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Leukotriene D4 induces association of active RhoA with phospholipase C-gamma1 in intestinal epithelial cells.

Thodeti, Charles Kumar; Massoumi, Ramin LU ; Bindslev, Lene and Sjölander, Anita LU (2002) In Biochemical Journal 365(Pt 1). p.157-163
Abstract
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 15 s, activation of RhoA by LTD4 led to an increased association of RhoA with G-protein betagamma (Gbetagamma) and phospholipase C-gamma1 (PLC-gamma1) 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... (More)
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 15 s, activation of RhoA by LTD4 led to an increased association of RhoA with G-protein betagamma (Gbetagamma) and phospholipase C-gamma1 (PLC-gamma1) 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 Gbeta and PLC-gamma1 immunoprecipitates within 15 s of LTD4 treatment. An interaction between RhoA, Gbetagamma and PLC-gamma1 is supported by our finding that a GST fusion protein of constitutively active RhoA (GST-RhoAV14) precipitated Gbetagamma and PLC-gamma1 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-gamma1 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-gamma1, which are essential for the PLC-gamma1-mediated calcium mobilization. (Less)
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keywords
Human, Heterotrimeric GTP-Binding Proteins : metabolism, Epithelial Cells : metabolism, Epithelial Cells : drug effects, Cell Line, Calcium Signaling : physiology, Calcium Signaling : drug effects, Confocal, Phospholipase C : metabolism, Recombinant Fusion Proteins : metabolism, Support, Non-U.S. Gov't, rhoA GTP-Binding Protein : metabolism, Intestines : drug effects, Intestines : metabolism, Isoenzymes : metabolism, Leukotriene D4 : pharmacology, Microscopy
in
Biochemical Journal
volume
365
issue
Pt 1
pages
157 - 163
publisher
Portland Press Limited
external identifiers
  • wos:000176879900018
  • pmid:12071848
  • scopus:0036628543
ISSN
0264-6021
DOI
10.1042/BJ20020248
language
English
LU publication?
yes
id
1a59bf29-ab86-4429-b0ef-ebb7435b8a12 (old id 108890)
alternative location
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12071848&dopt=Abstract
date added to LUP
2007-07-27 11:40:13
date last changed
2017-01-01 06:49:37
@article{1a59bf29-ab86-4429-b0ef-ebb7435b8a12,
  abstract     = {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 15 s, activation of RhoA by LTD4 led to an increased association of RhoA with G-protein betagamma (Gbetagamma) and phospholipase C-gamma1 (PLC-gamma1) 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 Gbeta and PLC-gamma1 immunoprecipitates within 15 s of LTD4 treatment. An interaction between RhoA, Gbetagamma and PLC-gamma1 is supported by our finding that a GST fusion protein of constitutively active RhoA (GST-RhoAV14) precipitated Gbetagamma and PLC-gamma1 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-gamma1 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-gamma1, which are essential for the PLC-gamma1-mediated calcium mobilization.},
  author       = {Thodeti, Charles Kumar and Massoumi, Ramin and Bindslev, Lene and Sjölander, Anita},
  issn         = {0264-6021},
  keyword      = {Human,Heterotrimeric GTP-Binding Proteins : metabolism,Epithelial Cells : metabolism,Epithelial Cells : drug effects,Cell Line,Calcium Signaling : physiology,Calcium Signaling : drug effects,Confocal,Phospholipase C : metabolism,Recombinant Fusion Proteins : metabolism,Support,Non-U.S. Gov't,rhoA GTP-Binding Protein : metabolism,Intestines : drug effects,Intestines : metabolism,Isoenzymes : metabolism,Leukotriene D4 : pharmacology,Microscopy},
  language     = {eng},
  number       = {Pt 1},
  pages        = {157--163},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Leukotriene D4 induces association of active RhoA with phospholipase C-gamma1 in intestinal epithelial cells.},
  url          = {http://dx.doi.org/10.1042/BJ20020248},
  volume       = {365},
  year         = {2002},
}