NHE3 activity is dependent on direct phosphoinositide binding at the N terminus of its intracellular cytosolic region

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Abstract

The small intestinal BB Na+/H+ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P3) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His6 proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P2 and PI(3,4,5)P 3 bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na +/H+ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P2 and PI(3,4,5)P3 binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P 2 and PI(3,4,5)P3 binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P2 or PI(3,4,5)P3 binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr501-Arg512 and Arg520-Arg552) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.

Original languageEnglish (US)
Pages (from-to)34566-34578
Number of pages13
JournalJournal of Biological Chemistry
Volume285
Issue number45
DOIs
StatePublished - Nov 5 2010

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Phosphatidylinositols
Amino Acids
Mutation
Lipids
Sodium-Hydrogen Antiporter
Water absorption
Nickel
Serum
Phosphatidylinositol 3-Kinases
Assays
Proteins
Fusion reactions
Sodium
phosphoinositide-3,4,5-triphosphate
Water

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

@article{cbca55a40c8c4f38a99dc2aab40ca32d,
title = "NHE3 activity is dependent on direct phosphoinositide binding at the N terminus of its intracellular cytosolic region",
abstract = "The small intestinal BB Na+/H+ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P3) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His6 proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P2 and PI(3,4,5)P 3 bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na +/H+ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P2 and PI(3,4,5)P3 binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P 2 and PI(3,4,5)P3 binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P2 or PI(3,4,5)P3 binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr501-Arg512 and Arg520-Arg552) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.",
author = "Sachin Mohan and Tse, {Chung Ming} and Gabelli, {Sandra B} and Rafiquel Sarker and Boyoung Cha and Kamau Fahie and Mythili Nadella and Nicholas Zachos and Becky Tu-Sekine and Raben, {Daniel Max} and Amzel, {Mario L} and Mark Donowitz",
year = "2010",
month = "11",
day = "5",
doi = "10.1074/jbc.M110.165712",
language = "English (US)",
volume = "285",
pages = "34566--34578",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "45",

}

TY - JOUR

T1 - NHE3 activity is dependent on direct phosphoinositide binding at the N terminus of its intracellular cytosolic region

AU - Mohan, Sachin

AU - Tse, Chung Ming

AU - Gabelli, Sandra B

AU - Sarker, Rafiquel

AU - Cha, Boyoung

AU - Fahie, Kamau

AU - Nadella, Mythili

AU - Zachos, Nicholas

AU - Tu-Sekine, Becky

AU - Raben, Daniel Max

AU - Amzel, Mario L

AU - Donowitz, Mark

PY - 2010/11/5

Y1 - 2010/11/5

N2 - The small intestinal BB Na+/H+ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P3) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His6 proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P2 and PI(3,4,5)P 3 bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na +/H+ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P2 and PI(3,4,5)P3 binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P 2 and PI(3,4,5)P3 binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P2 or PI(3,4,5)P3 binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr501-Arg512 and Arg520-Arg552) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.

AB - The small intestinal BB Na+/H+ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P3) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His6 proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P2 and PI(3,4,5)P 3 bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na +/H+ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P2 and PI(3,4,5)P3 binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P 2 and PI(3,4,5)P3 binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P2 or PI(3,4,5)P3 binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr501-Arg512 and Arg520-Arg552) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.

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U2 - 10.1074/jbc.M110.165712

DO - 10.1074/jbc.M110.165712

M3 - Article

C2 - 20736165

AN - SCOPUS:78049370026

VL - 285

SP - 34566

EP - 34578

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 45

ER -