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Biochimica et Biophysica Acta 1745 (2005) 131 – 144 http://www.elsevier.com/locate/bba

Identification and characterization of a novel tight junction-associated family of that interacts with a WW domain of MAGI-1

Kevin M. Patrie*

Department of Internal Medicine, University of Michigan, 1580 MSRB II, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0676, USA

Received 18 October 2004; received in revised form 24 May 2005; accepted 25 May 2005 Available online 15 June 2005

Abstract

The membrane-associated guanylate kinase , MAGI-1, has been shown to be a component of epithelial tight junctions in both Madin–Darby canine kidney cells and in intestinal epithelium. Because we have previously observed MAGI-1 expression in glomerular visceral epithelial cells (podocytes) of the kidney, we screened a glomerular cDNA library to identify the potential binding partners of MAGI- 1 and isolated a partial cDNA encoding a novel protein. The partial cDNA exhibited a high degree of identity to an uncharacterized human cDNA clone, KIAA0989, which encodes a protein of 780 amino acids and contains a predicted coiled-coil domain in the middle of the protein. In vitro binding assays using the partial cDNA as a GST fusion protein confirm the binding to full-length MAGI-1 expressed in HEK293 cells, as well as endogenous MAGI-1, and also identified the first WW domain of MAGI-1 as the domain responsible for binding to this novel protein. Although a conventional PPxY binding motif for WW domains was not present in the partial cDNA clone, a variant WW binding motif was identified, LPxY, and found to be necessary for interacting with MAGI-1. When expressed in Madin–Darby canine kidney cells, the full-length novel protein was found to colocalize with MAGI-1 at the tight junction of these cells and the coiled-coil domain was found to be necessary for this localization. Because of its interaction with MAGI-1 and its localization to cell–cell junctions, this novel protein has been given the name MAGI-1-associated coiled-coil tight junction protein (MASCOT). D 2005 Elsevier B.V. All rights reserved.

Keywords: MAGI-1; Tight junction; WW domain; Coiled-coil; MAGI-1-associated coiled-coil tight junction protein; AMOTL2

1. Introduction molecular seals. In addition to this ‘‘barrier’’ function, tight junctions are also responsible for maintaining the unique The epithelia and endothelia of organisms play a apical and basolateral membrane distribution of proteins and fundamental role in establishing a physical barrier between lipids that characterizes polarized cells. A specialized two extracellular compartments of different composition epithelial cell that is gaining interest because of its within tissues. The free diffusion of solutes between the importance in normal kidney function is the glomerular compartments is regulated by tight junctions that act as visceral epithelial cell, or podocyte. Its highly complex and unique morphology manifests itself in the interdigitating nature of its minor, or foot, processes as they wrap around Abbreviations: JAM, junctional adhesion molecule; MAGI-1, mem- capillaries within the glomerulus. A specialized cell–cell brane-associated guanylate kinase inverted-1; MAGUK, membrane-asso- ciated guanylate kinase; SH3, Src homology 3; PTB, phosphotyrosine junction called the slit diaphragm serves to laterally join the binding; PDZ, PSD-95/Dlg/ZO-1; YAP, Yes-associated protein; GuK, foot processes and potentially adds to the isoporous filtering guanylate kinase; DRPLA, dentatorubral and pallidoluysian atrophy; AIP, nature of the kidney. In addition, the slit diaphragm serves to atrophin-1 interacting protein; GST, glutathione-S-transferase; HEK293, delineate distinct apical and basal membrane domains in the human embryonic kidney 293; MDCK, Madin–Darby canine kidney; foot processes, thereby imparting a degree of polarization in JEAP, junctional-enriched and -associated protein; LCCP, Leman coiled- coil protein; AMOTL1, -like 1; AMOTL2, angiomotin-like 2 their plasma membranes [1]. The nature of the slit * Tel.: +1 734 615 2414; fax: +1 734 763 0982. diaphragm membrane domain is believed to be that of a E-mail address: [email protected]. modified epithelial adherens junction although it does

