scholarly journals Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3

2015 ◽  
Vol 26 (11) ◽  
pp. 2030-2043 ◽  
Author(s):  
Varsha Singh ◽  
Jianbo Yang ◽  
Boyoung Cha ◽  
Tiane-e Chen ◽  
Rafiquel Sarker ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Pdz Domain ◽  
Surface Expression ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Pdz Domains ◽  
Sorting Nexin ◽  
C Terminus ◽  
Cargo Proteins

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain–dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.

scholarly journals A novel adhesive complex at the base of intestinal microvilli

2021 ◽  
Author(s):  
Christian Hartmann ◽  
Eva-Maria Thüring ◽  
Birgitta E. Michels ◽  
Denise Pajonczyk ◽  
Sophia Leußink ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Pdz Domain ◽  
Basal Region ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Pdz Domains ◽  
Ternary Complex Formation ◽  
Adhesion Complex

AbstractIntestinal epithelial cells form dense arrays of microvilli at the apical membrane to enhance their functional capacity. Microvilli contain a protocadherin-based intermicrovillar adhesion complex localized at their tips which regulates microvillar length and packaging. Here, we identify a second adhesive complex in microvilli of intestinal epithelial cells. This complex is localized at the basal region of microvilli and consists of the adhesion molecule TMIGD1, the phosphoprotein EBP50 and the F-actin – plasma membrane cross-linking protein ezrin. Ternary complex formation requires unmasking of the EBP50 PDZ domains by ezrin binding and is strongly enhanced upon mutating Ser162 located in PDZ domain 2 of EBP50. Dephosphorylation of EBP50 at S162 is mediated by PP1α, a serine/threonine phosphatase localized at the microvillar base and involved in ezrin phosphocycling. Importantly, the binding of EBP50 to TMIGD1 enhances the dynamic turnover of EBP50 at microvilli in a Ser162 phosphorylation-dependent manner. We identify an adhesive complex at the microvillar base and propose a potential mechanism that regulates microvillar dynamics in enterocytes.

scholarly journals Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins

2021 ◽  
Author(s):  
Michael D Healy ◽  
Joanna Sacharz ◽  
Kerrie E McNally ◽  
Calum McConville ◽  
Ryan J Hall ◽  
...  
Keyword(s):  
Pdz Domain ◽  
Structural Basis ◽  
Binding Motif ◽  
Endosomal Trafficking ◽  
Pdz Domains ◽  
Sorting Nexin ◽  
Affinity Enrichment ◽  
C Terminus ◽  
Loop Sequence ◽  
Cargo Proteins

The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

scholarly journals Leukotriene D4 induces association of active RhoA with phospholipase C-γ1 in intestinal epithelial cells

2002 ◽  
Vol 365 (1) ◽  
pp. 157-163 ◽  
Author(s):  
Charles Kumar THODETI ◽  
Ramin MASSOUMI ◽  
Lene BINDSLEV ◽  
Anita SJÖLANDER
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Phospholipase C ◽  
Fluorescent Protein ◽  
Intestinal Epithelial Cells ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Green Fluorescent ◽  
Constitutively Active

It has been previously suggested that leukotriene-induced Ca2+ signalling is mediated through a Rho-dependent process, but neither direct activation of Rho nor a mechanism underlying such signalling has been reported. Accordingly, we used the Rhotekin binding assay to assess RhoA activation in intestinal epithelial cells and observed that RhoA was activated by leukotriene D4 (LTD4). We also found that, within 15s, activation of RhoA by LTD4 led to an increased association of RhoA with G-protein βγ (Gβγ) and phospholipase C-γ1 (PLC-γ1) in the plasma membrane, as evidenced by the results of co-immunoprecipitation, glutathione S-transferase (GST) pulldown assays, and confocal microscopy. Amounts of RhoA increased in both Gβ and PLC-γ1 immunoprecipitates within 15s of LTD4 treatment. An interaction between RhoA, Gβγ and PLC-γ1 is supported by our finding that a GST fusion protein of constitutively active RhoA (GST-RhoAV14) precipitated Gβγ and PLC-γ1 from cell lysates in an agonist-dependent manner. Such an association is also substantiated by our confocal immunofluorescence results, which revealed that LTD4 induction increased co-localization of constitutively active RhoA and PLC-γ1 to the plasma membrane of cells transfected with enhanced green fluorescent protein L63RhoA. Furthermore, microinjection of neutralizing RhoA antibodies, but not control antibodies, significantly reduced LTD4-induced Ca2+ mobilization. Our results are the first to demonstrate a LTD4-induced activation of RhoA and more importantly its association with PLC-γ1, which are essential for the PLC-γ1-mediated calcium mobilization.

