BIOLOGICAL OVERVIEW

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. The Drosophila homolog (termed Coracle) of protein 4.1 has been identified and characterized: such work has advanced an understanding of the developmental role and a genetic analysis of the cellular functions involving this membrane-skeletal protein (Fehon, 1994).

Coracle is a component of septate junctions, structures that serve as selective-permeability barriers, separating the apical from the basal regions in sheets of epithelial cells. For more information about the Drosophila septate boundary see Neurexin. Another component of Drosophila septate junctions is the protein Discs large. The region of a human erythroid Discs large protein (p55) that interacts with mammalian protein 4.1 has also been identified. A novel sequence located between the SH3 motif and the guanylate kinase domain of p55 is the binding site for the 30kDa domain of protein 4.1. A characteristic of this 39-amino acid sequence is the presence of a cluster of lysine residues located in the C-terminal half of the protein. This sequence is conserved in the human Discs large protein (hDlg) and in Drosophila Discs large. Protein 4.1, and its Drosophila homolog Coracle may provide a membrane localization site for Discs large homologs (Marfatia, 1995).

Mammalian protein 4.1 is known to interact with the Drosophila cytoskeletal proteins actin and spectrin). The cytoskeletal interaction domain of Mammalian protein 4.1 is not conserved in Coracle; neither actin nor spectrin associate with septate junctions in Drosophila. Thus, while the presumed function of mammalian erythrocyte protein 4.1 may be to mediate interactions between the transmembrane protein glycophorin and the cytoskeletal proteins actin and spectrin, this function is not conserved in Drosophila. Nevertheless, the N-terminal 350 amino acids of the protein 4.1 homolog (Coracle) is highly conserved in both Drosophila and mammalian proteins. This domain serves to localize these proteins to their respective membranes. This implies that Coracle may be important for junctional structure in Drosophila, a role that is clearly not relevant for erythrocytes. Studies of human 4.1 in non-erythoid cells have reported that 4.1 is assocated with membranes in regions of cell-cell contacts, indicating that Coracle function in junctions could be conserved in vertebrates (Fehon, 1994).

Coracle is required for Dorsal closure, one of the more dramatic of all the morphogenetic movements taking place during Drosophila embryogenesis. The closure event occurs late in embryogenesis, and results in the establishment of the dorsal epidermis. Cells in the two lateral epidermal primordia change shape and spread over the amnioserosa, a membranous structure covering the dorsal side of the embryo. Epithelial cells elongate in this spreading, covering process. Neither cell proliferation nor cell rearrangement occurs. Included among the Drosophila genes known to be involved in dorsal closure are Rac1, a low molecular weight GTPase; basket, also known a Jun N-terminal kinase, and zipper, Drosophila's non-muscle myosin. A role for Coracle in dorsal closure is unknown, although it is known that human protein 4.1 can interact with muscle and nonmuscle myosin (Fehon, 1994 and references).

The original coracle mutations were identified based on their ability to dominantly suppress the Ellipse gain of function hypermorphic allele of the Drosophila Epidermal growth factor receptor homolog. The Ellipse mutation produces a rough eye phenotype in Drosophila. It is not known whether this genetic interaction represents a direct interaction between these two genes. However, it is known the mammalian EGF receptor tyrosine kinase can phosphorylate protein 4.1 and that phosphorylation affects the functions of 4.1 family proteins. Thus EgfR regulation of Coracle could play a role in cell dynamics during Drosophila eye morphogenesis (Fehon, 1994).

The N-terminal 383 amino acids of Coracle define an essential functional domain possessing membrane-organizing properties. The full range of functions provided by this highly conserved domain has been investigated and the domain has been found to be sufficient to rescue all embryonic defects associated with loss of coracle function. In addition, this domain is sufficient to rescue the reduced cell proliferation defect in imaginal discs, although it is incapable of rescuing null mutants to the adult stage. This result suggests the presence of a second functional domain within Coracle, a notion supported by molecular characterization of a series of coracle alleles (Ward, 2001).