0167-4889/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2005.05.011 132 K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144 contain molecular components found in tight junctions as din preferentially recognized the second WW domain of well [2]. Therefore, the exact nature of the slit diaphragm is MAGI-1 and demonstrated the first complete character- best viewed at this point as a specialized cell–cell junction ization of a MAGI family member binding to another structure. protein via a WW domain interaction. At the molecular level, epithelial tight junctions consist From the same screen that identified synaptopodin as a of a growing number of transmembrane and submembra- MAGI-1 binding partner, an additional binding partner for nous proteins whose functions are only now being realized the WW domains of MAGI-1 has been identified and is in the context of this plasma membrane microdomain (for a reported here. The isolated protein belongs to a novel family detailed review see [3,4]). Transmembrane proteins of tight of poorly characterized molecules that share the distinction junctions consist mainly of the tetraspan proteins claudin of having a predicted coiled-coil domain in the middle of the and occludin and a few members of the JAM family of protein with no other known protein domains. In addition to immunoglobulin (Ig) domain-containing adhesion mole- the protein identified here, one other member of this protein cules. Tight junction-associated submembranous proteins family is capable of binding specifically to the first WW are more numerous and diverse than the transmembrane domain of MAGI-1. Finally, the two members of this proteins and can be divided into two discreet groups based protein family can self-associate through their coiled-coil on the presence or absence of a protein–protein interaction domain and are observed to localize to the tight junction of domain referred to as the PSD-95/dlg/ZO-1 (PDZ) domain. epithelial cells. Many of the PDZ containing proteins of the tight junction belong to a large family of molecules referred to as the membrane-associated guanylate kinases (MAGUKs) 2. Experimental procedures that include ZO-1 [5], ZO-2 [6] and ZO-3 [7], as well as Pals1 [8], MAGI-1 [9] and MAGI-3 [10]. The MAGI 2.1. Expression cloning proteins consist of three members that together make up a MAGUK subfamily. We and others have found that MAGI- Details on the screening of the glomerular cDNA library 1 is a component of tight junctions in MDCK cells [9,11] with the GST-MAGI-1 WW12 probe (amino acids 219–416 and it is found localized in a podocyte-like fashion in kidney of mouse MAGI-1) has been described elsewhere [11]. Four glomeruli [12]. MAGUK proteins share a common struc- of the 11 total independent clones isolated in the screen tural organization and are proposed to function as molecular comprise the same region of mouse MASCOT/AMOTL2 scaffolds within cells [3,13]. The unique organization of and are represented in this study by clone 18.1. protein–protein interaction domains seen in conventional MAGUK proteins is inverted in the MAGI family of 2.2. Plasmid constructs proteins. In addition, two WW domains in the MAGI proteins replace the SH3 domain observed in the conven- Clone 18.1 was cloned into pGEX-4T and myc-RK5 [15] tional MAGUKs. Many of these MAGUK proteins are to make GST-clone 18.1 and myc-clone 18.1, respectively. found at special subcellular regions such as post-synaptic The proline within the LPxY motif of myc-clone 18.1 was densities within neurons as well as the tight and adherens mutated to an alanine (myc-clone 18.1 P105A) with the junctions of epithelial cells and are believed to play a role in QuickChangei kit (Stratagene, La Jolla, CA) using the the structure and function of these specialized complexes. manufacturer’s instructions. Although the binding of a number of proteins to the PDZ Full-length human and mouse MASCOT/AMOTL2 as domains and GuK domain of MAGI-1, MAGI-2 and well as human JEAP/AMOTL1 expressed sequence tag MAGI-3 has been reported, there is little data on proteins (EST) cDNAs were obtained from commercial sources that interact with the WW domains of MAGI proteins. In a (ATCC, Manassas, VA; ResGeni, Invitrogen Corp., screen to identify interacting partners for the DRPLA Carlsbad, CA) (human MASCOT/AMOTL2, clone product atrophin-1, partial cDNAs containing the WW CS0DI067YC01; mouse MASCOT/AMOTL2, IMAGE domains of MAGI-1 (AIP-3) and MAGI-2 (AIP-1) were ID# 4191248; human JEAP/AMOTL1, IMAGE ID# isolated [14], but no further characterization on these 3533422). All cDNAs were sequenced in their entirety. interactions has yet been reported. This lack of identified The entire coding sequences for human MASCOT/ binding proteins for the MAGI WW domains and our AMOTL2 and human JEAP/AMOTL1 were isolated by finding that MAGI-1 is expressed in a glomerular podocyte- PCR and cloned into Flag-pcDNA3.1(À) and 3Â HA- like fashion prompted us to screen a mouse glomerular pcDNA3.1(À) to make Flag-MASCOT/AMOTL2, Flag- cDNA library using the two WW domains of MAGI-1 as a JEAP/AMOTL1, HA-MASCOT/AMOTL2 and HA-JEAP/ probe. We have previously reported on the initial results of AMOTL1. The following deletion constructs of human this screen that identified a region of the actin-bundling MASCOT/AMOTL2 were generated by PCR using primers protein synaptopodin (containing its two PPxY motifs) as an flanking the region to be isolated: MASCOT/AMOTL2 1– interacting partner for MAGI-1 [11]. A more thorough 307, MASCOT/AMOTL2 1–578, MASCOT/AMOTL2 characterization of this interaction showed that synaptopo- 308–780, and MASCOT/AMOTL2 579–780. The deletion K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144 133 constructs were then directly cloned into the 3Â HA- 2.5. Tissue and cell lysates pcDNA3.1(À) expression vector. The following MAGI-1 constructs used in this study are Adult mouse brains were placed in a dounce homoge- described in detail elsewhere [11]: myc-MAGI-1 FL, GST- nizer along with a high salt Triton lysis buffer (50 mM MAGI-1 WW12, GST-MAGI-1 WW1, GST-MAGI-1 Hepes pH 7.5, 500 mM NaCl, 1.5 mM MgCl2,1mM WW2, myc-MAGI-1 WW12, myc-MAGI-1 WW1, myc- EGTA, 10% glycerol, 1% Triton X-100) plus protease MAGI-1 WW2. inhibitors (Complete protease inhibitor cocktail tablets; Roche Molecular Biochemicals) and homogenized with 2.3. Antibodies 25–30 strokes. The lysates were centrifuged at 20,000Âg for 30 min at 4 -C and the resulting supernatants transferred Anti-MAGI-1 polyclonal antibody UM223 is described to fresh tubes and stored at À20 -C until use. Glomerular previously [11]. Anti-myc antibody 9E10 was obtained from extracts were prepared in RIPA lysis buffer (50 mM Hepes mouse ascites fluid produced at the Hybridoma Core facility pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% at the University of Michigan. Anti-HA antibodies 3F10 (rat glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% monoclonal) and 12CA5 (mouse monoclonal) were from sodium dodecyl sulfate) plus protease inhibitors as des- Roche Molecular Biochemicals (Indianapolis, IN). Anti- cribed previously [12]. ZO-1 mouse monoclonal antibody, ZO1-1A12, and affinity Transiently transfected HEK293 cells were washed once purified rabbit anti-occludin, 71–1500, were from Zymed in ice-cold phosphate-buffered saline (PBS) and scraped in Laboratories, Inc. (San Francisco, CA). Anti-Flag mono- 400 Al Triton lysis buffer (with 150 mM NaCl), transferred clonal antibody M2 was from Sigma (St. Louis, MO). An to microcentrifuge tubes, vortexed briefly and incubated on anti-MASCOT/AMOTL2 polyclonal antibody (UM441) a rocking platform at 4 -C for 15 min. The lysates were was generated in rabbits by immunizing them with a GST centrifuged at 20,000Âg for 20 min at 4 -C and the resulting fusion protein containing amino acids 216–334 of mouse supernatants transferred to fresh tubes and stored at À20 -C MASCOT/AMOTL2. The resulting rabbit antiserum was until use. then affinity purified using a His-tagged version of the For MDCK, Caco-2 and SK-BR-3 cell lysates, two 15-cm MASCOT/AMOTL2 construct immobilized on Sepharose dishes for each were grown to confluency. The cells from 4B beads. AlexaFluor\ secondary antibodies for immuno- each dish were washed twice with PBS and scraped in 1 ml fluorescence were from Molecular Probes, Inc. (Eugene, of Triton lysis buffer, transferred to microcentrifuge tubes, OR). Horseradish-peroxidase conjugated goat anti-rabbit vortexed briefly and incubated on a rocking platform for 15 and sheep anti-mouse were from Zymed Laboratories, Inc. min at 4 -C. The lysates were centrifuged at 20,000Âg for 20 and Sigma, respectively. min at 4 -C and the resulting supernatants transferred to fresh tubes and referred to as the Triton-soluble fraction. The 2.4. Cell culture and transfections Triton-insoluble pellets were resuspended in 2 ml RIPA buffer by vortexing and sonicating briefly then incubating on HEK293 cells and MDCK cells were grown in Dulbec- a rocking platform for 15 min at 4 -C. These lysates were co’s modified Eagle’s medium (DMEM) (Invitrogen, clarified by centrifugation at 20,000Âg for 20 min at 4 -C Carlsbad, CA) supplemented with 10% fetal calf serum, 2 and the resulting supernatants transferred to fresh tubes and mM l-glutamine, 100 units/ml penicillin G and 100 Ag/ml referred to as the Triton-insoluble fraction. streptomycin. Human intestinal Caco-2 cells and human breast adenocarcinoma SK-BR-3 cells were grown in media 2.6. Western blotting, GST fusion protein precipitation formulations suggested by their supplier (ATCC). (pulldowns) and immunoprecipitations For transient transfections, HEK293 cells were grown to 50–80% confluency in 10-cm dishes and transfected with Approximately 80 Ag of protein from the Triton-soluble epitope-tagged expression constructs using Superfect Trans- fraction of MDCK, Caco-2 and SK-BR-3 cells was separated fection Reagent (Qiagen Inc., Valencia, CA). Cells were on a 7% SDS-polyacrylamide gel and transferred to a allowed to recover for 24–48 h prior to harvesting for nitrocellulose membrane (initial Western blotting using the lysates. Stable cell lines of MDCK were obtained by UM441 anti-MASCOT/AMOTL2 antibody revealed that the transfecting them at 50% confluency in 6-cm dishes with majority of MASCOT/AMOTL2 in these cells was found in Superfect Transfection Reagent. Twenty-four hours after the Triton-soluble fraction). The presence of MASCOT/ transfection, the cells were trypsinized and one-tenth of the AMOTL2 was detected with the anti-MASCOT/AMOTL2 volume of trypsinized cells transferred to 15-cm dishes UM441 antibody and the appropriate HRP conjugated containing complete media supplemented with Geneticin secondary antibody. Lysates (4 Al) from HEK293 cells (Life Technologies, Grand Island, NY) at 600 Ag/ml final transfected with HA-MASCOT/AMOTL2 or HA-JEAP/ concentration. After approximately 10 days in selection AMOTL1 were subjected to SDS-PAGE in conjunction with media, colonies were isolated, expanded and subsequently the above cell line lysates to serve as controls for the assayed for expression by immunofluorescence. specificity of the anti-MASCOT/AMOTL2 antibody. 134 K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144

