scholarly journals Targeting of Transmembrane Protein Shrew-1 to Adherens Junctions Is Controlled by Cytoplasmic Sorting Motifs

2006 ◽  
Vol 17 (8) ◽  
pp. 3397-3408 ◽  
Author(s):  
Viktor Jakob ◽  
Alexander Schreiner ◽  
Ritva Tikkanen ◽  
Anna Starzinski-Powitz
Keyword(s):  
Amino Acids ◽  
Epithelial Cells ◽  
Mutational Analysis ◽  
Transmembrane Protein ◽  
Adherens Junctions ◽  
Basolateral Membrane ◽  
Adaptor Protein ◽  
Sorting Signals ◽  
A Subunit ◽  
Polarized Epithelial Cells

We recently identified transmembrane protein shrew-1 and showed that it is able to target to adherens junctions in polarized epithelial cells. This suggested shrew-1 possesses specific basolateral sorting motifs, which we analyzed by mutational analysis. Systematic mutation of amino acids in putative sorting signals in the cytoplasmic domain of shrew-1 revealed three tyrosines and a dileucine motif necessary for basolateral sorting. Substitution of these amino acids leads to apical localization of shrew-1. By applying tannic acid to either the apical or basolateral part of polarized epithelial cells, thereby blocking vesicle fusion with the plasma membrane, we obtained evidence that the apically localized mutants were primarily targeted to the basolateral membrane and were then redistributed to the apical domain. Further support for a postendocytic sorting mechanism of shrew-1 was obtained by demonstrating that μ1B, a subunit of the epithelial cell-specific adaptor complex AP-1B, interacts with shrew-1. In conclusion, our data provide evidence for a scenario where shrew-1 is primarily delivered to the basolateral membrane by a so far unknown mechanism. Once there, adaptor protein complex AP-1B is involved in retaining shrew-1 at the basolateral membrane by postendocytic sorting mechanisms.

scholarly journals Novel Membrane Protein shrew-1 Targets to Cadherin-Mediated Junctions in Polarized Epithelial Cells

2004 ◽  
Vol 15 (1) ◽  
pp. 397-406 ◽  
Author(s):  
Sanita Bharti ◽  
Heike Handrow-Metzmacher ◽  
Silvia Zickenheiner ◽  
Andreas Zeitvogel ◽  
Rudolf Baumann ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Protein ◽  
Transmembrane Protein ◽  
Adherens Junctions ◽  
Direct Interaction ◽  
Potential Candidate ◽  
Putative Open Reading Frame ◽  
Polarized Epithelial Cells ◽  
E Cadherin

While searching for potential candidate molecules relevant for the pathogenesis of endometriosis, we discovered a 2910-base pair cDNA encoding a novel putative 411-amino acid integral membrane protein that we called shrew-1. The putative open-reading frame was confirmed with antibodies against shrew-1 peptides that labeled a protein of ∼48 kDa in extracts of shrew-1 mRNA-positive tissue and also detected ectopically expressed shrew-1. Expression of epitope-tagged shrew-1 in epithelial cells and analysis by surface biotinylation and immunoblots demonstrated that shrew-1 is indeed a transmembrane protein. Shrew-1 is able to target to E-cadherin-mediated adherens junctions and interact with the E-cadherin–catenin complex in polarized MCF7 and Madin-Darby canine kidney cells, but not with the N-cadherin–catenin complex in nonpolarized epithelial cells. Direct interaction of shrew-1 with β-catenin in in vitro pull-down assay suggests that β-catenin might be one of the proteins that targets and/or retains shrew-1 in the adherens junctions. Interestingly, shrew-1 was partially translocated in response to scatter factor (ligand of receptor tyrosine kinase c-met) from the plasma membrane to the cytoplasm where it still colocalized with endogenous E-cadherin. In summary, we introduce shrew-1 as a novel component of adherens junctions, interacting with E-cadherin–β-catenin complexes in polarized epithelial cells.

