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)P(2)) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P(3)) 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. His(6) 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)P(2) 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)P(2) and PI(3,4,5)P(3) 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)P(3) binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P(2) or PI(3,4,5)P(3) binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr(501)-Arg(512) and Arg(520)-Arg(552)) 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.
Highlights
34566 JOURNAL OF BIOLOGICAL CHEMISTRY expanding list includes voltage-gated Kϩ channels, inwardly rectifying Kϩ channels, and members of the TRP channel family, ENaC, NHE1, and NBCe1 [1,2,3,4,5,6,7,8]
Expanding list includes voltage-gated Kϩ channels, inwardly rectifying Kϩ channels, and members of the TRP channel family, ENaC, NHE1, and NBCe1 [1,2,3,4,5,6,7,8]. This regulation has been explained on the basis of two contrasting mechanisms. (i) There is direct phosphoinositide interaction with specific amino acids in the transport protein, explained either on the basis of charge (8 –13) or presence of canonical lipid binding domains such as pleckstrin homology domains [1, 8, 14]. Common to these direct interaction studies has been the demonstration that mutagenesis of specific amino acids leads to changes in molecular interactions with phosphoinositides that lead to subsequent changes in physiologic channel or transporter activity. (ii) An indirect mechanism involves an additional intermediate, either protein or lipids, that binds via a phosphoinositide-dependent mechanism [5, 11]
We demonstrate that the N-terminal part of the NHE3 C terminus is involved in regulation of basal and serumstimulated NHE3 activity by interaction with the phosphoinositides PI[3,4,5]P3 and/or PI[4,5]P2
Summary
34566 JOURNAL OF BIOLOGICAL CHEMISTRY expanding list includes voltage-gated Kϩ channels, inwardly rectifying Kϩ channels, and members of the TRP channel family, ENaC, NHE1, and NBCe1 [1,2,3,4,5,6,7,8] This regulation has been explained on the basis of two contrasting mechanisms. (i) There is direct phosphoinositide interaction with specific amino acids in the transport protein, explained either on the basis of charge (8 –13) or presence of canonical lipid binding domains such as pleckstrin homology domains [1, 8, 14]. The aim of this study was to understand the mechanism of NHE3 regulation by phosphoinositides by the following: (i) investigating whether NHE3 can directly bind phosphoinositides; (ii) identifying regions and amino acids that are necessary for this interaction, and (iii) studying the physiologic relevance of this interaction
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