The production and purification of GST fusion proteins (PBS-G) and placed on cells overnight at 30 -C in a humid from DH5a bacteria cells are described elsewhere [15]. chamber. The filters were washed three times with PBS-G, 5 Tissue or cell lysates were combined with ¨10 Ag of GST min each wash. Fluorophore-conjugated anti-rabbit, anti-rat fusion protein bound to glutathione-agarose beads and or anti-mouse secondary antibodies diluted in PGS-G were incubated on a rocking platform at 4 -C overnight. The incubated on the filters for 2 h at 30 -C in a humid chamber. beads were then washed twice with ice-cold HNTG (20 mM Filters were then washed four times with PBS-G and Hepes pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% mounted on glass slides with ProLong Antifade mounting glycerol) and once with ice-cold HNT (20 mM Hepes pH medium (Molecular Probes, Inc.). Immunofluorescence 7.5, 150 mM NaCl, 0.1% Triton X-100). Forty microliters of images were obtained with a Zeiss Axiovert 100 M confocal 2Â SDS sample buffer was added to the beads and then microscope. placed at 100 -C for 5 min. Proteins eluted off of the beads were subjected to SDS-PAGE, transferred to a nitrocellulose membrane and blotted with the appropriate primary and 3. Results horseradish peroxidase (HRP)-conjugated secondary anti- bodies. Blots were then developed with chemiluminescence 3.1. Expression cloning of a novel protein that interacts with reagents (Perkin Elmer, Boston, MA) and exposed to X-ray one of the WW domains of MAGI-1 film. For immunoprecipitations, lysates (100 Al) from trans- In a previous effort to identify proteins in the kidney that fected HEK293 cells were brought up to a total volume of 1 interact with the MAGUK protein MAGI-1, a cDNA ml with HNTG and placed on a rocking platform overnight expression library made from a glomerular-enriched prep- at 4 -C with anti-myc antibodies. The following day, Protein aration of mouse kidneys was screened with the WW A-sepharose beads were added to the samples and rocked domains of MAGI-1 [11]. Of the eleven positive clones for an additional 2 h at 4 -C. The beads were then washed isolated in this screen, four contained inserts that were and processed in the same manner as the GST pulldown identical to each other in their nucleotide sequence and samples above. coded for a novel protein, represented in this study by clone 18.1. Clone 18.1 encoded a protein of 127 amino acids that 2.7. Northern analysis is 91% identical to an amino-terminal region of a previously uncharacterized protein encoded by the human cDNA A mouse multiple tissue polyA+ RNA northern blot was KIAA0989 (Fig. 1). Clone 18.1 therefore appears to contain obtained from a commercial source (OriGene Technologies, a portion of the mouse ortholog of KIAA0989. An initial Inc., Rockville, MD). The insert from clone 18.1 was used as confirmation of the interaction between clone 18.1 and a probe for MASCOT/AMOTL2 and a 472 SacI MAGI-1 was conducted using a GST pulldown assay with fragment from a mouse EST cDNA (IMAGE clone ID# clone 18.1 fused to GST (GST-clone 18.1). When lysates of 514276) was used as the probe for JEAP/AMOTL1. A HEK293 cells overexpressing a myc-tagged construct human h-actin cDNA probe was also used as a loading containing both WW domains of MAGI-1, myc-MAGI-1 control (OriGene). All DNAs were labeled with 32P-dCTP WW12, were incubated with GST-clone 18.1, the GST (NEN, Boston, MA) using the Rediprime II random prime fusion protein was able to efficiently pull down myc-MAGI- labeling system (Amersham Pharmacia Biotech Inc., Piscat- 1 WW12 whereas GST alone did not, thereby supporting the away, NJ). The blot was prehybridized in ULTRAhybi expression cloning results (Fig. 2A). By carrying out the solution (Ambion Inc., Austin, TX) overnight at 42 -C. The same type of analysis with myc-tagged regions of MAGI-1 blot was then hybridized with 1Â106 cpm/ml of probe in containing either WW domain 1 or WW domain 2 alone, ULTRAhybi solution for 8 h at 42 -C. The blot was washed myc-MAGI-1 WW1 and myc-MAGI-1 WW2, respectively, twice with 2Â SSC/0.1% SDS at 65 -C then twice with 0.1Â it was determined that WW domain 1 showed a degree of SSC/0.1% SDS at 42 -C, 15 min each wash. The hybridized binding similar to both WW domains together whereas WW blot was then exposed to X-ray film for 16–48 h at À70 -C. domain 2 did not (Fig. 2A). This indicates that the first WW domain of MAGI-1 is responsible for binding to clone 18.1. 2.8. Immunofluorescence The GST-clone 18.1 fusion protein was also found to interact with a full-length myc-MAGI-1 construct expressed Wild-type MDCK cells or stable MDCK cell lines were in HEK293 cells (Fig. 2B) as well as endogenous MAGI-1 seeded onto Transwell filters (Corning Costar Corp., Cam- from brain lysates (Fig. 2C). bridge, MA) and allowed to reach confluency. Cells were fixed in 4% paraformaldehyde (in PBS) for 15 min at room 3.2. Clone 18.1 and KIAA0989 constitute a member of a temperature then solubilized with 1% SDS (in PBS) for 3 novel family of coiled-coil containing proteins min at room temperature. Blocking was performed in 50% goat serum (diluted in PBS) for 6 h at 30 -C in a humid The full-length KIAA0989 cDNA is 4412 base pairs in chamber. Primary antibodies were diluted in 2% goat serum length and contains an open reading frame of 780 amino K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144 135

Fig. 1. Diagram of the MASCOT/AMOTL2 family of proteins. Each known MASCOT/AMOTL2 family member highlighting the domains and motifs found in each protein is diagrammed. The potential coiled-coil domain found in each member is shown in red. Conventional PPxY binding motifs for Group I WW domains are indicated with a blue rectangle and those of the unconventional LPxY binding motif indicated with a green rectangle. The clone isolated in the expression cloning, Clone 18.1, is shown in yellow below KIAA0989 at a position relative to KIAA0989, to where it is found in the full-length protein. The partial cDNA LCCP is shown in yellow below KIAA0989, again in a position relative to where it would be found in the full-length protein. Numbers flanking each MASCOT/AMOTL2 member denote amino acid numbers at the extreme amino and carboxy-termini.

