scholarly journals A Regulated Complex of the Scaffolding Proteins PDZK1 and EBP50 with Ezrin Contribute to Microvillar Organization

2010 ◽  
Vol 21 (9) ◽  
pp. 1519-1529 ◽  
Author(s):  
David P. LaLonde ◽  
Damien Garbett ◽  
Anthony Bretscher
Keyword(s):  
Epithelial Cells ◽  
Complex Formation ◽  
Ternary Complex ◽  
Apical Membrane ◽  
Pdz Domain ◽  
Scaffolding Proteins ◽  
Pdz Domains ◽  
Zona Occludens

PDZK1 and ezrin, radixin, moesin binding phosphoprotein 50 kDa (EBP50) are postsynaptic density 95/disc-large/zona occludens (PDZ)-domain–containing scaffolding proteins found in the apical microvilli of polarized epithelial cells. Binary interactions have been shown between the tail of PDZK1 and the PDZ domains of EBP50, as well as between EBP50 and the membrane–cytoskeletal linking protein ezrin. Here, we show that these molecules form a regulated ternary complex in vitro and in vivo. Complex formation is cooperative because ezrin positively influences the PDZK1/EBP50 interaction. Moreover, the interaction of PDZK1 with EBP50 is enhanced by the occupancy of EBP50's adjacent PDZ domain. The complex is further regulated by location, because PDZK1 shuttles from the nucleus in low confluence cells to microvilli in high confluence cells, and this regulates the formation of the PDZK1/EBP50/ezrin complex in vivo. Knockdown of EBP50 decreases the presence of microvilli, a phenotype that can be rescued by EBP50 re-expression or expression of a PDZK1 chimera that is directly targeted to ezrin. Thus, when appropriately located, PDZK1 can provide a function necessary for microvilli formation normally provided by EBP50. By entering into the ternary complex, PDZK1 can both enhance the scaffolding at the apical membrane as well as augment EBP50's role in microvilli formation.

PDZ domain-mediated interaction of rabbit podocalyxin and Na+/H+ exchange regulatory factor-2

2002 ◽  
Vol 282 (6) ◽  
pp. F1129-F1139 ◽  
Author(s):  
Yong Li ◽  
Jian Li ◽  
Samuel W. Straight ◽  
David B. Kershaw
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Pdz Domain ◽  
Type I ◽  
Apical Surface ◽  
Regulatory Factor ◽  
Vitro Analysis ◽  
Zona Occludens

The transmembrane sialoglycoprotein podocalyxin is thought to be essential in the fine interdigitating foot process structure of the podocyte. The intracellular COOH-terminal amino acids Asp-Thr-His-Leu (DTHL) of podocalyxin comprise a putative ligand for a type I PSD95-Dlg-zona occludens-1 (PDZ) domain. A 20-amino acid synthetic peptide containing this motif was used to screen a cDNA library, and clones of rabbit Na+/H+ exchange regulatory factor-2 (NHERF-2) were obtained. In vitro analysis demonstrated that each PDZ domain of NHERF-2 could bind podocalyxin independently. NHERF-2 coprecipitated from glomerular extracts with podocalyxin, and podocalyxin and NHERF-2 colocalized in the glomerular capillary loops, indicating that podocalyxin and NHERF-2 may interact in vivo. Podocalyxin peptide missing the terminal leucine (−DTHL) failed to interact with NHERF-2 in vitro. Podocalyxin localized to the apical membrane of transfected Madin-Darby canine kidney (MDCK) cells. However, mutant podocalyxin (missing a functional DTHL COOH-terminal motif) showed cytoplasmic and apical membrane localization in transfected cells and was also less stable at the apical membrane, as assessed by confocal microscopy and biotinylation studies. Mutant podocalyxin did lower the transepithelial resistance of MDCK cell monolayers, albeit to a lesser extent than full-length podocalyxin. We conclude that podocalyxin can interact with both PDZ domains of NHERF-2 and that this interaction requires the intact COOH terminus of podocalyxin, which is also responsible for the efficient apical localization of podocalyxin in transfected MDCK cells. These results suggest that the interaction of podocalyxin with NHERF-2 may function to efficiently retain podocalyxin at the apical surface of the podocyte and provide a mechanism linking podocalyxin to the actin cytoskeleton.