Visualization of transcytosis in MDCK cells using laser scanning confocal microscopy

1994 ◽  
Vol 52 ◽  
pp. 220-221
Author(s):  
Greg Martin ◽  
Rohit Cariappa ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Laser Scanning ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Polarized Epithelial Cells

The plasma membrane of polarized epithelial cells is composed of two structurally and functionally distinct domains -- the apical and basolateral -- that also differ in molecular composition. The routes followed by integral membrane proteins from their site of synthesis to their site of function varies between different kinds of epithelia. Madin-Darby canine kidney (MDCK) cells deliver plasma membrane proteins directly to the correct domain, while polarized hepatocytes deliver all newly synthesized plasma membrane proteins initially to the basolateral membrane, then retrieve and redirect the apical membrane proteins. We are studying the targeting signals and delivery routes of DPPIV, a single transmembrane protein whose destination is the apical domain in polarized epithelial cells.DPPIV transfected into MDCK cells is delivered to the basolateral plasma membrane after long (13hr) treatment with Brefeldin A (BFA). After BFA’s removal these molecules are retrieved from the basolateral membrane and transcytosed to the apical plasma membrane. This protocol provides a useful model for studies of the indirect route of protein sorting in polarized epithelial cells, since DPPIV at the basolateral surface can be labeled with specific antibody and then subsequently followed in living cells.

scholarly journals Myosin VI is required for sorting of AP-1B–dependent cargo to the basolateral domain in polarized MDCK cells

2007 ◽  
Vol 177 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Josephine Sui-Yan Au ◽  
Claudia Puri ◽  
Gudrun Ihrke ◽  
John Kendrick-Jones ◽  
Folma Buss
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Adaptor Protein ◽  
Site Directed Mutagenesis ◽  
Myosin Vi ◽  
Tail Domain ◽  
Functional Complex ◽  
Polarized Epithelial Cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B–dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.

scholarly journals Sorting of Marburg Virus Surface Protein and Virus Release Take Place at Opposite Surfaces of Infected Polarized Epithelial Cells

2001 ◽  
Vol 75 (3) ◽  
pp. 1274-1283 ◽  
Author(s):  
Christian Sänger ◽  
Elke Mühlberger ◽  
Elena Ryabchikova ◽  
Larissa Kolesnikova ◽  
Hans-Dieter Klenk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Surface Protein ◽  
Hemorrhagic Fever ◽  
Marburg Virus ◽  
Virus Surface ◽  
Mdckii Cells ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

ABSTRACT Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein.

Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium

1999 ◽  
Vol 276 (1) ◽  
pp. C91-C101 ◽  
Author(s):  
Kurt Amsler ◽  
Scott K. Kuwada
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Basolateral Membrane ◽  
Growth Factor Receptor ◽  
Membrane Receptor ◽  
Membrane Receptors ◽  
Receptor Interaction ◽  
Signaling Proteins ◽  
Polarized Epithelial Cells

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-γ (PLC-γ) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-γ protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

scholarly journals Junction Protein Shrew-1 Influences Cell Invasion and Interacts with Invasion-promoting Protein CD147

2007 ◽  
Vol 18 (4) ◽  
pp. 1272-1281 ◽  
Author(s):  
Alexander Schreiner ◽  
Mika Ruonala ◽  
Viktor Jakob ◽  
Jan Suthaus ◽  
Eckhard Boles ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Hela Cells ◽  
Adherens Junctions ◽  
Basolateral Membrane ◽  
Cellular Invasion ◽  
Junction Protein ◽  
Fibrosarcoma Cells ◽  
Interfering Rna ◽  
Darby Canine Kidney

