Many extracellular proteins with diverse functions contain domains similar to epidermal growth factor (EGF). These include the coagulation factors IX and X, proteins with two EGF-like domains, the first of which contains the consensus residues. The first EGF-like domain of human factor IX contains a calcium-binding site, which is believed to be responsible for one of the high-affinity sites detected in this protein. Similar results have been obtained for bovine factor X. Handford (1991) has defined residues involved in a new type of calcium-binding site and provides strong circumstantial evidence for calcium-binding motifs in many extracellular proteins, including the developmentally important proteins of Drosophila, Notch, Delta and Crumbs (Handford, 1991).
Androgen-binding protein (ABP) and sex hormone-binding globulin (SHBG) are extracellular steroid-binding proteins that are homologous to the COOH-terminal domain of vitamin K-dependent protein S, a protein important in blood clotting. The sequences of ABP, SHBG, and protein S are also similar to two basement membrane proteins, Laminin and Merosin, and to Crumbs, an integral membrane protein. These latter three proteins have important roles in regulating differentiation and development. The sequence similarity corresponds to the G domain of laminin A chain, which binds heparin and type IV collagen. Analysis of a multiple alignment of these proteins reveals one well-conserved segment corresponding to the part of SHBG that binds to its membrane receptor and another corresponding to the part of protein S that binds to C4b-binding protein (Joseph, 1992).
Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of diseases that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. To isolate candidate genes for chorioretinal diseases, cDNAs specifically or preferentially expressed in the human retina and the retinal pigment epithelium (RPE) were cloned through a novel suppression subtractive hybridization (SSH) method. One of these cDNAs (RET3C11) maps to chromosome 1q31-q32.1, a region harboring a gene involved in a severe form of autosomal recessive RP characterized by a typical preservation of the para-arteriolar RPE (RP12). The full-length cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A G-like domains and a C-type lectin domain. This protein is homologous to the Drosophila melanogaster protein Crumbs (Crb), and is denoted CRB1 (crumbs homolog 1). In ten unrelated RP patients with preserved para-arteriolar RPE, a homozygous AluY insertion disrupting the ORF, five homozygous missense mutations and four compound heterozygous mutations were identified in CRB1. The similarity to Crb suggests a role for CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration (den Hollander, 1999).
Mutations in the human Crumbs homolog 1 (CRB1) gene cause severe retinal dystrophies, ranging from retinitis pigmentosa to Leber congenital amaurosis. The CRB1 gene is expressed specifically in human retina and brain and encodes a protein homologous to the Drosophila Crumbs protein. In crumbs mutant embryos, apico-basal polarity of epithelial cells is lost, leading to widespread epidermal cell death. The small cytoplasmic domain of Crumbs organizes an intracellular protein scaffold that defines the assembly of a continuous zonula adherens. The crumbs mutant phenotype can be partially rescued by expression of just the membrane-bound cytoplasmic domain, and overexpression of this domain in a wild-type background results in a multilayered epidermis. A striking difference between CRB1 and Crumbs is that the latter contains a transmembrane region and a 37 amino acid cytoplasmic domain. An alternative splice variant of human CRB1 is described that encodes a cytoplasmic domain 72% similar to that of Drosophila Crumbs. Two intracellular subdomains that are necessary for function in Drosophila are absolutely conserved. Rescuing and overexpression studies in Drosophila show that the cytoplasmic domains are functionally related between these distant species. This suggests that CRB1 organizes an intracellular protein scaffold in the human retina. Human homologs of proteins binding to Crumbs may be part of this complex and represent candidate genes for retinal dystrophies (den Hollander, 2001).
