shotgun: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - shotgun

Synonyms - DE-cadherin

Cytological map position - 57B13-14

Function - cell adhesion

Keyword(s) - epithelial junction protein, tumor suppressor

Symbol - shg

FlyBase ID:FBgn0003391

Genetic map position -

Classification - E-cadherin

Cellular location - surface transmembrane protein



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Vertebrates and invertebrates share common mechanisms for regulation of cell-cell adhesion, including a family of proteins known as cadherins. Cadherins are important in setting up boundaries between epithelial compartments, preventing cell populations with different fates from mixing with one another. They control orderly release of cells from epithelium during epithelial-mesenchymal transitions. shotgun, or DE-cadherin is a homolog of classic vertebrate cadherins. The level of shotgun, expressed from maternal mRNA throughout the entire blastoderm [Image], is high enough to sustain embryonic development except for severe disruption of Malpighian tubules (Uemura, 1996). Zygotic shg is expressed in all epithelial tissue except for presumptive mesoderm cells prior to gastrulation [Image].

The presence of shg expression in neuroectoderm, and its absence in delaminated neuroblasts, suggests an involvement of SHG in the critical process of delamination: the migration of neuroblasts from the ectoderm into the ventral nervous system (Tepass, 1996). Another cadherin, Cadherin-N shows a complementary expression pattern. It is expressed primarily in the nervous system but not in epithelial precursors of neural cells (Iwai, 1997).

E-cadherin is a cell adhesion molecule that provides one constituent of the adherens junction, a cellular junction localized at the apicolateral boundary of an epithelial cell. E-cadherin recruits other proteins to the region of cell-cell contact, such as Crumbs, a transmembrane protein involved in organizing the adherens junction, and Armadillo, a junction associated protein coded for by a pair rule gene involved in wingless signaling.

shotgun is involved in Malpighian tubule morphogenesis. Malpighian tubules are initiated by a local invagination from the hindgut primordia. In shg mutants, the Malpighian tubules are rounded and eventually disintegrate. SHG is concentrated at the apical poles of epithelial cell-cell junctions (Oda, 1994). Interestingly, the apical polarity of the cells is preserved in shg mutants as reflected in Crumbs protein distribution (Uemura, 1996).

Tracheal branch outgrowth and fusion are similarly defective in shotgun mutants. A "stall" phenotype is apparent, exhibiting delayed branch extention. armadillo mutants have similar effects on tracheal development. The role of Armadillo in tracheal morphogenesis is partly separate from that of Wingless, because the formation of dorsal tracheal trunks is only partially impaired in wg mutants. This means that the connection of wingless signaling to occurances at the apical junction is not sufficient to completely disrupt morphogenesis in wingless mutants (Uemura, 1996).

The primary role of shotgun is preservation of the integrity of Drosophila epithelium and it is downregulated during critical periods of epithelial-mesenchymal transition such as gastrulation and neurogenesis. As an epithelial cadherin, it is the central element of the adherens junction, a multiprotein complex that holds cells together and transduces adhesion signals across membranes. It is involved as the nucleation site of cytoskeleton to cell junctions. In zygotic shotgun mutants the level of alpha-catenin and Armadillo at the adherens junction is dramatically reduced, pointing to the importance of Drosophila E-cadherin in assembling the adherens junction (Tepass, 1996).

The molecular structures of classic cadherins show clear differences between chordate and nonchordate metazoans. Although nonchordate classic cadherins have cadherin superfamily-specific extracellular repeats (CRs) and a highly conserved cytoplasmic domain (CP), these cadherins have a unique extracellular domain that is absent from vertebrate and ascidian (phylum Urochordata) classic cadherins. This domain is termed here the primitive classic cadherin domain (PCCD). The PCCD consist of three motifs. The first is unique to nonchordate classic cadherins, and has been named the NC (nonchordate motif). The second is rich in cysteine residues and rather resembles EGF repeats, and has been named the CE (cysteine-rich EGF repeat-like motif). The third is similar to the laminin A globular domain, and is named the LAG (laminin A globular domain-like motif). These motifs constitute a domain that constitutes the primitive classic cadherin domain (PCCD). To understand the roles of the PCCD, a series of mutant forms of the Drosophila classic cadherin Shotgun were constructed. Biochemical analyses indicate that the last two CRs and PCCD form a special structure with proteolytic cleavage. Mutations in the PCCD do not eliminate the cell-cell-binding function of Shotgun in cultured cells, but prevent the cadherin from efficiently translocating to the plasma membrane in epithelial cells of the developing embryo. In addition, genetic rescue assays suggest that although CP-mediated control plays a central role in tracheal fusion, the role of the PCCD in efficient recruitment of Shotgun to apical areas of the plasma membranes is also important for dynamic epithelial morphogenesis. It is proposed that there is a fundamental difference in the mode of classic cadherin-mediated cell-cell adhesion between chordate and nonchordate metazoans (Oda, 1999).