scholarly journals The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation

2015 ◽  
Vol 26 (20) ◽  
pp. 3615-3627 ◽  
Author(s):  
Cécile Sauvanet ◽  
Damien Garbett ◽  
Anthony Bretscher
Keyword(s):  
Pdz Domain ◽  
Duplication Event ◽  
Scaffolding Protein ◽  
Gene Duplication Event ◽  
Pdz Domains ◽  
Tail Region ◽  
Dynamics And Function ◽  
And Function

We examine the dynamics and function of the apical scaffolding protein E3KARP/NHERF2, which consists of two PDZ domains and a tail containing an ezrin-binding domain. The exchange rate of E3KARP is greatly enhanced during mitosis due to phosphorylation at Ser-303 in its tail region. Whereas E3KARP can substitute for the function of the closely related scaffolding protein EBP50/NHERF1 in the formation of interphase microvilli, E3KARP S303D cannot. Moreover, the S303D mutation enhances the in vivo dynamics of the E3KARP tail alone, whereas in vitro the interaction of E3KARP with active ezrin is unaffected by S303D, implicating another factor regulating dynamics in vivo. A-Raf is found to be required for S303 phosphorylation in mitotic cells. Regulation of the dynamics of EBP50 is known to be dependent on its tail region but modulated by PDZ domain occupancy, which is not the case for E3KARP. Of interest, in both cases, the mechanisms regulating dynamics involve the tails, which are the most diverged region of the paralogues and probably evolved independently after a gene duplication event that occurred early in vertebrate evolution.

scholarly journals Domain-swapped Dimerization of the Second PDZ Domain of ZO2 May Provide a Structural Basis for the Polymerization of Claudins

2007 ◽  
Vol 282 (49) ◽  
pp. 35988-35999 ◽  
Author(s):  
Jiawen Wu ◽  
Yinshan Yang ◽  
Jiahai Zhang ◽  
Peng Ji ◽  
Wenjing Du ◽  
...  
Keyword(s):  
Solution Structure ◽  
Pdz Domain ◽  
Three Dimensional ◽  
Structural Basis ◽  
Pdz Domains ◽  
Zonula Occludens ◽  
Associated Proteins ◽  
High Conservation

Zonula occludens proteins (ZOs), including ZO1/2/3, are tight junction-associated proteins. Each of them contains three PDZ domains. It has been demonstrated that ZO1 can form either homodimers or heterodimers with ZO2 or ZO3 through the second PDZ domain. However, the underlying structural basis is not well understood. In this study, the solution structure of the second PDZ domain of ZO2 (ZO2-PDZ2) was determined using NMR spectroscopy. The results revealed a novel dimerization mode for PDZ domains via three-dimensional domain swapping, which can be generalized to homodimers of ZO1-PDZ2 or ZO3-PDZ2 and heterodimers of ZO1-PDZ2/ZO2-PDZ2 or ZO1-PDZ2/ZO3-PDZ2 due to high conservation between PDZ2 domains in ZO proteins. Furthermore, GST pulldown experiments and immunoprecipitation studies demonstrated that interactions between ZO1-PDZ2 and ZO2-PDZ2 and their self-associations indeed exist both in vitro and in vivo. Chemical cross-linking and dynamic laser light scattering experiments revealed that both ZO1-PDZ2 and ZO2-PDZ2 can form oligomers in solution. This PDZ domain-mediated oligomerization of ZOs may provide a structural basis for the polymerization of claudins, namely the formation of tight junctions.

scholarly journals Plasmolipin regulates basolateral-to-apical transcytosis of ICAM-1 and leukocyte adhesion in polarized hepatic epithelial cells