Fig. 2. GST fusion protein pulldown assays identifying the first WW domain of MAGI-1 as the domain responsible for binding to Clone 18.1 and JEAP/ AMOTL1. Lysates of HEK293 cells expressing various myc-tagged constructs of MAGI-1 and Clone 18.1 or HA-tagged full-length JEAP/AMOTL1 and lysates of mouse brain were incubated with GST alone or the GST fusion proteins GST-clone 18.1, GST-MAGI-1 WW12, GST-MAGI-1 WW1 and GST-MAGI-1 WW2 as indicated. Resulting protein blots were incubated with anti-myc, anti-HA or anti-MAGI-1 primary antibodies followed by the appropriate secondary antibodies and then developed with chemiluminescence reagents. The following lysates were used: HEK293 cells expressing myc-MAGI-1 WW12, myc-MAGI- 1 WW1 and myc-MAGI-1 WW2 (A), myc-MAGI-1 FL (B), HA-JEAP/AMOTL1 (D), myc-clone 18.1 WT and myc-clone 18.1 P105A (E); mouse brain for endogenous proteins (C). Lysate lanes represent 10% of that which was used in the pulldowns. Molecular weight markers are indicated on the left of each panel. 136 K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144

acids encoding a protein with a predicted Mr of ¨86 kDa. A published regarding the three members of this novel protein search of a non-redundant database with the KIAA0989 family [18]. In this report, an amino acid alignment between nucleotide sequence revealed that the 3V half of KIAA0989 these three proteins from different species was presented is identical to a previously uncharacterized human cDNA that illustrates the high degree of conservation found in the referred to as Leman coiled-coil protein (LCCP) (Fig. 1). coiled-coil portion and PDZ-binding motif of this protein Therefore, KIAA0989 appears to be a more complete cDNA family. Because of its interaction with MAGI-1 and of LCCP. The encoded carboxy-terminal portion of association with epithelial tight junctions (see below), KIAA0989 is also similar to, yet distinct, from a recently KIAA0989/LCCP has been renamed MAGI-1-associated described protein named angiomotin, which was reported to coiled-coil tight junction protein (MASCOT). During the interact with the angiogenesis inhibitor angiostatin [16].A course of this study, the gene and protein for KIAA0989/ search of an EST database using the KIAA0989 protein LCCP have been deposited into databases under the name sequence revealed a number of cDNAs from different angiomotin-like 2 (AMOTL2). Again, to avoid potential species that corresponded to a single protein exhibiting a confusion, MASCOT will be referred to as MASCOT/ significant degree of identity to KIAA0989. During the AMOTL2 from here on. initial stages of this present study characterizing the interaction of clone 18.1 with MAGI-1, this homologous 3.3. MASCOT/AMOTL2 binds to MAGI-1 through a variant protein was cloned and identified in a screen for proteins WW domain binding motif associating with the tight junction of epithelial cells and was named junctional-enriched and -associated protein (JEAP) Upon closer examination of its amino acid sequence, [17]. Because of its close similarity to angiomotin, JEAP is clone 18.1 did not contain any of the consensus binding also referred to as angiomotin-like 1, and the symbol motifs for WW domains. Within the last few years however, AMOTL1 has been deposited into databases for the gene three studies have shown that Group I WW domains are able and protein. Therefore, to avoid confusion, JEAP will be to bind a variant motif with the sequence LPxY at its core referred to as JEAP/AMOTL1 from here on. With its [19–21]. In one of these studies, a WW domain that was relatively high degree of similarity with clone 18.1, it was actually the first WW domain of human MAGI-1 was hypothesized that JEAP/AMOTL1 may also possess the shown to recognize a ligand with the consensus sequence of ability to interact with MAGI-1. This was tested by GST LPxYxEVA (where x is any amino acid, V is an aromatic pulldown experiments on HEK293 cell lysates expressing residue and A is a hydrophobic residue) [21]. Both clone an HA-tagged full-length EST cDNA of JEAP/AMOTL1. 18.1 and MASCOT/AMOTL2 (as well as JEAP/AMOTL1) GST fusion proteins consisting of either both WW domains contain the sequence LPTYEEAK, which matches the of MAGI-1 (GST-MAGI-1 WW12) or just the first WW consensus sequence for this variant Group I WW domain domain of MAGI-1 (GST-MAGI-1 WW1) were able to ligand. To determine if this sequence was responsible for the efficiently pull down HA-JEAP/AMOTL1 whereas WW interaction of clone 18.1 with the WW domains of MAGI-1, domain 2 (GST-MAGI-1 WW2) showed only a weak a point mutant was introduced into clone 18.1 that changed interaction (Fig. 2D). Therefore, the first WW domain of the proline in the consensus sequence to alanine. The mutant MAGI-1 appears to mediate binding to both clone 18.1 and clone 18.1, P105A, as well as wild-type clone 18.1 was its close homolog JEAP/AMOTL1. expressed as a myc-tagged protein in HEK293 cells and the A domain search of the KIAA0989 protein using the resulting lysates incubated with GST-MAGI-1 WW12. web-based SMART and COILS2 programs found only a When compared to wild-type clone 18.1, the mutant was potential coiled-coil region from amino acids 308–581. unable to bind to the GST fusion protein indicating that the Likewise, the 882 amino acid JEAP/AMOTL1 protein LPxY motif is responsible for the interaction with MAGI-1 contains a coiled-coil region in its middle third portion (Fig. 2E). and angiomotin is predicted to have a coiled-coil region spanning most of its length as well. In their amino-terminal 3.4. Expression of MASCOT/AMOTL2 and JEAP/AMOTL1 halves, KIAA0989 and JEAP/AMOTL1 contain conven- transcripts in mouse tissues and endogenous tional PPxY motifs that would be predicted to bind to Group MASCOT/AMOTL2 protein in various cell lines I WW domains (Fig. 1) and they exhibit variable lengths of a poly-glutamine stretch of amino acids. Finally, all three The expression patterns of both MASCOT/AMOTL2 and proteins, KIAA0989/LCCP, JEAP/AMOTL1 and angiomo- JEAP/AMOTL1 were determined using a commercially tin, contain amino acid sequences at their extreme carboxy- available mouse Northern blot. A major ¨4.5 kb MAS- terminus that are predicted to form a PDZ binding motif for COT/AMOTL2 transcript was expressed at high levels in Class II PDZ domains: KIAA0989/LCCP, EILI; JEAP/ heart, kidney, liver, lung and skin (Fig. 3A). Moderate to low AMOTL1, EVLI; angiomotin, EYLI. Together, the three levels of the 4.5 kb MASCOT/AMOTL2 transcript were proteins discussed above form a new family of molecules detected in all other tissues present on the Northern blot whose functions are relatively unknown at this time. During indicating that MASCOT/AMOTL2 is a ubiquitously the final preparations of this manuscript, a report was expressed gene. In the brain and testis, minor transcripts of K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144 137