Three coracle transcripts have been precisely mapped, and Northern blot analysis indicates the presence of several others. coracle cDNA 1 (isoform 1) encodes a protein that is predicted to be 1698 amino acids (aa) in length, while cDNAs 2 and 3 (isoforms 2 and 3) encode much shorter products of 889 and 703 aa, respectively. Isoforms 2 and 3 differ primarily from isoform 1 in that they lack coding region E, a large (2427 bp), alternatively spliced exonic region. To determine the number of introns and the precise intron/exon boundaries of the coracle gene, genomic DNA from the coracle locus was PCR amplified and sequenced and this genomic sequence was compared to the known cDNA isoforms. On the basis of these comparisons, coracle is composed of 17 exons and 16 introns. In addition, this analysis reveals a complex pattern of splicing that is due to the use of alternative splice acceptor sequences that in some cases are not separated by intervening intronic sequences. Three introns are flanked by alternate splice acceptor sites that result in mRNAs with different coding sequences. The first of these regions, at intron 8, results in alternative splicing involving regions B and C. Region B contains an alternative splice acceptor that inserts a 12-bp coding region and is used in isoform 3. Region C, which is contiguous with region B in the genomic DNA, is present in isoforms 2 and 3, while in isoform 1, splicing spans from the 5' end of intron 8, across regions B and C to the 3' end of the 711-bp intron 9, thereby excising these coding regions. A similar behavior is observed around intron 11, which falls in between regions D and E in the coding sequence. Surprisingly, region E, the largest of the alternatively spliced regions in coracle (2427 bp), is not immediately bounded by introns on both ends. Rather, the 3' end of region E is defined by another alternative splice acceptor site within the large exon that encodes regions E and F. Thus, splicing in this area can span just intron 11 (353 bp; isoform 1), intron 11 plus region E (2780 bp; isoform 2), or intron 11, regions E and F, plus intron 12 (3717 bp; isoform 3). Similar behavior is observed around exon H, although in this case there does not appear to be any alternative splice acceptor within the exon, thereby simplifying the splicing pattern (Ward, 2001).

The observed complex pattern of alternative splicing raises the possibility that different isoforms may have different functions during development. This notion is supported by the observation that the cor¹ and cor² alleles are associated with nonsense mutations that should affect only isoform 1. The observation that these mutations display a fully penetrant, embryonic lethal phenotype implies that isoform 1 encodes functions that are not present in the other isoforms and, therefore, that region E is likely to contain an essential functional domain. Alternatively, it is possible that while functionally equivalent, the expression level of the other isoforms is too low to provide sufficient coracle function for viability, consistent with previous observations that the alternative mRNA splice forms are expressed at lower levels than isoform 1 (Ward, 2001).

To distinguish between qualitative and quantitative functional differences between the three Coracle isoforms, P-element-based transgenes encoding isoforms 1, 2, and 3 under the control of the Drosophila Ubiquitin promoter were constructed. Use of the Ubiquitin promoter ensures that all three isoforms are expressed at roughly equivalent levels and that they are expressed in all tissues throughout embryonic development. To test the ability of these isoforms to rescue coracle lethal mutations, independent insertion lines of each were crossed into the appropriate coracle mutant background, and viability of the homozygous mutant class was scored. All three of the tested isoforms provided sufficient coracle function to rescue >67% of the expected coracle mutant offspring bearing null alleles, indicating that all encode the essential coracle functions when expressed ubiquitously and at sufficient levels. In all cases both males and females were fertile, allowing the maintainance of stocks of rescued homozygous mutant animals. In comparison to isoform 1, isoform 2 lacks coding sequences within regions E (2427 bp) and H (102 bp), and isoform 3 lacks, in addition, region F (570 bp). In comparison to isoform 3, isoform 1 lacks coding sequences within regions B (12 bp) and C (102 bp). Thus, the observation that isoforms lacking these regions can rescue severe loss-of-function coracle mutations indicates that, although in combination regions B, C, E, F, and H encode 1071 amino acid residues, they do not contain any essential functional domains. Likewise, the observation that each of these isoforms rescues cor⁵, a null coracle allele, indicates that each encodes all essential coracle functions and thus that these functions are restricted to coding regions A, D, G, and I (Ward, 2001).