Plasma membrane Ca2+-ATPase associates with CLP36, α-actinin and actin in human platelets

2007 ◽  
Vol 97 (04) ◽  
pp. 587-597 ◽  
Author(s):  
Larry Bozulic ◽  
Mohammad Malik ◽  
David Powell ◽  
Adrian Nanez ◽  
Andrew Link ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pdz Domain ◽  
Gel Filtration ◽  
Human Platelets ◽  
Cytoskeletal Proteins ◽  
Pdz Domains ◽  
Glutathione S Transferase ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Domain Interactions

SummaryThe plasma membrane Ca2+-ATPase (PMCA) plays an essentialrole in maintaining low cytosolic Ca2+ in resting platelets. Earlier studies demonstrated that the 4b isoform of PMCA interacts viaits C-terminal end with the PDZ domains of membrane-associated guanylate kinase proteins. Activation of saponin-permeabilized platelets in the presence of a peptide composed of the lastten residues of the PMCA4b C-terminus leads to a significant decrease of PMCA associated with the cytoskeleton, suggesting that PDZ domain interactions play a role in tethering the pumpto the cytoskeleton. Here we present experiments conducted to evaluate the mechanism of this association. Co-immunoprecipitationassays coupled with liquid chromatography/tandemmass spectrometry analysis and immunoblotting were used to identify proteins that interact with PMCA in the resting platelet. Our results indicate that the only PDZ domain-containing proteinassociated with PMCA is the LIM family protein, CLP36. Glutathione-S-transferase pull-down from a platelet extractusing a fusion protein containing the C-terminal PDZ domainbinding motif of PMCA confirmed binding of CLP36 to PMCA. Gel filtration chromatography of detergent-solubilized plateletsdemonstrated the existence of a 1,000-kDa complex containingPMCA and CLP36, and in addition, α -actinin and actin. Immunoflourescencemicroscopy confirmed the co-localization ofPMCA with CLP36 in resting and activated platelets. Taken togetherthese results suggest that PMCA is localized in non-filamentousactin complexes in resting platelets by means of PDZdomain interactions and then associates with the actin cytoskeletonduring cytoskeletal rearrangement upon platelet activation. Thus, in addition to the reversible serine/threonine andtyrosine phosphorylation events previously described in humanplatelets, PMCA function may be regulated by interactions withanchoring and cytoskeletal proteins.

scholarly journals The NHE3 Juxtamembrane Cytoplasmic Domain Directly Binds Ezrin: Dual Role in NHE3 Trafficking and Mobility in the Brush Border

2006 ◽  
Vol 17 (6) ◽  
pp. 2661-2673 ◽  
Author(s):  
Boyoung Cha ◽  
Ming Tse ◽  
Chris Yun ◽  
Olga Kovbasnjuk ◽  
Sachin Mohan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Half Life ◽  
Double Mutant ◽  
Pdz Domain ◽  
Point Mutations ◽  
Surface Expression ◽  
Triple Mutant ◽  
C Terminus ◽  
A Site

Based on physiological studies, the epithelial brush-border (BB) Na+/H+ antiporter3 (NHE3) seems to associate with the actin cytoskeleton both by binding to and independently of the PDZ domain containing proteins NHERF1 and NHERF2. We now show that NHE3 directly binds ezrin at a site in its C terminus between aa 475-589, which is separate from the PSD95/dlg/zonular occludens-1 (PDZ) interacting domain. This is an area predicted to be α-helical, with a positive aa cluster on one side (K516, R520, and R527). Point mutations of these positively charged aa reduced (NHE3 double mutant [R520F, R527F]) or abolished (NHE3 triple mutant [K516Q, R520F, R 527F]) ezrin binding. Functional consequences of these NHE3 point mutants included the following. 1) A marked decrease in surface amount with a greater decrease in NHE3 activity. 2) Decreased surface expression due to decreased rates of exocytosis and plasma membrane delivery of newly synthesized NHE3, with normal total expression levels and slightly reduced endocytosis rates. 3) A longer plasma membrane half-life of mutant NHE3 with normal total half-life. 4) Decreased BB mobile fraction of NHE3 double mutant. These results show that NHE3 binds ezrin directly as well as indirectly and suggest that the former is related to 1) the exocytic trafficking of and plasma membrane delivery of newly synthesized NHE3, which determines the amount of plasma membrane NHE3 and partially determines NHE3 activity, and 2) BB mobility of NHE3, which may increase its delivery from microvilli to the intervillus clefts, perhaps for NHE3-regulated endocytosis.

scholarly journals Positive cross talk between protein kinase D and β-catenin in intestinal epithelial cells: impact on β-catenin nuclear localization and phosphorylation at Ser552

2016 ◽  
Vol 310 (7) ◽  
pp. C542-C557 ◽  
Author(s):  
Jia Wang ◽  
Liang Han ◽  
James Sinnett-Smith ◽  
Li-Li Han ◽  
Jan V. Stevens ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Transgenic Mice ◽  
Nuclear Localization ◽  
Cross Talk ◽  
Intestinal Epithelial Cells ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Ang Ii ◽  
Potent Inducer

