Neurexin
Mammalian protein 4.1 is the prototype of a family of proteins
that include ezrin, talin, brain tumor suppressor merlin, and tyrosine
phosphatases. Protein 4.1 functions to link transmembrane proteins with the underlying spectrin/actin cytoskeleton. To permit a genetic analysis of
the developmental role and cellular functions of this membrane-skeletal protein, its Drosophila
homolog (termed D4.1) has been identified and characterized. D4.1 is localized to the septate junctions of epithelial cells and is encoded by the coracle gene, a new locus
whose primary mutant phenotype is a failure in dorsal closure. In addition, coracle mutations dominantly suppress Ellipse, a
hypermorphic allele of the Drosophila EGF-receptor homolog. These data indicate that D4.1 is associated with the septate junction,
and suggest that it may play a role in cell-cell interactions that are essential for normal development (Fehon, 1994).
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).
NRX 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 D4.1/Coracle except in the PNS and CNS where D4.1/Coracle is only expressed in a few cells.
No defects in the localization of Discs large protein is detected in Nrx mutants. However, D4.1/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 D4.1/Coracle to septate junctions, creating a parallel with red blood cell cytoskeletal anchoring proteins (Baumgartner, 1996).
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 DLG family
members (Marfatia, 1995). A Drosophila protein with homology to protein 4.1
is the recently discovered Inscuteable
(Kraut, 1996). 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
DLG: 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).
Septate junctions (SJs) in epithelial and neuronal cells play an important role in the formation and maintenance of charge and the size of selective barriers. They form the basis for the ensheathment of nerve fibers in Drosophila and for the attachment of myelin loops to axonal surface in vertebrates. The cell-adhesion molecules NRX IV/Caspr/Paranodin (NCP1), contactin and Neurofascin-155 (NF-155) are all present at the vertebrate axo-glial SJs. Mutational analyses have shown that vertebrate NCP1 and its Drosophila homolog, Neurexin IV
are required for the formation of SJs. The Drosophila homolog of vertebrate contactin, Contactin, has been genetically, molecularly, and biochemically characterized. Ultrastructural and dye-exclusion analyses of Cont mutant embryos show that Cont is required for organization of SJs and paracellular barrier function. Cont, Neuroglian (Nrg) (Drosophila homolog of NF-155) and Nrx IV are interdependent for their SJ localization and these proteins form a tripartite complex. Hence, these data provide evidence that the organization of SJs is dependent on the interactions between these highly conserved cell-adhesion molecules (Faivre-Sarrailh, 2004).
Analyses of cont, nrx IV and nrg mutants have shown that these genes display a common alteration of SJ phenotype and thus are required for the formation and/or organization of SJs. In addition, the immunohistochemical analysis of these mutants shows that Cont, Nrx IV and Nrg are dependent on each other for SJ localization. The phenotypic similarities raise the interesting issue of whether these proteins are part of a macromolecular protein complex that exists at SJs, and thus become dependent on each other for their localization. To determine whether these three proteins form a biochemical complex, microsomal NP-40 extracts were prepared from Drosophila embryos and used for co-immunoprecipitation experiments. Cont was efficiently co-immunoprecipitated with Nrx IV using either anti-Nrx IV or anti-Cont antibodies. Cont and Nrx IV were not co-precipitated by monoclonal anti-Nrg antibodies, owing to weak ability to immunoprecipitate Nrg complexes. However, by probing Western blots with the 3C1 anti-Nrg mAb, Nrg was detected in anti-Cont immunoprecipitates. Only the lower molecular weight Nrg167 isoform, which is expressed in epithelial cells, was co-immunoprecipitated with Cont. By contrast, the neuronal Nrg180 isoform could only be detected in the total NP-40 lysate. These results demonstrate that Cont, Nrx IV and Nrg are part of a protein complex and that the molecular interactions between these proteins may underlie the organization of the SJs (Faivre-Sarrailh, 2004).
Thus biochemical and immunohistochemical data show that Cont is part of a protein complex that includes Nrx IV and that Cont is mislocalized in nrx IV mutants. Furthermore, in these mutants, Cont protein is not expressed properly on the plasma membrane and is seen in intracellular vesicles in the epithelial cells. This raised the possibility that Nrx IV might be involved in the cell surface targeting or stabilization of Cont (Faivre-Sarrailh, 2004).
Since Drosophila S2 cells constitutively express Nrx IV and Cont, this question was addressed using neuroblastoma N2a cells as a heterologous expression system. In transfected N2a cells, Nrx IV is efficiently expressed at the cell surface. By contrast, in cells expressing Cont, the Cont protein appears to remain localized intracellularly and double-staining with the ER marker BiP indicates that Cont is retained in the ER. Immunofluorescence staining under permeabilizing conditions of N2a cells, which were co-transfected with Cont and Nrx IV, revealed that Cont is transported to and co-localized with Nrx IV at the plasma membrane. Similar results were obtained with intact living cells. Thus, these results indicate that Nrx IV mediates the cell-surface targeting of Cont. Next, Cont was co-immunoprecipitated with Nrx IV from co-transfected COS cell lysates, as further evidence that these proteins interact in cis. Such a cis-interaction has been reported for their vertebrate counterparts contactin and NCP1 (Peles, 1997) (Faivre-Sarrailh, 2004).
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