2022 ◽  
Vol 79 (1) ◽  
Author(s):  
Cristina Cacho-Navas ◽  
Natalia Reglero-Real ◽  
Natalia Colás-Algora ◽  
Susana Barroso ◽  
Gema de Rivas ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Intracellular Transport ◽  
Molecular Mechanisms ◽  
Apical Membrane ◽  
Membrane Domains ◽  
Adhesion Receptor ◽  
Epithelial Barriers

AbstractApical localization of Intercellular Adhesion Receptor (ICAM)-1 regulates the adhesion and guidance of leukocytes across polarized epithelial barriers. Here, we investigate the molecular mechanisms that determine ICAM-1 localization into apical membrane domains of polarized hepatic epithelial cells, and their effect on lymphocyte-hepatic epithelial cell interaction. We had previously shown that segregation of ICAM-1 into apical membrane domains, which form bile canaliculi and bile ducts in hepatic epithelial cells, requires basolateral-to-apical transcytosis. Searching for protein machinery potentially involved in ICAM-1 polarization we found that the SNARE-associated protein plasmolipin (PLLP) is expressed in the subapical compartment of hepatic epithelial cells in vitro and in vivo. BioID analysis of ICAM-1 revealed proximal interaction between this adhesion receptor and PLLP. ICAM-1 colocalized and interacted with PLLP during the transcytosis of the receptor. PLLP gene editing and silencing increased the basolateral localization and reduced the apical confinement of ICAM-1 without affecting apicobasal polarity of hepatic epithelial cells, indicating that ICAM-1 transcytosis is specifically impaired in the absence of PLLP. Importantly, PLLP depletion was sufficient to increase T-cell adhesion to hepatic epithelial cells. Such an increase depended on the epithelial cell polarity and ICAM-1 expression, showing that the epithelial transcytotic machinery regulates the adhesion of lymphocytes to polarized epithelial cells. Our findings strongly suggest that the polarized intracellular transport of adhesion receptors constitutes a new regulatory layer of the epithelial inflammatory response.

scholarly journals Coactivator OBF-1 Makes Selective Contacts with Both the POU-Specific Domain and the POU Homeodomain and Acts as a Molecular Clamp on DNA

1998 ◽  
Vol 18 (12) ◽  
pp. 7397-7409 ◽  
Author(s):  
Patrick Sauter ◽  
Patrick Matthias
Keyword(s):  
Complex Formation ◽  
Ternary Complex ◽  
Mobility Shift ◽  
Specific Domain ◽  
Pou Domain ◽  
Octamer Motif ◽  
Selective Interaction ◽  
Promoter Dna

ABSTRACT The lymphoid-specific transcriptional coactivator OBF-1 (also known as OCA-B or Bob-1) is recruited to octamer site-containing promoters by interacting with Oct-1 or Oct-2 and thereby enhances the transactivation potential of these two Oct factors. For this interaction the POU domain is sufficient. By contrast, OBF-1 does not interact with the POU domains of other POU proteins, such as Oct-4, Oct-6, or Pit-1, even though these factors bind efficiently to the octamer motif. Here we examined the structural requirements for selective interaction between the POU domain and OBF-1. Previous data have shown that formation of a ternary complex among OBF-1, the POU domain, and the DNA is critically dependent on residues within the octamer site. By methylation interference analysis we identified bases that react differently in the presence of OBF-1 compared to the POU domain alone, and using phosphothioate backbone-modified probes in electrophoretic mobility shift assays, we identified several positions influencing ternary complex formation. We then used Oct-1/Pit-1 POU domain chimeras to analyze the selectivity of the interaction between OBF-1 and the POU domain. This analysis indicated that both the POU specific domain (POUS) and the POU homeodomain (POUH) contribute to complex formation. Amino acids that are different in the Pit-1 and Oct-1 POU domains and are considered to be solvent accessible based on the Oct-1 POU domain/DNA cocrystal structure were replaced with alanine residues and analyzed for their influence on complex formation. Thereby, we identified residues L6 and E7 in the POUS and residues K155 and I159 in the POUH to be critical in vitro and in vivo for selective interaction with OBF-1. Furthermore, in an in vivo assay we could show that OBF-1 is able to functionally recruit two artificially separated halves of the POU domain to the promoter DNA, thereby leading to transactivation. These data allow us to propose a model of the interaction between OBF-1 and the POU domain, whereby OBF-1 acts as a molecular clamp holding together the two moieties of the POU domain and the DNA.

