Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95-Discs Large-zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (Pals)1 (see Drosophila Lin-7), also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. The L27N domain targets Pals1 to tight junctions by binding to a PDZ domain protein, Pals1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. Drosophila PATJ exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homolog, CRB1, colocalize with Pals1 to tight junctions, and CRB1 interacts with PATJ, albeit indirectly, via binding the Pals1 PDZ domain. In agreement, a Drosophila homolog of Pals1 participates in identical interactions with Drosophila Crumbs and. The Drosophila Pals1 homolog is Stardust, a crucial polarity gene in Drosophila. Thus, these data identify a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity (Roh, 2002).
In Drosophila, the Crumbs/Stardust/Patj complex is required during the establishment of polarized epithelia. Embryos that lack a component of this complex or overexpress Crumbs exhibit defects in epithelial morphogenesis. A novel mammalian epithelial Crumbs isoform, Crumbs3 (CRB3) has been cloned. CRB3 exists in a complex at tight junctions (TJs) with Pals1, the mammalian homologs of Stardust, and PATJ. Overexpression of CRB3 leads to delayed TJ formation in MDCK epithelial cell monolayers and disruption of polarity in MDCK cysts cultured in collagen. Both phenomena require the last four residues of CRB3. Next, a dominant-negative Myc-Lin-2-Pals1 chimeric protein, where the PDZ domain of Lin-2 was replaced with that of Pals1, was expressed in MDCK cells. TJ and apical polarity defects are observed in these cells. Collectively, this suggests that the CRB-Pals1 interaction is important for formation of TJs and polarized epithelia. These results provide insight into the function of the mammalian Crumbs complex during TJ formation and epithelial polarization (Roh, 2003).
Crumbs is an apical organizer crucial for the maintenance of epithelial polarity in Drosophila. It is known that Crumbs interacts with Discs lost (Dlt; now redefined as Drosophila Patj), a protein with four PDZ (PSD95/Discs Large/ZO-1) domains, and Stardust (Sdt), a protein of the MAGUK (membrane-associated guanylate kinase) family. Potential homologs of Dlt have been sought in human epithelial cells and one of them has been characterized in intestinal epithelial cells. Human INAD-like (hINADl) contains 8 PDZ domains, is concentrated in tight junctions, and is also found at the apical plasma membrane. Overexpression of hINADl disrupts the tight junctions localization of ZO-1 and 3. A partial cDNA coding the transmembrane and cytoplasmic domains of a new human crumbs (CRB3), expressed in Caco-2 cells, has been identified. This CRB3 is able to interact through its C-terminal end with the N-terminal domain of hINADl. Taken together, the data indicate that hINADl is likely to represent a Dlt homolog in mammalian epithelial cells and might be involved in regulating the integrity of tight junctions. It is thus proposed that hINADl be renamed PATJ, for protein associated to tight junctions (Lemmers, 2002).
Drosophila Crumbs is a transmembrane protein that plays an important role in epithelial cell polarity and photoreceptor development. Overexpression of Crumbs in Drosophila epithelia expands the apical surface and leads to disruption of cell polarity. Drosophila Crumbs also interacts with two other polarity genes, Stardust and Patj. Recent work has identified a human orthologue of Drosophila Crumbs, known as CRB1, that is mutated in the eye disorders, retinitis pigmentosa and Leber congenital amaurosis. CRB1 can form a complex with mammalian orthologues of Stardust and Patj, known as protein associated with Lin-7 (Pals1) (see Drosophila Lin-7) and Pals1 associated tight junction (PATJ), respectively. In the current report a full length cDNA has been cloned for a human paralogue of CRB1 called Crumbs3 (CRB3). In contrast to Drosophila Crumbs and CRB1, CRB3 has a very short extracellular domain but like these proteins it has a conserved intracellular domain that allows it to complex with Pals1 and PATJ. Mouse and human CRB3 have identical intracellular domains but divergent extracellular domains except for a conserved N-glycosylation site. CRB3 is localized to the apical surface and tight junctions but the conserved N linked glycosylation site does not appear to be necessary for CRB3 apical targeting. CRB3 is a specialized isoform of the Crumbs protein family that is expressed in epithelia and can tie the apical membrane to the tight junction (Markarova, 2003).
