Tight Junction Protein Par6 Interacts with an Evolutionarily Conserved Region in the Amino Terminus of PALS1/Stardust

Abstract

Tight junctions are the structures in mammalian epithelial cells that separate the apical and basolateral membranes and may also be important in the establishment of cell polarity. Two evolutionarily conserved multiprotein complexes, Crumbs-PALS1 (Stardust)-PATJ and Cdc42-Par6-Par3-atypical protein kinase C, have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. These two complexes have been linked physically and functionally by an interaction between PALS1 and Par6. Here we identify an evolutionarily conserved region in the amino terminus of PALS1 as the Par6 binding site and identify valine and aspartic acid residues in this region as essential for interacting with the PDZ domain of Par6. We have also characterized, in more detail, the amino terminus of Drosophila Stardust and demonstrate that the interaction mechanism between Stardust and Drosophila Par6 is evolutionarily conserved. Par6 interferes with PATJ in binding PALS1, and these two interactions do not appear to function synergistically. Taken together, these results define the molecular mechanisms linking two conserved polarity complexes.Epithelial cells possess asymmetry with respect to the apicobasal axis reflected by the differential distribution of proteins and lipids in the apical and basolateral surfaces (1). Polarized mammalian epithelial cells have a tight junctional seal, which serves as a physical barrier that separates apical and basolateral membranes. It has been shown that proteins containing the PDZ (postsynaptic density-95/discs large/ZO-1) domain play an important role during cell polarization (2), and multiple PDZ protein complexes are involved in the assembly and maintenance of tight junctions.One of the major groups of PDZ proteins is the membraneassociated guanylate kinase protein, which has one or more PDZ domains as well as an Src homology 3 domain and a noncatalytic guanylate kinase domain. Genetic and biochemical studies in Drosophila melanogaster have shown that membrane-associated guanylate kinase protein Stardust (Sdt) interacts with the transmembrane protein Crumbs (Crb) through its PDZ domain, and mutations in either Crb or Sdt cause polarity defects in Drosophila epithelia (3). The mammalian homologue of Sdt is PALS1 (protein associated with Lin seven) (4). Like Sdt, the PDZ domain of PALS1 binds the C-terminal tail of mammalian Crb isoforms, and PALS1 also interacts with a multi-PDZ domain protein, PATJ (PALS1-associated tight junction protein), through L27 (Lin-2 and Lin-7) domain dimerization (4, 5). The Crb-PALS1-PATJ complex localizes to the tight junctions of mammalian epithelial cells, and the disruption of the complex leads to defects in cell polarity (6). Similarly, the Drosophila PATJ homologue, formerly known as discs lost (Dlt) (7,8), can interact with the L27 domain of Sdt. Therefore, the analogous complex in D. melanogaster is Crb-Sdt-PATJ.Another evolutionarily conserved tight junction complex is composed of PDZ proteins Par3, Par6, and aPKC and the...

PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial Cells

“…Although this mutant does rescue the phenotype at 6 h, we did note that junctional rescue was delayed with this mutant if we looked at 3 h after calcium addition (data not shown). Nonetheless, this mutation that reduces the binding of Par6 to PALS1 in multiple studies (Wang et al, 2004) has at best minor effects on PALS1 function. We think that the interaction of the PALS1 complex Top, MDCKII cells.…”

“…Figure 2A depicts the domain structure of murine PALS1. The U1 region mutant V37G shows reduced binding with the polarity protein Par6 (Wang et al, 2004), the L27N domain mutant disrupts the interaction between PALS1 and PATJ (Roh et al, 2002), and the PALS1 L27C domain mutant does not bind Lin-7 (Kamberov et al, 2000). It has been shown that extensive intramolecular interactions exist between the C-terminal SH3 domain and the GUK domain of MAGUK proteins (McGee et al, 2001;Tavares et al, 2001).…”

“…The C-terminal L27 (L27C) domain of PALS1 interacts with an L27 domain within Lin-7 (Kamberov et al, 2000), and prior work in our laboratory has shown that an evolutionarily conserved region in the N terminus of PALS1 mediates its interaction with Par6. This interaction links the polarity complexes PALS1-PATJ-CRB and Par3-Par6 -aPKC together (Hurd et al, 2003;Wang et al, 2004). The function of the C-terminal SH3 domain and GUK domain of PALS1 is unknown.…”

Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes

“…The activity of these two complexes is counterbalanced by proteins such as Scribble-Lethal (2) giant larvaeDiscs large (Dlg), which localize to the basolateral domain. In the past, various studies have demonstrated that both apical complexes are rather dynamic and that their composition might be tissue-dependent and temporally and/or developmentally regulated (3)(4)(5)(6)(7)(8)(9).…”

“…Apical-basal polarization of epithelia is regulated by conserved complexes determining the apical versus the basolateral domain (1,2): apical to the adherens junctions (AJ), 3 the membrane-associated partitioning-defective (PAR)-atypical protein kinase C (aPKC) complex regulates assembly of the Crumbs (Crb) complex, which assembles more apically in the so-called subapical region. The activity of these two complexes is counterbalanced by proteins such as Scribble-Lethal (2) giant larvaeDiscs large (Dlg), which localize to the basolateral domain.…”