Interestingly, all coracle mutations that are predicted to affect the N-terminal functional domain are embryonic lethal, whereas cor¹⁴, which truncates the C-terminal domain, shows no embryonic lethality. This observation raises the possibility that the N-terminal functional domain is required to complete embryonic development, whereas the C-terminal domain is required at a later stage. Ectopic expression experiments using just the FERM domain strongly support this supposition. Embryos completely lacking coracle function display a range of defects including failure in dorsal closure, thinning of the cuticle, necrosis of the salivary glands, and an inability to inflate the trachea at the end of embryogenesis. Expression of the N-terminal 383 amino acids fully rescues all of these defects. Additionally, the cor⁸ and cor¹⁰ mutations, which affect the FERM domain and cause embryonic lethality, can be rescued to viable, fertile adults by the ubiquitous expression of just the FERM domain. In contrast, cor⁴ and cor⁶ mutant animals, which also have molecular lesions within the FERM domain, are not rescued by ubiquitous expression of COR^1-383. Interestingly, the cor⁴ and cor⁶ mutant proteins display abnormal subcellular localizations, raising the possibility that correct subcellular localization is as crucial for the function of the C-terminal domain as it is for the function of the FERM domain. Also, cor¹ and cor², which have an intact FERM domain display embryonic defects, but like most nonsense mutations these alleles show reduced protein expression (Ward, 2001).

These experiments strongly suggest the existence of an essential functional domain within the C-terminal region of Coracle. Even though cor¹⁴ is one of the weakest alleles tested in these experiments, ectopic expression of the FERM domain is incapable of rescuing cor¹⁴ animals. In contrast, cor¹⁴ is rescued by expression of a full-length coracle transgene, indicating that a region outside the FERM domain is necessary for viability. cor¹⁴ results from a nonsense mutation at Arg¹⁶⁰⁷, suggesting that the C-terminal functional domain includes sequences within the highly conserved C-terminal 100 amino acids (Ward, 2001).

The proposed modular organization of the functional domains within Coracle leads to the following prediction: alleles that specifically alter only one functional domain should complement alleles that affect only the other functional domain. Specifically, combinations involving cor⁸ and cor¹⁰ (specifically affecting the N-terminal domain) and cor¹⁴ (specifically affecting the C-terminal domain) support this hypothesis. Although all three of these alleles are recessive lethal (either homozygous or over a deficiency), cor¹⁰ is >75% viable when heterozygous with cor¹⁴, and cor⁸ weakly complements cor¹⁴. This result is in agreement with the results using molecular genetic approaches and strongly supports the conclusions on the modular nature of the functional domains within Coracle (Ward, 2001).

Most, if not all, of the embryonic defects associated with loss of coracle function are due to an inability to maintain a physiologically 'tight' epithelium. Ectopic expression of the FERM domain in coracle mutant embryos is sufficient to rescue all of the described embryonic defects, raising the possibility that this rescue is accomplished by restoring the integrity of the septate junction. Ultrastructural and physiological analyses confirm this hypothesis, demonstrating that this domain provides an essential structural function at the septate junction in embryonic epithelia (Ward, 2001).