Initially identified in Caenorhabditis elegans Lin-2 and Lin-7, L27 domain is a protein-protein interaction domain capable of organizing scaffold proteins into supramolecular assemblies by formation of heteromeric L27 domain complexes. L27 domain-mediated protein assemblies have been shown to play essential roles in cellular processes including asymmetric cell division, establishment and maintenance of cell polarity, and clustering of receptors and ion channels. The structural basis of L27 domain heteromeric complex assembly is controversial. The high-resolution solution structure of the prototype L27 domain complex formed by mLin-2 and mLin-7 was determined as well as the solution structure of the L27 domain complex formed by Patj and Pals1. The structures suggest that a tetrameric structure composed of two units of heterodimer is a general assembly mode for cognate pairs of L27 domains. Structural analysis of the L27 domain complex structures further showed that the central four-helix bundles mediating tetramer assembly are highly distinct between different pairs of L27 domain complexes. Biochemical studies revealed that the C-terminal alpha-helix responsible for the formation of the central helix bundle is a critical specificity determinant for each L27 domain in choosing its binding partner. These results provide a unified picture for L27 domain-mediated protein-protein interactions (Feng, 2005).
The Crumbs complex that also contains the cortical proteins Stardust and DPATJ (a homologue of PATJ), is crucial for the building of epithelial monolayers in Drosophila. Although loss of function of the Crumbs or Stardust genes prevents the stabilization of a belt of adherens junctions at the apico-lateral border of the cells, no phenotype has been described for the Drosophila Patj gene and its role in epithelial morphogenesis and polarity remains unknown. Downregulated PATJ stable lines of Caco2 have been produced to clarify its role in epithelial morphogenesis. In PATJ knockdown cells, Pals1 (a Stardust homologue) is no longer associated with tight junctions whereas Crumbs3 (Crb3) is accumulated into a compartment spatially close to the apical membrane and related to early endosomes. Furthermore, occludin and ZO-3, two proteins of tight junctions are mislocalized on the lateral membrane indicating that PATJ plays a novel role in the building of tight junctions by providing a link between their lateral and apical components. Thus, PATJ stabilizes the Crb3 complex and regulates the spatial concentration of several components at the border between the apical and lateral domains (Michel, 2005).
Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. This study found that that mammalian PATJ, studied in cell culture, is targeted to the apical region and tight junctions once cell polarization is initiated. Using RNAi techniques it has been shown that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases the understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity (Shin, 2005).
Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal diseases including Leber's congenital amaurosis (LCA) and retinitis pigmentosa type 12. The CRB1 transmembrane protein localizes at a subapical region (SAR) above intercellular adherens junctions between photoreceptor and Muller glia (MG) cells. The Crb1-/- phenotype, as shown in Crb1-/- mice, is accelerated and intensified in primary retina cultures. Immuno-electron microscopy showed strong Crb1 immunoreactivity at the SAR in MG cells but barely in photoreceptor cells, whereas Crb2, Crb3, Patj, Pals1 and Mupp1 were present in both cell types. Human CRB1, introduced in MG cells in Crb1-/- primary retinas, was targeted to the SAR. RNA interference-induced silencing of the Crb1-interacting-protein Pals1 (protein associated with Lin7; Mpp5) in MG cells resulted in loss of Crb1, Crb2, Mupp1 and Veli3 protein localization and partial loss of Crb3. It is concluded that Pals1 is required for correct localization of Crb family members and its interactors at the SAR of polarized MG cells (van Rossum, 2006).