Shrew-1 was previously isolated from an endometriotic cell line in our search for invasion-associated genes. It proved to be a membrane protein that targets to the basolateral membrane of polarized epithelial cells, interacting with E-cadherin–catenin complexes of adherens junctions. Paradoxically, the existence of adherens junctions is incompatible with invasion. To investigate whether shrew-1 can indeed influence cellular invasion, we overexpressed it in HT1080 fibrosarcoma cells. This resulted in enhanced invasiveness, accompanied by an increased matrix metalloprotease (MMP)-9 level in the supernatant, raising the question about the role of shrew-1 in this process. Logic suggested we looked for an interaction with CD147, a known promoter of invasiveness and MMP activity. Indeed, genetics-based, biochemical, and microscopy experiments revealed shrew-1– and CD147-containing complexes in invasive endometriotic cells and an interaction in epithelial cells, which was stronger in MCF7 tumor cells, but weaker in Madin-Darby canine kidney cells. In contrast to the effect mediated by overexpression, small interfering RNA-mediated down-regulation of either shrew-1 or CD147 in HeLa cells decreased invasiveness without affecting the proliferation behavior of HeLa cells, but the knockdown cells displayed decreased motility. Altogether, our results imply that shrew-1 has a function in the regulation of cellular invasion, which may involve its interaction with CD147.

scholarly journals A Novel Cytoplasmic Tail MXXXL Motif Mediates the Internalization of Prostate-specific Membrane Antigen

2003 ◽  
Vol 14 (12) ◽  
pp. 4835-4845 ◽  
Author(s):  
Sigrid A. Rajasekaran ◽  
Gopalakrishnapillai Anilkumar ◽  
Eri Oshima ◽  
James U. Bowie ◽  
He Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Protein ◽  
Interleukin 2 ◽  
Adaptor Protein ◽  
Cytoplasmic Tail ◽  
Prostate Specific Membrane Antigen ◽  
Amino Acid Residues ◽  
Terminal Amino ◽  
Specific Membrane

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the α-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative μ2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.

scholarly journals The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

2015 ◽  
Vol 211 (2) ◽  
pp. 287-294 ◽  
Author(s):  
Emily H. Stoops ◽  
Michael Hull ◽  
Christina Olesen ◽  
Kavita Mistry ◽  
Jennifer L. Harder ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Primary Cilium ◽  
Protein Delivery ◽  
Hot Spot ◽  
Basolateral Membrane ◽  
Protein A ◽  
Surface Proteins ◽  
Apical Surface ◽  
Directed Movement ◽  
Polarized Epithelial Cells

In polarized epithelial cells, newly synthesized cell surface proteins travel in carrier vesicles from the trans Golgi network to the apical or basolateral plasma membrane. Despite extensive research on polarized trafficking, the sites of protein delivery are not fully characterized. Here we use the SNAP tag system to examine the site of delivery of the apical glycoprotein gp135. We show that a cohort of gp135 is delivered to a ring surrounding the base of the primary cilium, followed by microtubule-dependent radial movement away from the cilium. Delivery to the periciliary ring was specific to newly synthesized and not recycling protein. A subset of this newly delivered protein traverses the basolateral membrane en route to the apical membrane. Crumbs3a, another apical protein, was not delivered to the periciliary region, instead making its initial apical appearance in a pattern that resembled its steady-state distribution. Our results demonstrate a surprising “hot spot” for gp135 protein delivery at the base of the primary cilium and suggest the existence of a novel microtubule-based directed movement of a subset of apical surface proteins.

scholarly journals Vesicular Stomatitis Virus Glycoprotein Does Not Determine the Site of Virus Release in Polarized Epithelial Cells

2002 ◽  
Vol 76 (8) ◽  
pp. 4103-4107 ◽  
Author(s):  
Gert Zimmer ◽  
Klaus-Peter Zimmer ◽  
Ina Trotz ◽  
Georg Herrler
Keyword(s):  
Epithelial Cells ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Recombinant Viruses ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

ABSTRACT In polarized epithelial cells, the vesicular stomatitis virus glycoprotein is segregated to the basolateral plasma membrane, where budding of the virus takes place. We have generated recombinant viruses expressing mutant glycoproteins without the basolateral-membrane-targeting signal in the cytoplasmic domain. Though about 50% of the mutant glycoproteins were found at the apical plasma membranes of infected MDCK cells, the virus was still predominantly released at the basolateral membranes, indicating that factors other than the glycoprotein determine the site of virus budding.

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