Basolateral targeting by leucine-rich repeat domains in epithelial cells

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure–function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.     

LET-413 is a basolateral protein required for the assembly of adherens junctions in Caenorhabditis elegans

Epithelial cells are polarized, with apical and basal compartments demarcated by tight and adherens junctions. Proper establishment of these subapical junctions is critical for normal development and histogenesis. We report the characterization of the gene let-413 which has a critical role in assembling adherens junctions in Caenorhabditis elegans. In let-413 mutants, adherens junctions are abnormal and mislocalized to more basolateral positions, epithelial cell polarity is affected and the actin cytoskeleton is disorganized. The LET-413 protein contains one PDZ domain and 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases. Strikingly, LET-413 localizes to the basolateral membrane. We suggest that LET-413 acts as an adaptor protein involved in polarizing protein trafficking in epithelial cells.     

ERBIN: a basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor

The ERBB receptors have a crucial role in morphogenesis and oncogenesis. We have identified a new PDZ protein we named ERBIN (ERBB2 interacting protein) that acts as an adaptor for the receptor ERBB2/HER2 in epithelia. ERBIN contains 16 leucine-rich repeats (LRRs) in its amino terminus and a PDZ (PSD-95/DLG/ZO-1) domain at its carboxy terminus, and belongs to a new PDZ protein family. The PDZ domain directly and specifically interacts with ERBB2/HER2. ERBIN and ERBB2/HER2 colocalize to the lateral membrane of human intestinal epithelial cells. The ERBIN-binding site in ERBB2/HER2 has a critical role in restricting this receptor to the basolateral membrane of epithelial cells, as mutation of the ERBIN-binding site leads to the mislocalization of the receptor in these cells. We suggest that ERBIN acts in the localization and signalling of ERBB2/HER2 in epithelia.     

The Erbin PDZ domain binds with high affinity and specificity to the carboxyl termini of delta-catenin and ARVCF

Erbin is a recently described member of the LAP (leucine-rich repeat and PDZ domain) protein family. We used a C-terminally displayed phage peptide library to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bound with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resembled the C-terminal PDZ domain-binding motifs of three p120-related catenins: delta-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or delta-catenin also demonstrated specific high affinity binding. We characterized the interactions between the Erbin PDZ domain and both ARVCF and delta-catenin in vitro and in vivo. The Erbin PDZ domain co-localized and coprecipitated with ARVCF or delta-catenin complexed with beta-catenin and E/N-cadherin. Mutagenesis and peptide competition experiments showed that the association of Erbin with the cadherin-catenin complex was mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and delta-catenin. Finally, we showed that endogenous delta-catenin and Erbin co-localized in and co-immunoprecipitated from neurons. These results suggest that delta-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains.     

The ERBB2/HER2 receptor differentially interacts with ERBIN and PICK1 PSD-95/DLG/ZO-1 domain proteins

Identification of protein complexes associated with the ERBB2/HER2 receptor may help unravel the mechanisms of its activation and regulation in normal and pathological situations. Interactions between ERBB2/HER2 and Src homology 2 or phosphotyrosine binding domain signaling proteins have been extensively studied. We have identified ERBIN and PICK1 as new binding partners for ERBB2/HER2 that associate with its carboxyl-terminal sequence through a PDZ (PSD-95/DLG/ZO-1) domain. This peptide sequence acts as a dominant retention or targeting basolateral signal for receptors in epithelial cells. ERBIN belongs to the newly described LAP (LRR and PDZ) protein family, whose function is crucial in non vertebrates for epithelial homeostasis. Whereas ERBIN appears to locate ERBB2/HER2 to the basolateral epithelium, PICK1 is thought to be involved in the clustering of receptors. We show here that ERBIN and PICK1 bind to ERBB2/HER2 with different mechanisms, and we propose that these interactions are regulated in cells. Since ERBIN and PICK1 tend to oligomerize, further complexity of protein networks may participate in ERBB2/HER2 functions and specificity.     

Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth,viability, and directional outgrowth of epithelial seam cells

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical–basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.     

Lano, a novel LAP protein directly connected to MAGUK proteins in epithelial cells.

Protein networks asymetrically distributed to basolateral and apical epithelial membranes maintain cell polarity and homeostasis of epithelial tissues. Genetic studies in non-vertebrates assigned two families of basolateral proteins, MAGUK (membrane-associated and guanylate kinase) and LAP (leucine-rich repeats and PDZ) proteins, to a common pathway crucial for the epithelial architecture and acting as a gatekeeper to malignancy. In mammals, three LAP proteins have been described, Densin-180, Erbin, and hScribble. Here, we identify a protein called Lano (LAP and no PDZ) only present in vertebrates and presenting strong identities with LAP proteins. Despite the lack of PDZ domain, Lano is located at the basolateral side of epithelial cells in a similar manner to Erbin and hScribble. Using in vitro and in vivo experiments, we demonstrate that Lano directly interacts with the PDZ domains of MAGUK proteins, including hDLG (human disc large), in epithelial cells. A second pool of Lano is complexed to Erbin. These LAP-MAGUK protein complexes coexist at the basolateral side of epithelial cells. We provide evidence for a direct interaction between LAP and MAGUK proteins, and we propose that various LAP-MAGUK networks targeted to the basolateral side of epithelial cells participate to homeostasis of epithelial tissues and tumor growth.     

PAPIN. A novel multiple PSD-95/Dlg-A/ZO-1 protein interacting with neural plakophilin-related armadillo repeat protein/delta-catenin and p0071

A neural plakophilin-related armadillo repeat protein (NPRAP)/delta-catenin interacts with one of Alzheimer disease-related gene products, presenilin 1. We have previously reported the interaction of NPRAP/delta-catenin with synaptic scaffolding molecule, which is involved in the assembly of synaptic components. NPRAP/delta-catenin also interacts with E-cadherin and beta-catenin and is implicated in the organization of cell-cell junctions. p0071, a ubiquitous isoform of NPRAP/delta-catenin, is localized at desmosomes in HeLa and A431 cells and at adherens junctions in Madin-Darby bovine kidney cells. We have identified here a novel protein interacting with NPRAP/delta-catenin and p0071 and named this protein plakophilin-related armadillo repeat protein-interacting PSD-95/Dlg-A/ZO-1 (PDZ) protein (PAPIN). PAPIN has six PDZ domains and binds to NPRAP/delta-catenin and p0071 via the second PDZ domain. PAPIN and p0071 are ubiquitously expressed in various tissues and are localized at cell-cell junctions in normal rat kidney cells and bronchial epithelial cells. PAPIN may be a scaffolding protein connecting components of epithelial junctions with p0071.     

Identification of three novel Smad binding proteins involved in cell polarity

A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par-3, and Dishevelled) were identified that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S-transferase) pull-down assays, Erbin, Par-3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full-length Smad 3 protein. Failure of Erbin, Par-3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain. Erbin, Par-3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity. While transforming growth factor beta (TGFbeta) has been suggested to alter cell polarity through a Smad-independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFbeta could alter cell polarity via a Smad-dependent signaling mechanism.     

The p120 family of cell adhesion molecules

p120 is the prototypic member of the p120 subfamily of armadillo-related proteins that includes p0071, delta-catenin/NPRAP, ARVCF and the more distantly related plakophilins 1-3. Like armadillo, beta-catenin and plakoglobin these proteins are involved in mediating cell-cell adhesion. Besides their junctional localization they also reveal a cytoplasmic and nuclear localization. Non-cadherin-associated, cytoplasmic p120 functions in Rho signaling and regulation of cytoskeletal organization and actin dynamics. The nuclear function remains largely unsolved. Some characteristics seem to be shared by the various members of the family but it seems unlikely that p120-related proteins have solely redundant functions and compete for interactions with identical binding partners. Stabilization of cadherins at the membrane seems a common function of p120, p0071, delta-catenin and ARVCF but it is not yet known if and how these proteins confer distinct properties to cellular junctions. Moreover, p0071, NPRAP and ARVCF have a C-terminal PDZ-binding motif that is lacking in p120 pointing to distinct roles of these proteins. PDZ domains are found in a series of proteins involved in establishing cell polarity in epithelial cells. Thus, p120 proteins may not only be master regulators of cadherin abundance and activity but play additional roles in regulating cell polarity. This review focuses on the putative roles of p120 proteins in cell polarity.     

Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW(-4)ETW(-1)V; IC(50) = approximately 0.15 microm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp(-1) are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the beta2-beta3 loop provides a binding site for the Trp(-4) side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.     

Increased IP3/Ca signaling compensates depletion of LET-413/DLG-1 in C. elegans epithelial junction assembly

The let-413/scribble and dlg-1/discs large genes are key regulators of epithelial cell polarity in C. elegans and other systems but the mechanism how they organize a circumferential junctional belt around the apex of epithelial cells is not well understood. We report here that IP₃/Ca²⁺ signaling is involved in the let-413/dlg-1 pathway for the establishment of epithelial cell polarity during the development in C. elegans. Using RNAi to interfere with let-413 and dlg-1 gene functions during post-embryogenesis, we discovered a requirement for LET-413 and DLG-1 in the polarization of the spermathecal cells. The spermatheca forms an accordion-like organ through which eggs must enter to complete the ovulation process. LET-413- and DLG-1-depleted animals exhibit failure of ovulation. Consistent with this phenotype, the assembly of the apical junction into a continuous belt fails and the PAR-3 protein and microfilaments are no longer localized asymmetrically. All these defects can be suppressed by mutations in IPP-5, an inositol polyphosphate 5-phosphatase and in ITR-1, an inositol triphosphate receptor, which both are supposed to increase the intracellular Ca²⁺ level. Analysis of embryogenesis revealed that IP₃/Ca²⁺ signaling is also required during junction assembly in embryonic epithelia.     

Human CASK/LIN-2 Binds Syndecan-2 and Protein 4.1 and Localizes to the Basolateral Membrane of Epithelial Cells

In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction.     

The apical disposition of the Caenorhabditis elegans intestinal terminal web is maintained by LET-413

We wish to understand how organ-specific structures assemble during embryonic development. In the present paper, we consider what determines the subapical position of the terminal web in the intestinal cells of the nematode Caenorhabditis elegans. The terminal web refers to the organelle-depleted, intermediate filament-rich layer of cytoplasm that underlies the apical microvilli of polarized epithelial cells. It is generally regarded as the anchor for actin rootlets protruding from the microvillar cores. We demonstrate that: (i) the widely used monoclonal antibody MH33 reacts (only) with the gut-specific intermediate filament protein encoded by the ifb-2 gene; (ii) IFB-2 protein accumulates near the gut lumen beginning at the lima bean stage of embryogenesis and remains associated with the gut lumen into adulthood; and (iii) as revealed by immunoelectron microscopy, IFB-2 protein is confined to a discrete circumferential subapical layer within the intestinal terminal web (known in nematodes as the "endotube"); this layer joins directly to the apical junction complexes that connect adjacent gut cells. To investigate what determines the disposition of the IFB-2-containing structure as the terminal web assembles during development, RNAi was used to remove the functions of gene products previously shown to be involved in the overall apicobasal polarity of the developing gut cell. Removal of dlg-1, ajm-1, or hmp-1 function has little effect on the overall position or continuity of the terminal web IFB-2-containing layer. In contrast, removal of the function of the let-413 gene leads to a basolateral expansion of the terminal web, to the point where it can now extend around the entire circumference of the gut cell. The same treatment also leads to concordant basolateral expansion of both gut cell cortical actin and the actin-associated protein ERM-1. LET-413 has previously been shown to be basolaterally located and to prevent the basolateral expansion of several individual apical proteins. In the present context, we conclude that LET-413 is also necessary to maintain the entire terminal web or brush border assembly at the apical surface of C. elegans gut cells, a dramatic example of the so-called "fence" function ascribed to epithelial cell junctions. On the other hand, LET-413 is not necessary to establish this apical location during early development. Finally, the distance at which the terminal web intermediate filament layer lies beneath the gut cell surface (both apical and basolateral) must be determined independently of apical junction position.     