and testis and moderate to low levels in the kidney, liver, lung, muscle and skin (Fig. 3A). The predominant transcript size for JEAP/AMOTL1 was ¨9.5 kb, but in testis, the only transcript detected was ¨3.8 kb. To determine the distribution and approximate size of endogenous MASCOT/AMOTL2 in various epithelial cells lines, the Triton X-100-soluble fractions of MDCK, Caco-2 and SK-BR-3 cells were subjected to Western analysis using a MASCOT/AMOTL2-specific antibody. For comparison, a full-length human MASCOT/AMOTL2 EST cDNA expressed with an HA tag in HEK293 cells was analyzed together with the cell lines. The left panel of Fig. 3B shows that MDCK cells contain a protein doublet of different intensities that migrates slightly faster than the cDNA expressed in HEK293 cells and has a relative molecular weight of 95–100 kDa. This is slightly larger than the predicted size of MASCOT/AMOTL2 based on the KIAA0989 amino acid sequence. The slower migration and therefore larger relative molecular weight of the HA-tagged MASCOT/AMOTL2 cDNA expressed in HEK293 cells may be partly due to the 3Â HA tag on this protein. Both Caco-2 and SK-BR-3 cells contain a triplet of proteins at roughly the same location as the doublet seen for MASCOT/AMOTL2 in Fig. 3. Tissue distribution of MASCOT/AMOTL2 and JEAP/AMOTL1 MDCK cells and exhibit an intensity equal to the less intense transcripts and expression of endogenous MASCOT/AMOTL2 in epithelial band seen in MDCK cells. Other bands could also be seen in cell lines. (A) A commercially available mouse multi-tissue Northern blot MDCK cell lysates at approximately 70 and 40 kDa, with the 32 was probed with the clone 18.1 cDNA that was radiolabeled with P 40 kDa band also found in Caco-2, SK-BR-3 and HEK293 (upper panel). The blot was then stripped and reprobed with a radiolabeled cell lysates (data not shown). The nature of these smaller portion of mouse JEAP/AMOTL1 (middle panel). Finally, the blot was reprobed with a h-actin cDNA as a loading control (lower panel). MASCOT/AMOTL2 proteins is unknown at this time but Molecular weight size markers are indicated to the right of each panel. may be due to degradation of the protein or perhaps products The difference in sizes between the MASCOT/AMOTL2 and JEAP/ of alternative splicing of the MASCOT/AMOTL2 transcript. AMOTL1 transcripts and the difference in their tissue distributions suggest The specificity of the anti-MASCOT/AMOTL2 antibody that the probes that were used are specific for their transcripts. (B) Triton X- was confirmed by its failure to recognize an HA-JEAP/ 100-soluble lysates from MDCK, Caco-2 and SK-BR-3 cells as well as HEK293 cells expressing HA-MASCOT/AMOTL2 or HA-JEAP/AMOTL1 AMOTL1 construct expressed in HEK293 cells (Fig. 3B, left were separated on a 7% SDS-polyacrylamide gel, transferred to nitro- panel). The membrane containing the cell lysates was cellulose and blotted with an anti-MASCOT/AMOTL2 polyclonal antibody stripped and re-blotted with an anti-HA antibody to show (UM441). Confirmation of the presence of both HA tagged proteins was that both HA-tagged proteins were present on the membrane performed by stripping the membrane and re-blotting with an anti-HA (Fig. 3B, right panel). antibody (right panel). Lysates from the cells lines analyzed are indicated above each blot. The appropriate molecular weight marker is indicated to the right of each panel. 3.5. MASCOT/AMOTL2 and JEAP/AMOTL1 interact with MAGI-1 in vivo and are able to self-associate through their coiled-coil domains ¨2.5 kb and ¨2.0 kb could also be detected. Because the largest transcript for MASCOT/AMOTL2 is ¨4.5 kb, it A further test to show that MASCOT/AMOTL2 and indicates that the KIAA0989 cDNA (4412 bp) is most likely a JEAP/AMOTL1 are binding partners for MAGI-1 is to full-length transcript and the coding sequence within this coimmunoprecipitate the endogenous complex from tissue cDNA encodes the entire protein coding region for this gene. or cell line lysates. However, endogenous MAGI-1 is tightly Although the previously published report on JEAP/ associated with the cell membrane and is insoluble from AMOTL1 contains a Northern analysis, the tissue expres- known sources using extraction buffers that preserve sion profile presented is not clear [17]. Therefore, the same protein–protein interactions (e.g. Triton X-100-based lysis commercial RNA blot used for MASCOT/AMOTL2 was buffers). Harsh extraction buffers (e.g. RIPA buffer) that stripped and reprobed with a JEAP/AMOTL1-specific solubilize MAGI-1 tend to disrupt even the best interactions probe. The JEAP/AMOTL1 Northern analysis performed between proteins. Therefore, the binding of MAGI-1 to here indicates that this gene exhibits a more restricted and MASCOT/AMOTL2 and JEAP/AMOTL1 was further different pattern of expression compared to MASCOT/ characterized by testing the interactions in vivo using AMOTL2, with relative high levels found in the brain, heart exogenously expressed constructs in HEK293 cells. EST 138 K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144 cDNAs containing full-length mouse MASCOT/AMOTL2 AMOTL2 (Fig. 4C). When the same assay was carried and human JEAP/AMOTL1 were obtained, sequenced and out with JEAP/AMOTL1, it was discovered that JEAP/ expressed together with myc-MAGI-1 in HEK293 cells and AMOTL1 was also capable of self-associating (Fig. 4D). the resulting cell lysates used in an immunoprecipitation Surprisingly however, MASCOT/AMOTL2 and JEAP/ assay. Immunoprecipitation of the cell lysates with anti-myc AMOTL1 were unable to bind to each other (Fig. 4E). antibodies pulled down not only myc-MAGI-1 but also To identify the region of MASCOT/AMOTL2 that is MASCOT/AMOTL2 and HA-JEAP/AMOTL1 from cell responsible for its self-association, HA-tagged deletion lysates expressing both proteins (Fig. 4A and B) indicating mutants of MASCOT/AMOTL2 were created (Fig. 5A) that both MASCOT/AMOTL2 and JEAP/AMOTL1 interact and expressed individually with full-length Flag-tagged with MAGI-1 in vivo. MASCOT/AMOTL2 in HEK293 cells. Co-immunoprecipi- The coiled-coil domain is a protein–protein interaction tation analysis with the resulting HEK293 lysates shows that domain that allows a protein to self-associate or bind to the region of MASCOT/AMOTL2 encompassing amino other proteins with coiled-coil regions. The presence of a acids 308–578 is necessary for its self-association (Fig. 5B). predicted coiled-coil domain in MASCOT/AMOTL2 and As expected, this region of MASCOT/AMOTL2 coincides JEAP/AMOTL1 suggests that MASCOT/AMOTL2 could with the predicted coiled-coil domain indicating that the possibly associate with itself or with JEAP/AMOTL1 to coiled-coil domain must play a role in this interaction. form homo-oligomers or hetero-oligomers, respectively. To test this, Flag-MASCOT/AMOTL2 and HA-MASCOT/ 3.6. Tight junction localization of MASCOT/AMOTL2 and AMOTL2 constructs were expressed by themselves or JEAP/AMOTL1 in MDCK cells and disruption of tight together in HEK293 cells and the resulting lysates from junction proteins by a MASCOT/AMOTL2 deletion mutant the cells used in immunoprecipitation assays with anti-Flag antibodies. MASCOT/AMOTL2 was found to be capable of The interaction of MASCOT/AMOTL2 with MAGI-1 associating with itself, as seen in lysates from cells would suggest that MASCOT/AMOTL2 could be one of a expressing both epitope tagged versions of MASCOT/ growing number of proteins associating with the epithelial

