This proposal is to test the hypothesis that NHE3, the epithelial brush border Na/H antiporter, undergoes acute regulation by acting as a scaffold for some of its regulatory proteins and that the assembled regulatory complexes form in highly organized domains of the NHE3 C-terminus. The long-term goal is to understand how intestinal Na absorption is regulated as necessary background for developing drug therapy for diarrheal diseases based on the goal of pharmacologically stimulating NHE3 activity. The hypothesis underlying this grant will be tested by study of NHE3 regulation which centers on its C-terminal domain between aa 475 and 585, which is close to the N-terminal transport domain, and emphasizes the role of direct ezrin binding to NHE3, which was identified during the previous grant period as being necessary for multiple aspects of trafficking of NHE3.
The Specific Aims of this grant are:
Aim I : To understand how the C-terminal domain to which ezrin directly binds is involved in regulation NHE3 activity. We propose to define the role of direct ezrin binding to NHE3 on acute stimulation and inhibition of NHE3. We have identified that the domain of NHE3 which directly binds ezrin also interacts with multiple components of the PI-3K system and we will use mutagenesis to identify the role of each component on NHE3 activity. We will test the hypothesis that NHE3 trafficking is dependent on binding to the motor protein myosin VI.
Aim II : We have identified that the phosphoinositides PIP2 and PIP3 appear to directly bind to NHE3 between aa 475 and 585. We will use mutagenesis and liposomal pull down assays to identify the domains of the NHE3 C-terminus through which interactions with the phosphoinositides occur and determine their role in NHE3 trafficking and basal and regulated activity. Studies will be carried out mostly in the polarized intestinal Na absorptive cell line, Caco-2 cells using mutagenesis, shRNA and dominant-negative constructs transiently infected via viral vectors, supplemented with commercially available knockout mice models of some of the NHE3 associating proteins with consequences determined using two-photon microscopy/SNARF-4F measurement of NHE3 activity in intact intestine.
The long-term goal of this project is to understand how intestinal Na absorption is regulated as necessary background for developing drug therapy for diarrheal diseases based on the goal of pharmacologically stimulating NHE3 activity, which is the epithelial brush border Na/H antiporter and is involved in intestinal Na absorption. We will study regulation of NHE3 by signaling complexes that form on NHE3 itself in highly organized intracellular domains. We suspect that this will provide insight in how normal Na absorption occurs as well as how Na absorption becomes abnormal in diarrheal diseases.
|Zachos, Nicholas C; Kovbasnjuk, Olga; Foulke-Abel, Jennifer et al. (2016) Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology. J Biol Chem 291:3759-66|
|Foulke-Abel, Jennifer; In, Julie; Yin, Jianyi et al. (2016) Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology. Gastroenterology 150:638-649.e8|
|In, Julie G; Foulke-Abel, Jennifer; Estes, Mary K et al. (2016) Human mini-guts: new insights into intestinal physiology and host-pathogen interactions. Nat Rev Gastroenterol Hepatol 13:633-642|
|Janecke, Andreas R; Heinz-Erian, Peter; Yin, Jianyi et al. (2015) Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea. Hum Mol Genet 24:6614-23|
|Chen, Tiane; Kocinsky, Hetal S; Cha, Boyoung et al. (2015) Cyclic GMP kinase II (cGKII) inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at three required sites: identification of a multifunctional phosphorylation site. J Biol Chem 290:1952-65|
|Zhu, Xinjun Cindy; Sarker, Rafiquel; Horton, John R et al. (2015) Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity. Am J Physiol Cell Physiol 308:C758-66|
|Yang, Jianbo; Sarker, Rafiquel; Singh, Varsha et al. (2015) The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity. Biochem J 470:77-90|
|Thiagarajah, Jay R; Donowitz, Mark; Verkman, Alan S (2015) Secretory diarrhoea: mechanisms and emerging therapies. Nat Rev Gastroenterol Hepatol 12:446-57|
|Singh, Varsha; Yang, Jianbo; Cha, Boyoung et al. (2015) Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3. Mol Biol Cell 26:2030-43|
|Cha, Boyoung; Chen, Tiane; Sarker, Rafiquel et al. (2014) Lysophosphatidic acid stimulation of NHE3 exocytosis in polarized epithelial cells occurs with release from NHERF2 via ERK-PLC-PKCÎ´ signaling. Am J Physiol Cell Physiol 307:C55-65|
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