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 PDZ interactions regulate rapid turnover of the scaffolding protein EBP50 in microvilli

2012 ◽  
Vol 198 (2) ◽  
pp. 195-203 ◽  
Author(s):  
Damien Garbett ◽  
Anthony Bretscher
Keyword(s):  
Cell Extract ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Pdz Domains ◽  
Kinase C ◽  
Zonula Occludens ◽  
Discs Large ◽  
Specific Proteins

Scaffolding proteins containing PDZ (postsynaptic density 95/discs large/zonula occludens-1) domains are believed to provide relatively stable linkages between components of macromolecular complexes and in some cases to bridge to the actin cytoskeleton. The microvillar scaffolding protein EBP50 (ERM-binding phosphoprotein of 50 kD), consisting of two PDZ domains and an ezrin-binding site, retains specific proteins in microvilli and is necessary for microvillar biogenesis. Our analysis of the dynamics of microvillar proteins in vivo indicated that ezrin and microvillar membrane proteins had dynamics consistent with actin treadmilling and microvillar lifetimes. However, EBP50 was highly dynamic, turning over within seconds. EBP50 turnover was reduced by mutations that inactivate its PDZ domains and was enhanced by protein kinase C phosphorylation. Using a novel in vitro photoactivation fluorescence assay, the EBP50–ezrin interaction was shown to have a slow off-rate that was dramatically enhanced in a PDZ-regulated manner by addition of cell extract to near in vivo levels. Thus, the linking of relatively stable microvillar components can be mediated by surprisingly dynamic EBP50, a finding that may have important ramifications for other scaffolding proteins.

scholarly journals The tails of apical scaffolding proteins EBP50 and E3KARP regulate their localization and dynamics

2013 ◽  
Vol 24 (21) ◽  
pp. 3381-3392 ◽  
Author(s):  
Damien Garbett ◽  
Cécile Sauvanet ◽  
Raghuvir Viswanatha ◽  
Anthony Bretscher
Keyword(s):  
Regulatory Protein ◽  
Full Length ◽  
Scaffolding Proteins ◽  
Pdz Domains ◽  
Binding Partner ◽  
Binding Partners ◽  
The Difference ◽  
High Dynamics ◽  
Zona Occludens

The closely related apical scaffolding proteins ERM-binding phosphoprotein of 50 kDa (EBP50) and NHE3 kinase A regulatory protein (E3KARP) both consist of two postsynaptic density 95/disks large/zona occludens-1 (PDZ) domains and a tail ending in an ezrin-binding domain. Scaffolding proteins are thought to provide stable linkages between components of multiprotein complexes, yet in several types of epithelial cells, EBP50, but not E3KARP, shows rapid exchange from microvilli compared with its binding partners. The difference in dynamics is determined by the proteins’ tail regions. Exchange rates of EBP50 and E3KARP correlated strongly with their abilities to precipitate ezrin in vivo. The EBP50 tail alone is highly dynamic, but in the context of the full-length protein, the dynamics is lost when the PDZ domains are unable to bind ligand. Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53). Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively. Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing

1993 ◽  
Vol 21 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Knut-Jan Andersen ◽  
Erik Ilsø Christensen ◽  
Hogne Vik
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Toxicity Testing ◽  
Renal Tissue ◽  
Epithelial Cell Line ◽  
Multicellular Spheroids ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cells

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.

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