Fig. 4. In vivo binding analysis of MAGI-1 with MASCOT/AMOTL2 family members and assessment of MASCOT/AMOTL2 and JEAP/AMOTL1 self- associations. (A–E) HEK293 cells were transiently transfected with the following combinations of constructs either alone or together: myc-MAGI-1 and full- length mouse MASCOT/AMOTL2 EST cDNA (A), myc-MAGI-1 and HA-JEAP/AMOTL1 (B), Flag- and HA-MASCOT/AMOTL2 (human) (C), Flag- and HA-JEAP/AMOTL1 (D), or Flag-MASCOT/AMOTL2 and HA-JEAP/AMOTL1 (E). Lysates from transfected cells were incubated with the indicated antibodies and Protein A- or Protein G-sepharose beads. Proteins bound to the beads were eluted off, separated on a 7% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane. The blot was developed with the indicated antibodies and visualized with chemiluminescence reagents. Input control lysate lanes (lower two panels) represent 5% of the amount that was used in the immunoprecipitations. IP: immunoprecipitating antibodies; Blot: blotting antibodies. K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144 139

Fig. 5. Identification of the region necessary for MASCOT/AMOTL2 self-association. (A) Diagram illustrating the deletion constructs of MASCOT/AMOTL2 used in the coimmunoprecipitation assays. (B) HEK293 cells were transiently transfected with a full-length Flag-MASCOT/AMOTL2 construct along with full-length HA-MASCOT/AMOTL2 or HA tagged MASCOT/AMOTL2 deletion constructs. Lysates from transfected cells were incubated with anti-Flag antibodies and Protein G-sepharose beads. Proteins bound to the beads were eluted off, separated on a 12% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane. The blot was developed with an anti-HA antibody and visualized with chemiluminescence reagents. Input control lysate lanes (left panels) represent 5% of the amount that was used in the immunoprecipitations. IP: immunoprecipitating antibodies; Blot: blotting antibodies. tight junction since MAGI-1 has been found to associate localization of JEAP/AMOTL1 confirms what others have with the tight junction of epithelial cells where it presum- found for this protein [17]. Both exogenously expressed ably functions as a scaffolding protein [9,11]. To discern proteins also show a slight punctate pattern that is found at or whether MASCOT/AMOTL2, like JEAP/AMOTL1, is also just below a level corresponding to the tight junction that may a component of tight junctions, MDCK cells were reflect an apical membrane localization or a cytoplasmic analyzed by indirect immunofluorescence for endoge- vesicular localization. nously and exogenously expressed MASCOT/AMOTL2. By using coimmunoprecipitation experiments, the Unfortunately, the available anti-MASCOT/AMOTL2 poly- coiled-coil domain was elucidated as the region of clonal antibody used successfully to detect MASCOT/ MASCOT/AMOTL2 that is necessary for its self-associa- AMOTL2 in the coimmunoprecipitation assays was not tion. To determine the region of MASCOT/AMOTL2 that capable of detecting MASCOT/AMOTL2 in this procedure. is responsible for its subcellular localization, the deletion Therefore, MDCK cells were transfected with HA-MAS- constructs employed in the coimmunoprecipitation assays COT/AMOTL2 and stable cell lines established for use in were used to establish stable MDCK cells lines and the immunofluorescence analysis using anti-HA antibodies. localization of these deletion constructs were evaluated by Confluent monolayers of cells expressing HA-MASCOT/ indirect immunofluorescence. The MASCOT/AMOTL2 AMOTL2 were found to have the construct localized to the deletion constructs 1–307 (Fig. 7A) and 1–578 (Fig. cell borders and this localization overlapped with endoge- 7D) were found mainly localized at areas of cell–cell nously expressed MAGI-1 (Fig. 6A–C). The tight junction junctions covering the entire lateral border of cells that colocalization of MASCOT/AMOTL2 with MAGI-1 pro- partly overlapped with endogenous MAGI-1 (Fig. 7C and vides further evidence for the legitimacy of this interaction. F) at the tight junction. In some instances, a diffuse pattern An HA-JEAP/AMOTL1 construct was also examined in of staining was also observed at the top of cells reflecting stable MDCK cells and was found to colocalize with MAGI-1 at a possible apical localization of these two deletion the tight junction as well (Fig. 6D–F). This tight junction constructs as well. The deletion construct containing amino 140 K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144

Fig. 6. Localization of MASCOT/AMOTL2 and JEAP/AMOTL1 in MDCK cells. MDCK cells were transfected with HA-MASCOT/AMOTL2 (A–C) or HA- JEAP/AMOTL1 (D–F) constructs and stable clones isolated. A single clonal cell line for each construct was used to seed 12-mm Transwell filters and the cells were allowed to reach confluency. Cells were fixed in 4% paraformaldehyde and permeablized with 1% SDS prior to blocking in 50% goat serum. Double labeling was performed with anti-HA rat monoclonal (A, D) (green) and anti-ZO-1 mouse monoclonal antibodies (B, E) (red). Alexa-Fluor secondary antibodies were used to detect the primary antibodies. A merged image of each co-labeling is depicted in panels C and F. Below each X –Y panel is the X –Z plane. Bar = 10 Am and the same magnification was used for each image. acids 579–780 of MASCOT/AMOTL2, the carboxy- previously uncharacterized protein encoded by the human terminal third of the protein, was found randomly cDNA, KIAA0989. Further in vitro and in vivo binding distributed throughout cells with some apparent concen- assays confirmed the expression cloning results and tration at sites where MAGI-1 is found (Fig. 7J–L). In identified the first WW domain of MAGI-1 as the domain contrast to the other deletion constructs, MASCOT/ responsible for the interaction with this novel protein. An AMOTL2 308–780, containing the coiled-coil domain alignment of the amino acid sequence of a full-length mouse with the carboxy-terminus, exhibited colocalization with cDNA (containing the region encoded by clone 18.1) with MAGI-1 at tight junctions within MDCK cells. In addition, KIAA0989 verified that this novel protein, which has been a punctuate pattern of localization for 308–780 is observed named MASCOT/AMOTL2, is the mouse ortholog of at the apical domain in these cells. KIAA0989. Together with the identification of the actin- bundling protein synaptopodin as a binding partner for the second WW domain of MAGI-1, the interaction of cognate 4. Discussion binding partners for both WW domains of MAGI-1 have now been reported. It is plausible, however, that these two Binding partners for the WW domains of MAGI-1 have proteins are not the only interacting protein partners for the not been identified to the extent as those for its PDZ WW domains of MAGI-1. domains. However, in a previously published report, we The only distinguishing features of MASCOT/AMOTL2 have provided a detailed analysis on the interaction of a are a centrally located coiled-coil domain, a putative PPxY WW domain of MAGI-1 with the actin-bundling protein binding motif for Group I WW domains and a short synaptopodin [11]. From this same screening procedure of a polyglutamine stretch in its N-terminal half. In addition, a mouse glomerular cDNA library, an additional binding potential Class II PDZ binding motif is found at its extreme partner for the WW domains of MAGI-1 has been identified C-terminus. Searches of protein databases reveal that and is further characterized in this report. The clone that was MASCOT/AMOTL2 has two close homologs in JEAP/ isolated and characterized in this study, clone 18.1, is a AMOTL1 and angiomotin. Although they share only ¨50% partial cDNA encoding a novel protein that was found to identity to MASCOT/AMOTL2 at the amino acid level, share a high degree of identity to the N-terminal region of a both JEAP/AMOTL1 and angiomotin share the same K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144 141