Although these results indicate that the FERM domain provides an essential structural function during embryonic development, it is less clear what role this domain plays in post-embryonic development. Loss of coracle function in imaginal epithelia results in a proliferative disadvantage that is ameliorated by expression of just the FERM domain, but does not alter overall epithelial integrity or polarity. A number of cell signaling pathways have been implicated in controlling cell proliferation and growth in imaginal epithelia. Included among these are the epidermal growth factor, Wingless, Notch, and the Dpp pathways. Mutations that perturb the proper transmission of these signals produce imaginal defects similar to those reported for coracle. Additionally, coracle was originally identified as a dominant suppressor of Egfr^Ellipse, a hypermorphic allele of the epidermal growth factor receptor homolog. It is possible that the FERM domain facilitates the transduction of one or more of these signaling cascades by binding to and thereby localizing an important intracellular factor or factors. Continuing efforts to identify additional genes that interact with coracle will undoubtedly help resolve the function of this domain in regulating proliferation (Ward, 2001).

What, then, is the role of the C-terminal domain? By analogy with Protein 4.1 and the ERM proteins, it is suspected that this region in Coracle contains a protein-binding domain. In the ERM proteins, the C-terminal domain is thought to regulate the function of the FERM domain via an intramolecular interaction. It is not known yet if similar interactions may occur in Coracle or Protein 4.1. In addition, recent experiments have identified several potential protein-protein interactions mediated by this domain. For example, the immunophilin FKBP13 interacts with the C-terminal region of Protein 4.1G, one of several Protein 4.1 paralogs in mammals. If such interactions occur via the Coracle C-terminal domain, they could function to anchor additional proteins to the region of the septate junction. In contrast, two recent studies have suggested interactions between this same domain of Protein 4.1 and proteins known to function in the nucleus. These proteins are NuMA, the nuclear mitotic apparatus protein, and PIKE, a putative regulator of PI₃ kinase activity in the nucleus. At the moment the functional significance of these interactions is not known, nor is it known if Coracle has similar interactions. Further experiments, particularly using genetic approaches, will be required to determine their functional significance. However, both these data and data regarding putative protein-protein interactions are consistent with a function for the C-terminal domain that is distinct from that of the FERM domain and may differ significantly from current ideas about Protein 4.1/Coracle functions (Ward, 2001).

GENE STRUCTURE

In addition to the 5.9 kb cDNA, two other cDNA clones have been analyzed. These other cDNAs are identical for 1596 bp at the 5' end but diverge by alternative splicing 3' to this point. One cDNA contains a 102 bp insert, and a second contains the same insert, plus a second one of 12 bp just 5' to the first. In both cases, a continuous open reading frame is maintained through these inserts to 3' sequences that are shared with the 5.9 kb cDNA. The sizes of the three cDNAs (5.9, 3.5 and 2.9 kb) determined by sequence analyis correlate well with the measured sizes of the mRNAs in Northern blots (Fehon, 1994).

PROTEIN STRUCTURE

Amino Acids - 1698

Structural Domains

The Drosophila, Xenopus and human sequences are 54% identical over a stretch of 350 aa in the N-terminal region. Within this region there is a significant but lower identity with other members of the protein 4.1 gene family, including ezrin (31% over 229 amino acids), talin (22% over 194 amino acids), moesin A and B (30% over 229 amino acids) and merlin (27% over 229 amino acids) and the two tyrosine phosphatase genes (37% over 287 amino acids). In addition, Coracle shares sequence similarity with the human and Xenopus 4.1 genes in a smaller region at the C terminus that is not shared with any other members of the 4.1 gene family. The intervening 1200 amino acids of Coracle sequence shows no similarity to any sequences in the GenBank database. Protein 4.1 homologs exhibit a predicted alpha-helical structure that extends for approximately 400 amino acids immediately C terminal to the highly conserved domain of Coracle. The additional 800 amino acids of sequence in Coracle that extend beyond this putative alpha-helical domain are predicted to be composed of beta-sheet structures. No such extended beta-sheet domain has been proposed for any of the other protein 4.1 family members. The N-terminal conserved region in Coracle appears to have several hydrophobic domains that could interact with the cell membrane. The region corresponding to the spectrin/actin binding domain in the vertebrate erythroid 4.1 protein is not conserved in any of the Coracle splice forms that have been characterized, although this region is highly conserved between the human and Xenopus genes (Fehon, 1994).

date revised: 2 December 2001  

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