The epithelial brush border Na/H antiporter NHE3 accounts for the majority of total sodium absorbed in intestine and renal proximal tubule. NHE3 is highly regulated by stimulatory and inhibitory processes post prandially. Identifying and understanding the signal transduction events that can regulate NHE3 is important for gaining in depth knowledge of this important molecule. NHE3 has an N-terminus region with 11 or 12 TM segments (including a cleavable signal peptide) and a C-terminus which is at least primarily cystosolic. The cytosolic region is a site where different kinases, growth factors and other molecules act to regulate NHE3.
During my PhD, I studied the mechanism of NHE3 regulation by phosphoinositides. In chapter 1, I present a bioinformatic analysis to look for novel phosphoinositides binding domains in NHE3. We found predicted lipid binding group in the proximal region of the C-terminus. In chapter 2, we tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. The phosphoinositides PI(3,4,5)P3 and PI(4,5)P2 binde and regulate multiple transporters, including NHE1 (Hilgemann et al. 2001 and Aharonovitz et al. 2000). In my studies, I found that PI(3,4,5)P3 and PI(4,5)P2 can bind directly with the NHE3 cytosolic region (aa475 – 589) where positively charged, bulky and hydrophobic amino acids play a crucial role in this interaction. I developed tools to purify NHE3 C – terminal fragments and to study lipid protein interactions by liposomal pull down assays. Using NHE3 deficient PS120 fibroblast cell lines for Na+/H+ exchange studies, I found that direct binding of phosphoinositides correlated with serum stimulation of NHE3.
In chapter 3, we showed that phospholipases C-gamma directly associates with the NHE3 C-terminus and that this association is dynamic and changes with alteration in intracellular calcium levels. In chapter 4, we studied phosphorylation of NHE3 by Casein Kinase 2. We found that NHE3 was phosphorylated at S719 by this kinase, and this was required for basal NHE3 activity. Mutagenesis of this site leads to a decrease in plasma membrane delivery of NHE3. In chapter 5, we examined the role of ezrin, a cytoskeletal linker protein in regulating NHE3 and found that it is necessary for exocytic trafficking of NHE3.
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 72/03, Dissertation Abstracts International|
|Keywords:||Casein kinase 2, Lipid binding, NHE3 regulation, Phosphoinositides, Protein interactions, Serum stimulation|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be