Fig. 7. Identification of the tight junction targeting region of MASCOT/AMOTL2. MDCK cells were transfected with the HA-MASCOT/AMOTL2 deletion constructs illustrated in Fig. 5 and clonal cell lines for each construct were isolated. A single clonal cell line for each construct was used to seed 12-mm Transwell filters and the cells allowed to reach confluency. HA-MASCOT/AMOTL2 1–307 (A–C), HA-MASCOT/AMOTL2 1–578 (D–F), HA-MASCOT/ AMOTL2 308–780 (G–I), and HA-MASCOT/AMOTL2 579–780 (J–L). Cells were fixed, permeablized and blocked as in Fig. 6. Double labeling was performed with anti-HA rat monoclonal (A, D, G, J) (green) and anti-MAGI-1 rabbit polyclonal antibodies (B, E, H, K) (red). A merged image of the co- labeling is shown for each construct (C, F, I, L). Below each X –Y image is the X –Z plane. Bar = 10 Am and the same magnification was used for each image. 142 K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144 features as described for MASCOT/AMOTL2 (JEAP/ be expressed exclusively at the tight junctions of exocrine AMOTL1 and mouse angiomotin contain an additional gland epithelial cells and not at the cell–cell junctions of PPxY motif in their N-terminal half). These three proteins intestinal epithelial cells or vascular endothelial cells [17]. are thus the founding members of a novel protein family This finding potentially rules out JEAP/AMOTL1 as the whose functions are not fully understood at this time. family member expressed in epithelial cells of kidney The portion of MASCOT/AMOTL2 represented by the glomeruli and tubules. Angiomotin on the other hand clone isolated in this screen and used in this study, clone appears to be only expressed in endothelial cells of blood 18.1, lacks any known conventional binding motif for WW vessels [16]. Based on this indirect evidence, MASCOT/ domains. Recent results of a peptide library screen for WW AMOTL2 may be the member of this protein family domain consensus binding motifs established an unconven- expressed in the glomerular podocyte; however, specific tional binding core sequence of LPxY for the WW domain antibodies that are capable of detecting MASCOT/ of WWP3 (now identified as a fragment of MAGI-1 AMOTL2 using tissue fixation/permeablization techniques containing only the first of the two WW domains of human will be required. Tissue expression profiles for MASCOT/ MAGI-1) [21]. Furthermore, using the SPOT technique, AMOTL2, JEAP/AMOTL1 and angiomotin show that they Chen et al. demonstrate that substitution of the first proline overlap in a subset of tissues. It may be that each member of in the PPxY core binding motif of WBP1 (a binding partner this protein family has a cell-type specific pattern of for YAP) with leucine, serine or valine resulted in reduced expression within the same tissue although their functions but appreciable binding to YAP, whereas all other amino may be very similar within the different cell types. The acid residues were not tolerated at this position [19]. apparent exclusive cell-type expression of MASCOT/ Additional evidence that leucine may substitute for the first AMOTL2/JEAP/angiomotin proteins may account for the proline in the PPxY motif comes from a substitutional inability of MASCOT/AMOTL2 to form hetero-oligomers analysis of a peptide ligand for the WW domain of YAP with JEAP/AMOTL1, as they are not normally expressed [20]. Clone 18.1, as well as MASCOT/AMOTL2, contains together in the same cells. such an LPxY motif, and through analysis of a point mutant Localization studies of full-length MASCOT/AMOTL2 that changed the leucine to alanine, it was established that in MDCK epithelial cells revealed that it is colocalized this motif is responsible for mediating the interaction with along with MAGI-1 at tight junctions. This finding provides MAGI-1 (Fig. 2). This LPxY motif (as well as the amino further confirmation of the legitimacy for its interaction with acids immediately flanking it) is also found in the same MAGI-1, a known component of tight junction-associated general location within JEAP/AMOTL1 and a newly protein complexes. In their efforts to identify novel tight identified N-terminal extension of angiomotin (Patrie, junction-associated proteins, Nishimura, et al. have shown K.M., unpublished observation) as it is in MASCOT/ that JEAP/AMOTL1 can be targeted to epithelial tight AMOTL2. The positional and amino acid conservation junctions, a finding that is confirmed here using exoge- and evolutionary retention of this variant WW binding motif nously expressed JEAP/AMOTL1 in MDCK cells. In in this family of proteins suggest that it has some addition to the lateral membrane tight junction staining, a physiological relevance or functional role in these proteins. fine punctate staining of MASCOT/AMOTL2 and JEAP/ By finding the LPxY motif in expressed transcripts and AMOTL1 is observed in these cells that may reflect a showing that it mediates interactions with WW domains, the cytoplasmic vesicular localization. Alternatively, the punc- MASCOT/AMOTL2 family of proteins are the first gene tate staining of MASCOT/AMOTL2 and JEAP/AMOTL1 products to be identified that utilize this variant binding in MDCK cells could be due to apical membrane local- motif and provides evidence that this motif is a legitimate ization as these punta are found predominantly in the same binding site for Group I WW domains. Conventional PPxY plane as the tight junctions. binding motifs are also present in the MASCOT/AMOTL2 Expressing deletion constructs of MASCOT/AMOTL2 in family of proteins (one in MASCOT/AMOTL2 and two MDCK cells indicates that the minimal region of the protein each in JEAP/AMOTL1 and mouse angiomotin), suggesting required for proper tight junction targeting is amino acids that other proteins with Group I WW domains may interact 308–780. This region encompasses the predicted coiled-coil with them. Although possible, it has not been tested whether domain and ends with the carboxy-terminus. The last one- these other PPxY sites can serve as binding sites to the WW third of MASCOT/AMOTL2 (amino acids 579–780) on the domains of MAGI-1. other hand is mostly found in the cytoplasm, suggesting that Because MASCOT/AMOTL2 was isolated from a the coiled-coil domain is the region directing the protein to glomerular-enriched cDNA library, it would be of interest the tight junction. Although it is indicative that the coiled-coil to observe its precise localization pattern within the kidney domain is necessary for proper tight junction targeting, it may as well as other tissues. However, as stated earlier, the not be sufficient, as the deletion construct containing the available polyclonal antibody against MASCOT/AMOTL2 coiled-coil domain and the amino-terminus of MASCOT/ is not useful in immunofluorescent or other tissue staining AMOTL2 (1–578) is targeted to the entire length of the procedures. Although originally isolated from an endothelial lateral membrane and not just to the tight junction. Therefore, cell line library, JEAP/AMOTL1 was subsequently found to proper subcellular targeting of MASCOT/AMOTL2 is K.M. Patrie / Biochimica et Biophysica Acta 1745 (2005) 131–144 143 dependant on both the coiled-coil domain and the carboxy- [2] J. Reiser, W. Kriz, M. Kretzler, P. Mundel, The glomerular slit terminal one-third, which contains a PDZ binding motif. It is diaphragm is a modified adherens junction, J. Am. Soc. Nephrol. 