Mutations in the human Crumbs homolog 1 (CRB1) gene cause severe retinal dystrophies. CRB1 is homologous to Drosophila Crumbs, a protein essential for establishing and maintaining epithelial polarity. The mouse orthologue, Crb1, has been isolated and its expression pattern has been analyzed in embryonic and post-natal stages. Crb1 is expressed exclusively in the eye, and the central nervous system. In the developing eye, expression of Crb1 is detected in the retinal progenitors, and later on becomes restricted to the differentiated photoreceptor cells where it remains active up to the adult stage. In the developing neural tube, expression of Crb1 is restricted to its most ventral structures, coinciding with the expression domain of Nkx2.2. In the adult brain, Crb1 expression is confined to areas where the production and migration of neurons occurs in adulthood (den Hollander, 2002).
The correct assembly of junction components, such as E-cadherin and beta-catenin, into the zonula adherens is fundamental for the function of epithelia, both in flies and in vertebrates. In C. elegans, however, the cadherin-catenin system is not essential for general adhesion, raising the question as to the genetic basis controlling junction morphogenesis in nematodes. dlg-1, the C. elegans homolog of the Drosophila tumor-suppressor gene discs-large, plays a crucial role in epithelial development. DLG-1 is restricted to adherens junctions of all embryonic epithelia, which contrasts with the localization of the Drosophila and vertebrate homologs in septate and tight junctions, respectively. Proper localization of DLG-1 requires the basolateral LET-413 protein (identified as the Drosophila scrib ortholog), but is independent of the cadherin-catenin system. Embryos in which dlg-1 activity is eliminated by RNA-mediated interference fail to form a continuous belt of junction-associated antigens and arrest development. Loss of dlg-1 activity differentially affects localization of proteins normally enriched apically to the zonula adherens. While the distribution of an atypical protein kinase C (PKC-3) and other cytoplasmic proteins (PAR-3, PAR-6: see Drosophila par-6) is not affected in dlg-1 (RNAi) embryos, the transmembrane protein encoded by crb-1, the C. elegans homolog of Drosophila crumbs, is no longer concentrated in this domain. In contrast to Drosophila, however, crb-1 and a second crb-like gene are not essential for epithelial development in C. elegans. Together the data indicate that several aspects of the spatial organization of epithelial cells and its genetic control differ between flies, worms, and vertebrates, while others are conserved. The molecular nature of DLG-1 makes it a likely candidate to participate in the organization of a protein scaffold that controls the assembly of junction components into the zonula adherens (Bossinger, 2001).
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).
In early vertebrate development, apicobasally polarised blastomeres divide to produce inner non-polarised cells and outer polarised cells that follow different fates. How the polarity of these early blastomeres is established is not known. The role of Crumbs3, Lgl2 and the apical aPKC in the polarisation of frog blastomeres was examined. Lgl2 localises to the basolateral membrane of blastomeres, while Crumbs3 localises to the apical and basolateral membranes. Overexpression aPKC and Crumbs3 expands the apical domain at the expense of the basolateral and repositions tight junctions in the new apical-basolateral interface. Loss of aPKC function causes loss of apical markers and redirects basolateral markers ectopically to the apical membrane. Cell polarity and tight junctions, but not cell adhesion, are lost and outer polarised cells become inner-like apolar cells. Overexpression of Xenopus Lgl2 phenocopies the aPKC knockout, suggesting that Lgl2 and aPKC act antagonistically. This was confirmed by showing that aPKC and Lgl2 can inhibit the localisation of each other and that Lgl2 rescues the apicalisation caused by aPKC. It is concluded that an instrumental antagonistic interaction between aPKC and Lgl2 defines apicobasal polarity in early vertebrate development (Chalmers, 2005).
