Molecular Cloning and Characterization of Pals, Proteins Associated with mLin-7

Abstract

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7.The proper targeting of proteins at the apical or basolateral surface of epithelial cells is crucial for cellular transport. Similarly, proper targeting of proteins to either axons or dendrites plays an important role in neurotransmission. Many studies have been directed at the molecular basis that controls these targeting decisions. In Caenorhabditis elegans, three proteins (Lin-2, Lin-7, and Lin-10) are crucial for the correct localization of the worm epidermal growth factor receptor, Let-23, to the basolateral surface of the body wall epithelium (1-3). The lin-10 gene has also been shown to have a role in localization of glutamate receptors in the C. elegans brain (4). A similar complex of mammalian homologues of Lin-2, Lin-7, and Lin-10 have been identified in mammalian brain (5, 6). In mammalian brain, the homologue of Lin-10 is known as X11␣ or Mint1 (7-9). X11␣/Mint1 contains one PTB 1 domain and two PDZ domains. PTB domains act as a phosphotyrosine binding domain in proteins such as Shc and insulin receptor substrate-1, but, in X11 and many other proteins, the PTB domain binding targets bind independent of phosphotyrosine (10). PDZ domain proteins are named for their first identification in PSD-95, Drosophila Discs Large, and zona occludens-1 proteins (11). They commonly bind to sequences at the extreme carboxyl terminus of proteins. In addition to PTB and PDZ domains, X11␣/Mint1 contains an extended amino terminus that can bind mLin-2/Cask and Munc-18 (9, 12).mLin-2/CASK is a membrane-associated guanylate kinase (Maguk) protein containing a guanylate kinase-like domain, an SH3 domain, a PDZ domain, and a calmodulin kinase-like domain (1...

Nok plays an essential role in maintaining the integrity of the outer nuclear layer in the zebrafish retina

“…Besides a PDZ domain, six additional protein-protein interaction domains have been identified in Nok and some of their targets have been identified in mammalian epithelial cell culture systems. Among these domains, the Nterminal conserved domain interacts with Par-6 (Hurd et al, 2003), L27N interacts with DLT (Roh et al, 2002), L27C interact with Lin7 (Kamberov et al, 2000). These proteins also likely interact with Nok in photoreceptors and assist in targeting Crumbs to the inner segments.…”

“…Such function of the nok gene has not been discovered before. The nok gene, zebrafish homolog of fly stardust and mouse pals1 (Hong et al, 2001;Kamberov et al, 2000), was initially identified for its function in retinal epithelial polarity and cellular pattern formation during early retinal development in zebrafish (Wei and Malicki, 2002). Our observation of Nok's continued expression in the junctional region between the inner segments of photoreceptors in fully developed retina promoted us to perform experiments to determine the function of Nok in photoreceptor adhesion.…”

PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial 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 PALS1-PATJ-CRB complex localizes to the tight junction of mammalian epithelial cells, and the disruption of the complex leads to defects in cell polarity (Straight et al, 2004). 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).…”

Isoform‐specific interaction of Flamingo/Starry Night with excess Bazooka affects planar cell polarity in the Drosophila wing

“…A second evolutionarily conserved protein complex, initially identified in epithelia of Drosophila, comprises the transmembrane protein Crumbs, which is linked by its C-terminal amino acids (ERLI) to the membrane-associated guanylate kinase (MAGUK) Stardust (Bachmann et al, 2001;Hong et al, 2001). Stardust recruits the scaffolding proteins DLin-7 and DPATJ into the complex (Kamberov et al, 2000;Roh et al, 2002;Bachmann et al, 2004). The proteins are localized in the subapical region, apical to the zonula adherens, a site that corresponds to that of tight junctions (which are not found in invertebrate epithelia) in vertebrate cells, and most of them are essential for the establishment and/or maintenance of epithelial polarity in the Drosophila embryo.…”