βpix up-regulates Na+/H+ exchanger 3 through a Shank2-mediated protein-protein interaction

Jung Soo Lee, Young Mee Lee, Joo Young Kim, Hyun Woo Park, Sergio Grinstein, John Orlowski, Eunjoon Kim, Kyung Hwan Kim, Min Goo Lee

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Na+/H+ exchanger 3 (NHE3) plays an important role in neutral Na+ transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of βPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and βPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, βPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of βPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in βPix-mediated NHE3 regulation. Knockdown of endogenous βPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that βPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.

Original languageEnglish
Pages (from-to)8104-8113
Number of pages10
JournalJournal of Biological Chemistry
Volume285
Issue number11
DOIs
Publication statusPublished - 2010 Mar 12

Fingerprint

Sodium-Hydrogen Antiporter
Up-Regulation
rho GTP-Binding Proteins
Proteins
Membranes
Epithelial Cells
Rats
Macromolecular Substances
Guanine Nucleotide Exchange Factors
Monomeric GTP-Binding Proteins
HEK293 Cells
Nucleotides
RNA Interference
Immunoprecipitation
Fluorescent Antibody Technique
Pancreas
Assays
Colon
Cells
RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Lee, Jung Soo ; Lee, Young Mee ; Kim, Joo Young ; Park, Hyun Woo ; Grinstein, Sergio ; Orlowski, John ; Kim, Eunjoon ; Kim, Kyung Hwan ; Lee, Min Goo. / βpix up-regulates Na+/H+ exchanger 3 through a Shank2-mediated protein-protein interaction. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 11. pp. 8104-8113.
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abstract = "Na+/H+ exchanger 3 (NHE3) plays an important role in neutral Na+ transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of βPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and βPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, βPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of βPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in βPix-mediated NHE3 regulation. Knockdown of endogenous βPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that βPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.",
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Lee, JS, Lee, YM, Kim, JY, Park, HW, Grinstein, S, Orlowski, J, Kim, E, Kim, KH & Lee, MG 2010, 'βpix up-regulates Na+/H+ exchanger 3 through a Shank2-mediated protein-protein interaction', Journal of Biological Chemistry, vol. 285, no. 11, pp. 8104-8113. https://doi.org/10.1074/jbc.M109.055079

βpix up-regulates Na+/H+ exchanger 3 through a Shank2-mediated protein-protein interaction. / Lee, Jung Soo; Lee, Young Mee; Kim, Joo Young; Park, Hyun Woo; Grinstein, Sergio; Orlowski, John; Kim, Eunjoon; Kim, Kyung Hwan; Lee, Min Goo.

In: Journal of Biological Chemistry, Vol. 285, No. 11, 12.03.2010, p. 8104-8113.

Research output: Contribution to journalArticle

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T1 - βpix up-regulates Na+/H+ exchanger 3 through a Shank2-mediated protein-protein interaction

AU - Lee, Jung Soo

AU - Lee, Young Mee

AU - Kim, Joo Young

AU - Park, Hyun Woo

AU - Grinstein, Sergio

AU - Orlowski, John

AU - Kim, Eunjoon

AU - Kim, Kyung Hwan

AU - Lee, Min Goo

PY - 2010/3/12

Y1 - 2010/3/12

N2 - Na+/H+ exchanger 3 (NHE3) plays an important role in neutral Na+ transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of βPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and βPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, βPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of βPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in βPix-mediated NHE3 regulation. Knockdown of endogenous βPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that βPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.

AB - Na+/H+ exchanger 3 (NHE3) plays an important role in neutral Na+ transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of βPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and βPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, βPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of βPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in βPix-mediated NHE3 regulation. Knockdown of endogenous βPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that βPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.

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