Mammalian Lin-7 forms a complex with several proteins, including PALS1, that have a role in polarity determination in epithelial cells. Loss of Lin-7 protein from the polarized epithelial cell line Madin-Darby canine kidney II by small hairpin RNA results in defects in tight junction formation as indicated by lowered transepithelial electrical resistance and mislocalization of the tight junction protein ZO-1 after calcium switch. The knock down of Lin-7 also resulted in the loss of expression of several Lin-7 binding partners, including PALS1 and the polarity protein PATJ. The effects of Lin-7 knock down were rescued by the exogenous expression of murine Lin-7 constructs that contained the L27 domain, but not the PDZ domain alone. Furthermore, exogenously expressed PALS1, but not other Lin-7 binding partners, also rescued the effects of Lin-7 knock down, including the restoration of PATJ protein in rescued cell lines. Finally, the effects of Lin-7 knock down appeared to be due to instability of PALS1 protein in the absence of Lin-7, as indicated by an increased rate of PALS1 protein degradation. Taken together, these results indicate that Lin-7 functions in tight junction formation by stabilizing its membrane-associated guanylate kinase binding partner PALS1 (Straight, 2006).
In human, mutations in tuberous sclerosis complex protein 1 or 2 (TSC1/2 or hamartin/tuberin) cause tuberous sclerosis characterized by the occurrence of multiple hamartomas. In contrast, mutations in the Crumbs homolog-1 (CRB1) gene cause retinal degeneration diseases including Leber congenital amaurosis and retinitis pigmentosa type 12. This study reports, using a two-hybrid assay, a direct molecular interaction between TSC2 C-terminal part and PDZ 2 and 3 of PATJ, a scaffold member of the Crumbs 3 (CRB 3) complex in human intestinal epithelial cells, Caco2. TSC2 interacts not only with PATJ, but also with the whole CRB 3 complex by GST-pull down assays. In addition, TSC2 co-immunoprecipitates and co-localizes partially with PATJ at the level of the tight junctions. Furthermore, depletion of PATJ from Caco2 cells induces an increase in mammalian Target Of Rapamycin Complex 1 (mTORC1) activity, which is totally inhibited by rapamycin. In contrast, in the same cells, inhibition of phosphoinositol-3 kinase (PI-3K) by wortmannin does not abolish rpS6 phosphorylation. These functional data indicate that the Crumbs complex is a potential regulator of the mTORC1 pathway, cell metabolism and survival through a direct interaction with TSC1/2 (Massey-Harroche, 2007).
Directional migration by epithelial cells is important in wound healing. Recent studies have shown that polarity proteins such as mammalian Partitioning-defective 6 (Par6), atypical protein kinase C (aPKC) and mammalian Discs large 1 (Dlg1) are crucial not only for epithelial apico-basal polarity, but also for directional movement. This study shows that PATJ, another evolutionarily conserved polarity protein, is also required for directional migration by using a wound-induced migration assay. In addition, aPKC and Par3 were found to localize to the leading edge during migration of epithelia, and PATJ regulates their localization. Furthermore, these results show that microtubule-organizing centre orientation is disrupted in PATJ RNA interference (RNAi) MDCKII (Madin-Darby canine kidney II) cells during migration. Together, these data indicate that PATJ controls directional migration by regulating the localization of aPKC and Par3 to the leading edge. The migration defect in PATJ RNAi cells seems to be due to the disorganization of the microtubule network induced by mislocalization of polarity proteins (Shin, 2007).
The E6 protein from high-risk human papillomavirus types interacts with and degrades several PDZ domain-containing proteins that localize to adherens junctions or tight junctions in polarized epithelial cells. The tight junction-associated multi-PDZ protein PATJ was identified as a novel binding partner and degradation target of high-risk types 16 and 18 E6. PATJ functions in the assembly of the evolutionarily conserved CRB-PALS1-PATJ and Par6-aPKC-Par3 complexes and is critical for the formation of tight junctions in polarized cells. The ability of type 18 E6 full-length to bind to, and the subsequent degradation of, PATJ is dependent on its C-terminal PDZ binding motif. The spliced 18 E6* protein, which lacks a C-terminal PDZ binding motif, associates with and degrades PATJ independently of full-length 18 E6. Thus, PATJ is the first binding partner that is degraded in response to both isoforms of 18 E6. The ability of E6 to utilize a non-E6AP ubiquitin ligase for the degradation of several PDZ binding partners has been suggested. This study demonstrates that 18 E6-mediated degradation of PATJ is not inhibited in cells where E6AP is silenced by shRNA. This suggests that the E6-E6AP complex is not required for the degradation of this protein target (Storrs, 2007).
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