The mammalian genome encodes three Crumbs isoforms (Crb1-3), which are single-pass transmembrane proteins, with an extracellular domain rich in EGF-repeats and a cytosolic tail that interacts with other components of the Crumbs complex. The link between epithelial polarity and Crb was first described in Drosophila epithelia, where Crb was exclusively confined to the apical membranes 66. Mutations of this protein caused drastic defects in the epithelium and were embryonically lethal. Conversely, overexpression of Crb was sufficient to confer apical characteristics to all the plasma membrane of Drosophila epithelial cells67. Even more strikingly, the cytoplasmic tail of Crb was shown to be the mediator of this effect
67. Two more proteins were found to have functions similar to Crb: Stardust 68 and Dpatj (also called Disc Lost) 69. Further characterization of these two proteins showed that they form a complex with Crb and are required to establish epithelial polarity.
CRB
Humans have 3 Crbs, named Crb1-3 and they differ by tissue distribution. Crb1 is expressed in brain, cornea and retina and mutations in this protein are associated with degenerative pathologies of the retina 70. Crb2 is more widely expressed 71. Also this protein plays a role in
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retinal development, as mutations or depletions of Crb2 are associated with retinal degeneration 71, 72. Both Crb1 and Crb2 have a large extracellular domain with several EGF-like domains and laminin repeats, a single transmembrane domain and a short 37 amino acid intracellular C-terminus containing one FERM (4.1, ezrin, radixin, moesin) and one PDZ protein-binding motif (ending with the aminoacids ERLI) 73. Crb3, instead, does not possess a large extracellular domain. However, its cytosolic tail is strongly conserved and it binds Pals1 (protein associated with Lin-7) and PATJ (Pals1 associated tight junction), the mammalian orthologues of Stardust and Dpatj 74. In addition to the FERM and the PDZ/ERLI domain, Crb3 has an SH3 domain 75. Crb3 is widely expressed in all epithelial tissues 75 and it localizes apically to the TJs 75, 76. Overexpression of Crb3 results in expansion of the apical membrane and delayed assembly of TJs 75, 76. Furthermore, MDCK cells overexpressing Crb3 were unable to correctly form cysts when growing in a 3D matrix 76, meaning that Crb3 overexpression disturbs apico-basal polarity. This could be due to the fact that Crb3 overexpression leads to uncontrolled expansion of the apical membrane, at the expenses of the baso-lateral membrane that does not form. The effect of Crb3 on TJ and apico-basal polarity was dependent on its ERLI domain. There are two proteins known to bind to this domain: Pals1 76 and Par6 75. However, it is still not clear which of these two proteins plays a major role downstream of Crb3. Par6 also interacts directly with Pals1 15. Hurd et al. showed that Crb3 can control Par6 localization in a Pals1 dependent manner 15. Thus, Crb3 could function via recruiting the Par complex to TJs. Crb3 was also shown to recruit the Par complex to cilia, where Crb3 and the Par protein work together in the process of ciliogenesis 77. A splice variant of Crb3 was described to localize to cilia and at the spindle poles during mitosis 78. This variant of Crb3 has a C-terminal sequence ending with CLPI (CRB3-CLP) instead of the highly conserved ERLI domain. Crb3-CLP does not bind to Pals1 or to Par6 78. Its knockdown leads to both a loss of cilia and a multinuclear phenotype associated with centrosomal and spindle abnormalities. Importin β1 was found to be a specific interaction partner of Crb3-CLP responsible for these abnormalities 78.
Pals1
Pals1 (protein associated with Lin-7) is the mammalian homolog of Drosophila’s Std. It is also known as MPP5 (membrane-associated palmitoylated protein 5) and belongs to the family of membrane-associated guanylate kinase (MAGUK) proteins. MAGUKs are scaffold proteins that contain PDZ, SH3 (Src homology 3), and guanylate kinase (GUK) domains. Pals1 also
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contains two L27 domains: an L27C domain that binds mLin-7 and an L27N domain that binds to the PDZ domain of PATJ and is needed for localization of Pals1 to TJs 79. Pals1 therefore localizes to TJs via PATJ and serves as a bridge between PATJ and Crb3 79. Pals1 interacts with and stabilizes PATJ 80, 81. Loss of Pals1 leads to polarity defects, but it is not possible to conclude if these defects are caused by the depletion of Pals1 or by the concomitant reduction in the levels of PATJ 80. As previously discussed, Pals1 interacts with Par6 15, 16 and it appears that binding to Par6 and PATJ is mutually exclusive 82.
PATJ
PATJ (Pals1 associated tight junction) contains 10 PDZ domains in tandem and is a paralogue of MUPP1 (multi PDZ domain protein). PATJ is the orthologue of the Drosophila protein Dpatj (or Disc Lost) 83 and it maintains the same structure of Disc Lost 81. PATJ and MUPP1 localize to TJs 83, 84 and their localization was shown to be dependent on binding to other TJ proteins such as JAMs and ZO-3 81, 83. Even though MUPP1 and PATJ share most of their interaction partners, MUPP1 appears to be dispensable for polarity establishment, whereas PATJ is of crucial importance to recruit both the Crumbs complex and the Par complex to TJs 85. PATJ and, with lower affinity, MUPP1, bind to Par6 85. The binding of PATJ to Par6 seems to be important for Par6 localization at TJs and depletion of PATJ results in mislocalization of Par6
85. Interestingly, overexpression of a constitutively active Cdc42 rescued TJ formation in the absence of PATJ. However, TJs were not concentrated apically, but they were extended laterally 85. From these observation a model could be drawn where the Crumbs complex and the Par complex are recruited to TJs and there the Crumbs complex restricts the localization of the Par complex. PATJ could participate in the recruitment of the Par complex, but active Cdc42 is upstream of it, meaning that the Crumbs complex is not crucial for Par complex localization.
Consisted with the above mentioned importance of Pals1 in epithelial polarity is the observation that Pals1 controls the stability of MUPP1 and PATJ 80, 81. MUPP1 competes with PATJ for the binding to Pals1 81 and binding to Pals1 increases the stability of MUPP1 and PATJ
80, 81. Therefore, when MUPP1 is downregulated, PATJ is stabilized and vice versa. Accordingly, downregulation of Pals1 will cause a decrease on protein level of both MUPP1 and PATJ 81. The close connection between these proteins hints that amplification or depletion of only one of these proteins, conditions encountered for example in cancer, will also affect the levels of the others, probably affecting cell polarity.
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