Different mutations in the human Crumbs homolog-1 (CRB1) gene cause a variety of retinal dystrophies, such as Leber congenital amaurosis, early onset retinitis pigmentosa (e.g., RP12), RP with Coats-like exudative vasculopathy, and pigmented paravenous retinochoroidal atrophy. Loss of Crb1 leads to displaced photoreceptors and focal degeneration of all neural layers attributable to loss of adhesion between photoreceptors and Muller glia cells. To gain insight into genotype-phenotype relationship, Crb1(C249W) mice were generated that harbor an amino acid substitution (Cys249Trp) in the extracellular sixth calcium-binding epidermal growth factor domain of Crb1. This analysis showed that Crb1(C249W) as wild-type protein traffics to the subapical region adjacent to adherens junctions at the outer limiting membrane (OLM). Hence, these data suggest correct trafficking of the corresponding mutant CRB1 in RP12 patients. Crb1(C249W) mice showed loss of photoreceptors in the retina, relatively late compared with mice lacking Crb1. Scanning laser ophthalmoscopy revealed autofluorescent dots that presumably represent layer abnormalities after OLM disturbance. Gene expression analyses revealed lower levels of pituitary tumor transforming gene 1 (Pttg1) transcripts in Crb1C249W/- knock-in and Crb1-/- knock-out compared with control retinas. Exposure to white light decreased levels of Pttg1 in Crb1 mutant retinas. Deregulation of Pttg1 expression is postulated attributable to a C249W substitution in the extracellular domain of Crb1 (van de Pavert, 2007).
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 (Pals1), also contain two L27 domains: an L27C domain that binds mLin-7 (see Drosophila Lin-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. PATJ is a homolog of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that 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 Discs Lost. This Drosophila Pals1 homolog represents 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-Discs-lost 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 and PATJ, the mammalian homologs of Stardust and Discs lost, respectively. 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).
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 Discs Lost. 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 Discs Lost, known as protein associated with Lin-7 (Pals1) 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).
The Crumbs complex that also contains the cortical proteins Stardust and DPATJ, 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 Dpatj 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).
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).
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).
Establishment of apical-basal cell polarity has emerged as an important process during development, and the Crumbs complex is a major component of this process in Drosophila. By comparison, little is known about the role of Crumbs (Crb) proteins in vertebrate development. The FERM protein Mosaic Eyes (Moe; see Drosophila Yurt) is a novel regulatory component of the Crumbs complex. Moe coimmunoprecipitates with Ome/Crb2a and Nok (Pals1; see Drosophila Stardust) from adult eye and in vitro interaction experiments suggest these interactions are direct. Morpholino knockdown of ome/crb2a phenocopies the moe mutations. Moe and Crumbs proteins colocalize apically and this apical localization requires reciprocal protein function. By performing genetic mosaic analyses, it is shown that moe minus rod photoreceptors have greatly expanded apical structures, suggesting that Moe is a negative regulator of Crumbs protein function in photoreceptors. It is proposed that Moe is a crucial regulator of Crumbs protein cell-surface abundance and localization in embryos (Hsu, 2006)
Polarity is an essential attribute of most eukaryotic cells. One of the most prominent features of cell polarity in many tissues is the subdivision of cell membrane into apical and basolateral compartments by a belt of cell junctions. The proper formation of this subdivision is of key importance. In sensory cells, for example, the apical membrane compartment differentiates specialized structures responsible for the detection of visual, auditory, and olfactory stimuli. In other tissues, apical specializations are responsible for the propagation of fluid flow. Despite its importance, the role of genetic determinants of apico-basal polarity in vertebrate embryogenesis remains poorly investigated. This study shows that zebrafish oko meduzy (ome) locus encodes a crumbs gene homolog, essential for the proper apico-basal polarity of neural tube epithelia. Two ome paralogs, crb2b and crb3a, promote the formation of apical cell features: photoreceptor inner segments and cilia in renal and auditory systems. The motility of cilia is defective following the impairment of crb2b function. Apical surface defects in ome- and crb2b-deficient animals are associated with profound disorganization of neuronal architecture and with the formation of pronephric cysts, respectively. Unexpectedly, despite differences in their structure and expression patterns, crumbs genes are, at least partially, functionally interchangeable. It is concluded that ome and related crumbs genes are necessary for the formation of gross morphological features in several organs, including the CNS and the renal system. On the cellular level, crumbs genes regulate the formation of both ciliary and nonciliary apical membrane compartment (Omori, 2006).
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