Given the fundamental role of β-catenin signaling in intestinal epithelial cell proliferation and the growth-promoting function of protein kinase D1 (PKD1) in these cells, we hypothesized that PKDs mediate cross talk with β-catenin signaling. The results presented here provide several lines of evidence supporting this hypothesis. We found that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II (ANG II), a potent inducer of PKD activation, promoted endogenous β-catenin nuclear localization in a time-dependent manner. A significant increase was evident within 1 h of ANG II stimulation ( P < 0.01), peaked at 4 h ( P < 0.001), and declined afterwards. GPCR stimulation also induced a marked increase in β-catenin-regulated genes and phosphorylation at Ser552 in intestinal epithelial cells. Exposure to preferential inhibitors of the PKD family (CRT006610 or kb NB 142-70) or knockdown of the isoforms of the PKD family prevented the increase in β-catenin nuclear localization and phosphorylation at Ser552 in response to ANG II. GPCR stimulation also induced the formation of a complex between PKD1 and β-catenin, as shown by coimmunoprecipitation that depended on PKD1 catalytic activation, as it was abrogated by cell treatment with PKD family inhibitors. Using transgenic mice that express elevated PKD1 protein in the intestinal epithelium, we detected a marked increase in the localization of β-catenin in the nucleus of crypt epithelial cells in the ileum of PKD1 transgenic mice, compared with nontransgenic littermates. Collectively, our results identify a novel cross talk between PKD and β-catenin in intestinal epithelial cells, both in vitro and in vivo.

Oocyte proteomics: localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs

2004 ◽  
Vol 16 (2) ◽  
pp. 69 ◽  
Author(s):  
S. A. Coonrod ◽  
M. E. Calvert ◽  
P. P. Reddi ◽  
E. N. Kasper ◽  
L. C. Digilio ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Indirect Immunofluorescence ◽  
Zona Pellucida ◽  
Surface Expression ◽  
Surface Proteins ◽  
2D Electrophoresis ◽  
Dependent Manner ◽  
Two Dimensional ◽  
Phase Partitioning ◽  
Proteomic Approach

In order to gain a deeper understanding of the molecular underpinnings of sperm–egg interaction and early development, we have used two-dimensional (2D) electrophoresis, avidin blotting and tandem mass spectrometry to identify, clone and characterise abundant molecules from the mouse egg proteome. Two-dimensional avidin blots of biotinylated zona-free eggs revealed an abundant approximately 75-kDa surface-labelled heterogeneous protein possessing a staining pattern similar to that of the zona pellucida glycoprotein, mouse ZP3 (mZP3). In light of this observation, we investigated whether mZP3 specifically localises to the plasma membrane of mature eggs. Zona pellucidae of immature mouse oocytes and mature eggs were removed using acid Tyrode’s solution, chymotrypsin or mechanical shearing. Indirect immunofluorescence using the mZP3 monoclonal antibody (mAb) IE-10 demonstrated strong continuous staining over the entire surface of immature oocytes and weak microvillar staining on ovulated eggs, regardless of the method of zona removal. Interestingly, in mature eggs, increased fluorescence intensity was observed following artificial activation and fertilisation, whereas little to no fluorescence was observed in degenerated eggs. The surface localisation of ZP3 on mature eggs was supported by the finding that the IE-10 mAb immunoprecipitated an approximate 75-kDa protein from lysates of biotinylated zona-free eggs. To further investigate the specificity of the localisation of mZP3 to the oolemma, indirect immunofluorescence was performed using the IE-10 mAb on both CV-1 and CHO cells transfected with full-length recombinant mZP3 (re-mZP3). Plasma membrane targeting of the expressed re-mZP3 protein was observed in both cell lines. The membrane association of re-mZP3 was confirmed by the finding that biotinylated re-mZP3 (approximately 75 kDa) is immunoprecipitated from the hydrophobic phase of Triton X-114 extracts of transfected cells following phase partitioning. Immunoprecipitation assays also demonstrated that surface re-mZP3 was released from transfected CV-1 in a time-dependent manner. These results demonstrate that ZP3 is specifically associated with the surface of mature eggs and its subsequent release from the cell surface may represent one mechanism by which ZP3 is secreted. Furthermore, the increase in ZP3 surface expression following fertilisation suggests that ZP3 may have a functional role during sperm–oolemma binding and fusion. These results also validate the usefulness of using the 2D proteomic approach to identify and characterise egg-surface proteins.

scholarly journals Lipid rafts mediate internalization of β1-integrin in migrating intestinal epithelial cells

2008 ◽  
Vol 295 (5) ◽  
pp. G965-G976 ◽  
Author(s):  
Elena V. Vassilieva ◽  
Kirsten Gerner-Smidt ◽  
Andrei I. Ivanov ◽  
Asma Nusrat
Keyword(s):  
Epithelial Cells ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Intestinal Epithelial Cells ◽  
Focal Adhesions ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Caveolin 1 ◽  
Cell Matrix

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of β1-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, β1-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent β1-integrin internalization. However, β1-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized β1-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased β1-integrin endocytosis. Our data suggest that, in migrating IEC, β1-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.

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