When the amino acid sequences of the PCCDs of nonchordate classic cadherins are aligned, the PCCD of Shotgun shows 39.8%, 31.1%, and 27.5% amino acid sequence identity to those of Shotgun, LvG-cadherin (sea urchin), and HMR-1 (C. elegans), respectively. The PCCDs of Shotgun and Drosophila N-cadherin are the most similar to each other in all combinations. A phylogenetic tree was constructed based on the PCCDs, consistent with the tree based on the CPs. The PCCD consists of the NC, CE, and LAG. No significant similarity within the NC is found to sequences of other known proteins. The CEs have 10 or 12 conserved cysteine residues, and the LAGs have 4 conserved cysteine residues. Although sequences similar to the LAGs are found in a variety of proteins, including Drosophila Slit, Fat, Crumbs and rat neurexin I, the first two of the four cysteine residues are not conserved in proteins other than the nonchordate classic cadherins. These amino acid sequence data suggest that the PCCD forms a structure unique to nonchordate classic cadherins (Oda, 1999).

When full-length Shotgun tagged with green fluorescent protein (GFP) at the carboxyl terminus is expressed in Drosophila S2 cells, 150- and 110-kDa polypeptides named distal and proximal polypeptides (DP and PP) are detected in the cell lysates by anti-Shotgun monoclonal antibody DCAD2 and anti-GFP antibody, respectively, in addition to a small amount of the precursor (about 230 kDa). DP corresponds to the polypeptide detected in embryonic lysates by DCAD2, which recognizes the region between CR0 and CR1. A CP-deleted mutant molecule named dCPc3 is separated into a DP of the same size (150-kDa) and a PP of reduced size (80-kDa). Sequence analysis shows that protolytic removal of the signal peptide and proteolytic cleavage between G (aa 1010) and S (aa 1011) residues in the NC result in DP (aa 70-1010) and PP (aa 1011-1507). Deletion analysis indicates that the region covering CR4, CR5, NC, and CE (aa 738-1108) is necessary for NC cleavage, but this is not the case for most other regions, including the transmembrane segment (TM) (Oda, 1999).

The CR domain, PCCD, and CP are essential for the morphogenetic function of Shotgun. The morphogenetic abilities of Shotgun mutant molecules are not necessarily correlated with their adhesive abilities in S2 cells. Various extracellular-domain deletion mutants, dCR3h, dCR4h, and dEx, which have no cell-cell-binding ability, can produce positive morphogenetic effects on tracheal fusion, but dCPc3, dNc, dCL3, and inNcHA show no positive morphogenetic effects, despite having cell-cell-binding ability in S2 cells. These results indicate that CP-mediated control plays a central role in tracheal fusion. The CP binds to the catenins, which may mediate interaction with the actin cytoskeleton. This coupling is expected to translate the force of actin bundle contraction into cell shape changes. Even if cell-cell binding function is removed from Shotgun, such mechanical forces may induce incomplete tracheal fusion. Despite the intact CP, some mutations in the PCCD lead to complete loss of the morphogenetic ability of Shotgun. These mutant molecules fail in prompt translocation to the plasma membrane when expressed in epithelial cells of the developing embryo. Efficient recruitment of Shotgun to appropriate sites of the plasma membrane may be important for dynamic epithelial morphogenesis during development (Oda, 1999).

Shotgun protein must be translocated from the ER to the contact sites of the plasma membrane to participate in cell-cell binding. Exogenously expressed normal Shotgun immediately accumulates at apicolateral borders of the plasma membrane in tracheal cells and many other epithelial cells. Mutational analyses reveal important aspects of Shotgun translocation involving the last two CRs, PCCD, and CP. Mutations in the PCCD lead to strong accumulation of the molecules in subdomains of the cytoplasm probably corresponding to the ER. Deletion of the large extracellular region encompassing CRs 1-5 and the PCCD also reduce the efficiency of translocation. These observations favor the idea that the extracellular region, including the PCCD, has an active role in translocation out of the ER to the plasma membrane. Deficiencies in deletion mutants dCR3h or dCR4h have little effect on translocation, but dCR4 exhibits a defect similar to that of the PCCD mutant molecules. These observations suggested that the last two CRs are functionally linked with the PCCD, and that this set is required for normal translocation. It is intriguing that NC cleavage, which presumably takes place in the ER, also requires the same region. However, the results from mNcGSP and dCL3 indicate that NC cleavage is not a prerequisite for translocation and occurs even under conditions in which translocation is blocked. It is speculated that CRs 4 and 5 and the PCCD are an overlapping region required for NC cleavage and for translocation, but these two events occur independently. There are three possible explanations for the mechanism of the involvement of the PCCD in translocation. (1) The unique structure of the last two CRs and PCCD may interact in the ER lumen with some machinery that carries out protein sorting and directional transport. (2) The PCCD structure may be involved in a lateral interaction (between the synthesized cadherin proteins) that facilitates translocation. (3) The PCCD may have a role in folding the extracellular part of the synthesized cadherin in the ER lumen to form the compact structure necessary for efficient translocation. It is hypothesized that the common ancestor of chordate classic cadherins adopted an alternative translocation mechanism to the PCCD-mediated mechanism coincident with the structural change to small size (Oda, 1999).