11 (2000) 1–8. possible then that an interaction of a PDZ domain containing [3] L. Gonzalez-Mariscal, A. Betanzos, A. Avila-Flores, MAGUK protein with the PDZ binding domain of MASCOT/ proteins: structure and role in the tight junction, Semin. Cell Dev. AMOTL2 facilitates its proper localization within a cell. Biol. 4 (2000) 315–324. Because MASCOT/AMOTL2 308–780 does not contain the [4] F. D’Atri, S. Citi, Molecular complexity of vertebrate tight junctions, LPxY motif needed to bind to MAGI-1 and is still properly Mol. Membr. Biol. 19 (2002) 103–112. [5] B.R. Stevenson, J.D. Siliciano, M.S. Mooseker, D.A. Goodenough, targeted within MDCK cells, an interaction between MAGI-1 Identification of ZO-1: a high molecular weight polypeptide asso- and MASCOT/AMOTL2 is apparently not required for ciated with the tight junction (zonula occludens) in a variety of MASCOT/AMOTL2 targeting to the tight junction. Recip- epithelia, J. Cell Biol. 103 (1986) 755–766. rocally, MAGI-1 localization does not appear be dependent [6] B. Gumbiner, T. Lowenkopf, D. Apatira, Identification of a 160 kDa on its interaction with MASCOT/AMOTL2, as the two polypeptide that binds to the tight junction protein ZO-1, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 3460–3464. MASCOT/AMOTL2 deletion constructs containing the [7] J. Haskins, L. Gu, E.S. Wittchen, J. Hibbard, B.R. Stevenson, ZO-3, a LPxY motif, 1–307 and 1–578, do not effect the tight novel member of the MAGUK protein family found at the tight junction localization of endogenous MAGI-1 in MDCK cells. junction, interacts with ZO-1 and cingulin, J. Cell Biol. 141 (1998) Taken together, this data seems to indicate that the interaction 199–208. between MAGI-1 and MASCOT/AMOTL2 most likely takes [8] M.H. Roh, O. Makarova, C.J. Liu, K. Shin, S. Lee, S. Laurinec, M. Goyal, R. Wiggins, B. Margolis, The Maguk protein, Pals1, functions place after the tight junction has formed, and therefore, the as an adapter, linking mammalian homologues of Crumbs and Discs interaction between these two proteins may play a role in the Lost, J. Cell Biol. 157 (2002) 161–172. maintenance or stabilization of the tight junction. [9] N. Ide, Y. Hata, H. Nishioka, K. Hirao, I. Yao, M. Deguchi, A. The precise role that MAGI-1 and other MAGUK Mizoguchi, H. Nishimori, T. Tokino, Y. Nakamura, Y. Takai, Local- proteins play in the function and structure of epithelial ization of membrane-associated guanylate kinase (MAGI)-1/BAI- associated protein (BAP) 1 at tight junctions of epithelial cells, cells, in general, and glomerular podocytes, specifically, is Oncogene 18 (1999) 7810–7815. an area of increasing interest. The scaffolding nature of [10] Y. Wu, D. Dowbenko, S. Spencer, R. Laura, J. Lee, Q. Gu, L.A. MAGI-1 makes it an interesting target for study as it has the Lasky, Interaction of the tumor suppresser PTEN/MMAC with a potential to bind to a number of proteins of which only a PDZ domain of MAGI 3, a novel membrane-associated guanylate few have been identified and fully characterized. To fully kinase, J. Biol. Chem. 275 (2000) 21477–21485. [11] K.M. Patrie, A.J. Drescher, A. Welihinda, P. Mundel, B. Margolis, appreciate the function of a scaffolding protein like MAGI- Interaction of two actin-binding proteins, synaptopodin and alpha- 1, it would be advantageous to identify all the protein actinin-4, with the tight junction protein MAGI-1, J. Biol. Chem. 277 components that may be assembled on it within the context (2002) 30183–30190. of a single cell type or tissue. The identification of the [12] K.M. Patrie, A.J. Drescher, M. Goyal, R.C. Wiggins, B. Margolis, The MASCOT/AMOTL2 proteins as binding partners for membrane-associated guanylate kinase protein MAGI-1 binds megalin and is present in glomerular podocytes, J. Am. Soc. Nephrol. 12 MAGI-1 will clearly add to our understanding of the (2001) 667–677. function not only of MAGI-1 but also of the novel family [13] S.D. Dimitratos, D.F. Woods, D.G. Stathakis, P.J. Bryant, Signaling of proteins of which MASCOT/AMOTL2 is a member of. pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family, Bioessays 21 (1999) 912–921. [14] J.D. Wood, J. Yuan, R.L. Margolis, V. Colomer, K. Duan, J. Acknowledgements Kushi, Z. Kaminsky, J.J. Kleiderlein, A.H. Sharp, C.A. Ross, Atrophin-1, the DRPLA gene product, interacts with two families The author wishes to thank Dr. Ben Margolis (Howard of WW domain-containing proteins, Mol. Cell. Neurosci. 11 (1998) Hughes Medical Institute Investigator, University of Mich- 149–160. [15] J.-P. Borg, J. Ooi, E. Levy, B. Margolis, The phosphotyrosine igan), in whose lab this work was originally started, and Dr. interaction domains of X11 and FE65 bind to distinct sites on the Marius Sudol for helpful insights into WW domain structure YENPTY motif of amyloid precursor protein, Mol. Cell. Biol. 16 and function. (1996) 6229–6241. This manuscript was supported in part by a National [16] B. Troyanovsky, T. Levchenko, G. Mansson, O. Matvijenko, L. Kidney Foundation Postdoctoral Research Fellowship Holmgren, Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation, J. Cell Biol. 152 (2001) (5F32DK09912-02), a Pfizer Laboratories Young Investi- 1247–1254. gator Grant of the National Kidney Foundation, and [17] M. Nishimura, M. Kakizaki, Y. Ono, K. Morimoto, M. Take- Smokler Fund. uchi, Y. Inoue, T. Imai, Y. Takai, JEAP, a novel component of tight junctions in exocrine cells, J. Biol. Chem. 277 (2002) 5583–5587. [18] A. Bratt, W.J. Wilson, B. Troyanovsky, K. Aase, R. Kessler, E.G.V. References Meir, L. Holmgren, Angiomotin belongs to a novel protein family with conserved coiled-coil and PDZ binding domains, Gene 298 [1] D. Kerjaschki, Caught flat-footed: podocyte damage and the (2002) 69–77. molecular basis of focal glomerulosclerosis, J. Clin. Invest. 108 [19] H.I. 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