Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6.

Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. Here we show by in vitro analysis that, similar as in vertebrates, the single PDZ domain of Drosophila DmPar-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to DmPar-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit DmPar-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins.

[Role of Crumbs proteins in the control of epithelial cell and photoreceptor morphogenesis]. / Rôle des protéines Crumbs dans le contrôle de la morphogenèse des cellules épithéliales et des photorécepteurs.

Degeneration of retina can have many causes and among the genes involved, CRB1 has been shown to be associated with Retinitis pigmentosa (RP) group 12 and Leber congenital amaurosis (LCA), two dramatic pathologies in young patients. CRB1 belongs to a family of genes conserved from Caenorhabditis elegans to human. In Drosophila melanogaster, for example, crb is essential both for the formation of the adherens junctions in epithelial cells of ectodermal origin during gastrulation and for the morphogenesis of photoreceptors in the eye. Crumbs is a transmembrane protein with a short cytoplasmic domain that interacts with scaffold proteins, Stardust and Discs lost, and with the apical cytoskeleton made of moesin and betaheavy-spectrin. The extracellular domain of Crumbs is essential for its function in photoreceptors but so far there are no known proteins interacting with it. In human, there are three known crb homologues, CRB1, 2 and 3, and CRB1 is expressed in the retina and localizes to the adherens junctions of the rods. Based on the model drawn from Drosophila, CRB1 could be involved in maintaining the morphology of rods to ensure a normal function of the retina. This is supported by the fact that the homologues of the known partners of Crumbs are also conserved in human and expressed in the retina. Understanding the precise molecular mechanism by which CRB1 acts will help to find new therapies for patients suffering from RP12 and LCA.

CRB3 binds directly to Par6 and regulates the morphogenesis of the tight junctions in mammalian epithelial cells.

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.

Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells.

The formation of a belt-like junctional complex separating the apical from the lateral domain is an essential step in the differentiation of epithelial cells. Thus protein complexes regulating this event are of first importance for the development of cell polarity and physiological functions of epithelial tissues. In Drosophila, the discovery of a gene, crb, controlling the coalescence of the spots of zonula adherens (ZA) into a adhesive ring around the cells was a major step. We know now that Crumbs, the product of crb is an apical transmembrane protein conserved in mammals and that it interacts by its cytoplasmic domain with two cortical modular proteins, Stardust (Sdt) and Discs lost (Dlt) that are also essential for the correct assembly of the ZA. These two proteins are also conserved in mammals and it is most likely that the Crumbs complex plays a similar role in very different species. Recently, we have shown that Crumbs interacts with the cortical cytoskeleton made of DMoesin and beta heavy-Spectrin and this connection could explain in part the role of Crumbs in building the ZA. Future work will help to understand several aspects of the Crumbs complex that are still unknown, like the role of the large extracellular domain or the precise function of Sdt and Dlt in the building of the ZA. Finding an answer to these questions will help to find new therapies for Retinitis pigmentosa and other retina degeneration in which CRB1, the human homologue of crb, has been involved.

Crumbs interacts with moesin and beta(Heavy)-spectrin in the apical membrane skeleton of Drosophila.

The apical transmembrane protein Crumbs is necessary for both cell polarization and the assembly of the zonula adherens (ZA) in Drosophila epithelia. The apical spectrin-based membrane skeleton (SBMS) is a protein network that is essential for epithelial morphogenesis and ZA integrity, and exhibits close colocalization with Crumbs and the ZA in fly epithelia. These observations suggest that Crumbs may stabilize the ZA by recruiting the SBMS to the junctional region. Consistent with this hypothesis, we report that Crumbs is necessary for the organization of the apical SBMS in embryos and Schneider 2 cells, whereas the localization of Crumbs is not affected in karst mutants that eliminate the apical SBMS. Our data indicate that it is specifically the 4.1 protein/ezrin/radixin/moesin (FERM) domain binding consensus, and in particular, an arginine at position 7 in the cytoplasmic tail of Crumbs that is essential to efficiently recruit both the apical SBMS and the FERM domain protein, DMoesin. Crumbs, Discs lost, betaHeavy-spectrin, and DMoesin are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. We propose that Crumbs stabilizes the apical SBMS via DMoesin and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity.

hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells.

dCrumbs is an apical organizer crucial for the maintenance of epithelial polarity in Drosophila (1). It is known that dCrumbs interacts with Discs lost (Dlt), a protein with four PDZ (PSD95/Discs Large/ZO-1) domains (2), and Stardust (Sdt), a protein of the MAGUK (membrane-associated guanylate kinase) family (3, 4). We have searched for potential homologs of Dlt in human epithelial cells and characterized one of them 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 disrupted the tight junctions localization of ZO-1 and 3. We also identified a partial cDNA coding the transmembrane and cytoplasmic domains of a new human crumbs (CRB3) expressed in Caco-2 cells. This CRB3 was 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. We thus propose to rename hINADl PATJ for protein associated to tight junctions.