Basolateral Localization of the Caenorhabditis elegans Epidermal Growth Factor Receptor in Epithelial Cells by the PDZ Protein LIN-10

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.     

PDZ domains of Par-3 as potential phosphoinositide signaling integrators

Multiple PDZ domain scaffold protein Par-3 and phosphoinositides (PIPs) are required for polarity in diverse cell types. We show that the second PDZ domain of Par-3 binds to phosphatidylinositol (PI) lipid membranes with high affinity. We further demonstrate that a large subset of PDZ domains in mammalian genomes are capable of binding to PI lipid membranes, indicating that lipid binding is the second most prevalent interaction mode of PDZ domains known to date. The biochemical and structural basis of Par-3 PDZ2-mediated membrane interaction is characterized in detail. The membrane binding capacity of Par-3 PDZ2 is critical for epithelial cell polarization. Interestingly, the lipid phosphatase PTEN directly binds to the third PDZ domain of Par-3. The concatenation of the PIP-binding PDZ2 and the lipid phosphatase PTEN-binding PDZ3 endows Par-3 as an ideal scaffold protein for integrating PIP signaling events during cellular polarization.     

Folliculin,the Product of the Birt-Hogg-Dube Tumor Suppressor Gene,Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion

Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd^flox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.     

Proteome identification of binding-partners interacting with cell polarity protein Par3 in Jurkat cells

The evolutionarily conserved cell polarity protein Par3, a scaffold-like PDZreontaining protein, plays a critical role in the establishment and maintenance of epithelial cell polarity. Although the role of Par3 in establishing cell polarity in epithelial cells has been intensively explored, the function of Par3 in hematopoietic cells remains elusive. To address this issue, we generated GST-fusion proteins of Par3 PDZ domains. By combiningthe GST-pull-down approach with liquid chromatography-tandem mass spectrometry, we identified 10 potential novel binding proteins of PDZ domains of Par3 in Jurkat cells (a T-cell line). The interaction of Par3 with three proteins—nuclear transport protein importin-α4 and proteasome activators PA28β and PA28γ—was confirmed using in vitro binding assay, co-immunoprecipitation assay and immunofluorescence microscopy. Our results have the potential to uncover novel functions of the cell polarity protein Par3 in blood cells.     

A role for Erbin in the regulation of Nod2-dependent NF-kappaB signaling

Nod2 is an intracellular sensor of a specific bacterial cell wall component, muramyl dipeptide, and activation of Nod2 stimulates an inflammatory response. Specific mutations of Nod2 have been associated with two inflammatory diseases, Crohn disease and Blau syndrome, and are thought to contribute to disease susceptibility through altering Nod2 signaling. Association of disease with inappropriate activation of Nod2 highlights the importance of proper regulation of Nod2 activity. However, little is known about specific regulation of the Nod2 pathway. We performed a biochemical screen to discover potential regulators of Nod2 and identified Erbin, a protein involved in cell polarity, receptor localization, and regulation of the mitogen-activated protein kinase pathway, as a novel Nod2-interacting protein. In our studies, we demonstrate specific interaction of Erbin and Nod2 both in vitro and in vivo and characterize the regions required for interaction in both proteins. We found that Nod2-dependent activation of NF-kappaB and cytokine secretion is inhibited by Erbin overexpression, whereas Erbin-/- mouse embryo fibroblasts show an increased sensitivity to muramyl dipeptide. These studies identify Erbin as a regulator of Nod2 signaling and demonstrate a novel role for Erbin in inflammatory responses.