but also for understanding of neuron-glial interactions in the mammalian nervous system (Genova, 2003).

To identify additional components of the Drosophila SJ, a collection of P element insertion mutations was screened for a phenotype attributable to a loss of the paracellular barrier. Two genes, Na pump alpha subunit (Atpalpha) and Nervana 2 (Nrv2), which encodes the ß subunit of the Na+/K+ ATPase) were identified as essential for the barrier function of the SJ. In addition, Neuroglian (Nrg), which is homologous to known components of the PSJ, and Gli, which is necessary for the blood-brain barrier, were tested and found to be necessary for the paracellular barrier. Direct immunostaining, epitope-tagged expression constructs, and GFP-tagged proteins indicate that Nrv2, ATPalpha, and Nrg localize to the SJ, and that they are interdependent for this localization. In keeping with this finding, the existence of a protein complex containing Cora, Nrx, Nrg, and Nrv is demonstrated. Taken together, these results suggest a novel complex involving the Na+/K+ ATPase that is necessary for establishing and maintaining the primary paracellular barrier in invertebrate epithelia, the SJs. Thus these studies provide new insights into the structure and function of SJs in both invertebrate epithelial cells and in the homologous PSJ of the vertebrate nervous system (Genova, 2003).

Cora has been shown to bind to the cytoplasmic tail of Nrx in the SJ. Studies of the PSJ have shown that the mammalian homologs of Nrx and Nrg interact via their extracellular domains. Together, these observations suggest the existence of a multiprotein complex at the SJ in which Cora binds to Nrx, which in turn binds to Nrg. The finding that Nrx and Nrg coimmunoprecipitate when either anti-Cora or anti-Nrg antibodies are used to immunoprecipitate is consistent with this model. Because Drosophila epithelial cells express all three proteins, it is not possible to rigorously distinguish whether this interaction occurs within the same cell or between adjacent cells. However, the observation that wild-type cells are unable to efficiently assemble Cora and Nrx at the boundary with cora^- cells suggests that intercellular interaction with the same complex on adjacent cells is required for SJ assembly. In addition, Nrv is found to coimmunoprecipitates with both Cora and Nrx. Nrg has not been detected in this complex, suggesting that the interaction between NRV2 and the Cora-Nrx complex occurs independently of Nrg, perhaps on the cytoplasmic side of the membrane. Although these results imply the possibility of an interaction between Cora and the cytoplasmic tail of NRV2, this seems unlikely in light of observations that NRV1, 2.1, and 2.2 all localize to the SJ, despite having different cytoplasmic tails. Thus, it is more likely that the interaction between Cora and the ATPase occurs either through Nrx or the alpha subunit (Genova, 2003).

Somatic mosaic analysis has demonstrated that this complex of Cora, Nrx, Nrv, ATPalpha, and Nrg can be disrupted without affecting overall polarity, or other components of the SJ. No component essential for the paracellular barrier has been identified that is unaffected in mutant cells, suggesting that the substrate upon which this complex assembles has yet to be found. Previous studies have demonstrated that Ankyrin binds both the cytoplasmic domain of Nrg and, as has been described in mammalian cells, the alpha subunit of NA⁺/K⁺ ATPase. In addition, Ankyrin colocalizes with Nrg at points of Nrg-induced S2 cell adhesion complexes. Thus, one candidate for a substrate upon which this complex assembles is Ankyrin, a well-known member of the membrane skeleton (Genova, 2003).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.