CRB3 Binds Directly to Par6 and Regulates the Morphogenesis of the Tight Junctions in Mammalian Epithelial Cells

Abstract

Crumbs is an apical transmembrane protein crucial for epithelial morphogenesis in Drosophila melanogaster embryos. A protein with all the characteristics for a Crumbs homologue has been identified from patients suffering from retinitis pigmentosa group 12, but this protein (CRB1) is only expressed in retina and some parts of the brain, both in human and mouse. Here, we describe CRB3, another Crumbs homologue that is preferentially expressed in epithelial tissues and skeletal muscles in human. CRB3 shares the conserved cytoplasmic domain with other Crumbs but exhibits a very short extracellular domain without the EGF-and laminin A-like G repeats present in the other Crumbs. CRB3 is localized to the apical and subapical area of epithelial cells from the mouse and human intestine, suggesting that it could play a role in epithelial morphogenesis. Indeed, expression of CRB3 or of a chimera containing the extracellular domain of the neurotrophin receptor p75NTR and the transmembrane and cytoplasmic domains of CRB3 led to a slower development of functional tight junctions in Madin-Darby canine kidney cells. This phenotype relied on the presence of CRB3 four last amino acids (ERLI) that are involved in a direct interaction with Par6, a regulator of epithelial polarity and tight junction formation. Thus, CRB3, through its cytoplasmic domain and its interactors, plays a role in apical membrane morphogenesis and tight junction regulation. INTRODUCTIONThe acquisition of polarity in epithelial cells is a fundamental mechanism crucial for the development of multicellular organisms. This process involves transmembrane proteins acting as cell surface organizers that nucleate cortical proteins, thus providing a scaffold for the building of junctional complexes that physically separate the apical surface from the basolateral domain of the plasma membrane (Knust, 2000;Tepass et al., 2001). Sorting of newly synthesized proteins is then necessary to maintain the polarized distribution of apical and basolateral proteins Nelson and Yeaman, 2001). Cell surface organizers such as integrins and cadherins have been identified in mammalian epithelial cells (Yeaman et al., 1999), but in the last few years, genetics in Drosophila proved to be instrumental in opening new perspectives on protein complexes involved in epithelial polarity (Muller, 2000). Among such complexes, the Crumbs complex stands out because it contains an apical transmembrane protein crucial for cell morphogenesis (Rashbass and Skaer, 2000;Tepass et al., 2001). Crumbs is a large (2139-aa) glycoprotein with an extracellular domain containing 30 EGF-like repeats and four laminin A-like G domains, a transmembrane domain, and a short cytoplasmic domain of 37 amino acids (Tepass et al., 1990). Expression of crb starts at gastrulation, and the protein is accumulated at the subapical region (marginal zone) of ectodermal epithelial cells where it colocalizes with two other gene products involved in epithelial polarity, Stardust (sdt) and Discs lost (dlt). These three genes, crb, s...

Extensions of PDZ domains as important structural and functional elements

“…The PDZ domain of Par-6 is one of the few known PDZ domains that has a binding groove capable of interacting with an internal protein segment (e.g., an N-terminal region of Pals1, a scaffold protein associated with tight junctions); but, it is also known to bind more classically to C-terminal motifs (e.g., the C terminus of Crb3, a small transmembrane protein) (Hurd et al, 2003;Lemmers et al, 2004;Penkert et al, 2004). In COS7 cells, activated forms of Cdc42 can modulate the binding of Par-6 to Pals1, suggesting that there may be some functional connection between the CRIB motif and the PDZ domain of Par-6 (Hurd et al, 2003).…”

Molecular signatures of metaplastic carcinoma of the breast by large-scale transcriptional profiling: identification of genes potentially related to epithelial–mesenchymal transition

“…BMP2, which also has documented function related to EMT (Ma et al, 2005), was identified among genes upregulated in MCBs. In contrast, genes encoding proteins related to maintaining epithelial phenotype, including cytoskeleton (KRT18 and KRT19), cell-cell adhesion molecules (CDH1, CEA-CAM6 and TACSTD1 (epithelial adhesion molecule)) and tight junction (EPPK1 (epiplakin 1), CLDN7, CRB3 and F11R (junctional adhesion molecule 1)), were downregulated in MCBs (Liu et al, 2000;Fujiwara et al, 2001;Lemmers et al, 2004). ESR1 and ERBB2 were also downregulated in MCBs, consistent with universal lack of ERa and HER-2/neu expression in MCBs as compared to DCBs (Carter et al, 2006).…”

The Apical Polarity Determinant Crumbs 2 Is a Novel Regulator of ESC-Derived Neural Progenitors

“…The Par complex consists of the three homologues of Par6 (Par6a, Par6b, and Par6d), Par3 (Pard3 and Pard3b), the two atypical protein kinase C (aPKC) homologues, aPKCk and aPKCf, and Cdc42. The Crumbs complex and Par complex interact with each other by binding Par6 to either Crumbs [7] or Pals1 [8] (schematic summary shown in Fig. 2A).…”