The CP is another part essential for normal translocation of Shotgun. Although the CP-deleted molecule tends to translocate to the apical plasma membrane, it is not so actively concentrated into the sites of cell-cell contact. Despite the reduced efficiency of translocation, the mutant molecule lacking the large extracellular region appears to be recruited to the apical zones of cell-cell contact once it leaves the ER. It seems that the CP-mediated control plays an important role in efficient and directional translocation of Shotgun from the ER to the contact sites of the plasma membrane. Shotgun mutant molecules accumulating in the cytoplasm due to the lack of translocation appear to bind to Armadillo and alpha-catenin. This raises the possibility that normal Shotgun joins Armadillo and alpha-catenin before arriving at the plasma membrane (Oda, 1999).

In addition to the subcellular distributions of the classic cadherins, there are marked differences between chordates and nonchordates in junctional complexes. Adherens junctions (AJs) and septate junctions (SJs) are observed in a wide range of nonchordate metazoans by electron microscopy. The AJ is located at the apical-most portion of the cell, followed by the SJ. In chordates (including vertebrates and ascidians) tight junctions (TJs), in addition to AJs, are frequently found, but SJs have not been found. Exceptionally, in Amphioxus cephalochordata, neither TJs nor SJs have been observed. The TJ is positioned at the apical end of the lateral plasma membrane in chordate epithelial cells, followed by the AJ. These observations indicate that there are fundamental differences between chordate and nonchordate metazoans not only in cadherin-based cell-cell adhesion but also in other mechanisms of cell-cell connection (Oda, 1999).


PROTEIN STRUCTURE

Amino Acids - 1507

Structural Domains

The Drosophila homolog of classic vertebrate cadherins is DE-cadherin. The extracellular domain of DE-cadherin has six cadherin-specific repeats, although the first repeat seems to be cleaved off upon maturation, and the cytoplasmic domain shows significant identity to that of classic vertebrate cadherins. DE-cadherin is distinguishable from its vertebrate counterparts by a large insertion between the last cadherin repeat and the transmembrane domain, with local sequence similarity to Fat, Laminin A chain, Slit, and Neurexin I at the proximal region of the extracellular domain. This domain has two motifs, one a cysteine rich domain similar to Fat EGF repeat 3. The other is a laminin A globular-domain (G-domain) repeat (Oda, 1994).

The enormous size of Cadherin-N is primarily due to the presence of 15 cadherin repeats in the extracellular region, presenting a contrast to the 4 repeats in all vertebrate classic cadherins and the 6 repeats in Shotgun, the Drosophila E-cadherin. Both Drosophila cadherins have insertions of similar sequences between the last extracellular cadherin repeat and the membrane-spanning segment. The insert contains a series of subdomains: an Fcc box (fly classic cadherin box), a cysteine-rich segment (C-rich 1), a laminin A (See Drosophila Laminin A) globular segment (LmA-G), and another cysteine-rich segment (C-rich 2). The Fcc box, comprising 170 amino acids is defined as such because database searches with its sequences identify only the comparable regions of Shotgun as a relative. Similar sequences are not found in vertebrate cadherins. The whole Cadherin-N LmA-G displays 25% sequence identity to mouse laminin A and to the presynaptic transmembrane protein Neurexin. The Cadherin-N cytoplasmic domain is much more similar to those domains of Shotgun and vertebrate classic cadherins with respect to both size and sequence. The intracellular domains of the two Drosophila cadherins and mouse N-cadherin range between 157 and 160 amino acids in length, and have 37% and 46% sequence identity in any combination among them, with the higher figure representing the Cadherin-N identity to murine N-cadherin, and the lower representing Shotgun identity to murine N-cadherin. The degree of sequence conservation between the two Drosophila cadherins (41% identity) is lower than the 63% identity between N- and E-cadherin in the same vertebrate species, as for example, in mice (Iwai, 1997).


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

date revised:  20 APR 97

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