title: PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class I PDZ-binding motifs.
abstract: NHE3 directly binds Na+/H+exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.
authors.affiliation: Departments of Physiology and Medicine, Gastroenterology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Boyoung
authors.initials: B
authors.lastname: Cha
authors.affiliation: Departments of Physiology and Medicine, Gastroenterology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yang
authors.affiliation: Departments of Physiology and Medicine, Gastroenterology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Varsha
authors.initials: V
authors.lastname: Singh
authors.affiliation: Departments of Physiology and Medicine, Gastroenterology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Zachos
authors.affiliation: Departments of Physiology and Medicine, Gastroenterology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Rafiquel I
authors.initials: RI
authors.lastname: Sarker
authors.affiliation: Departments of Physiology and Medicine, Gastroenterology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Tian-E
authors.initials: TE
authors.lastname: Chen
authors.affiliation: Departments of Physiology and Medicine, Gastroenterology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Molee
authors.initials: M
authors.lastname: Chakraborty
authors.affiliation: Departments of Physiology and Medicine, Gastroenterology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Chung-Ming
authors.initials: CM
authors.lastname: Tse
authors.affiliation: Departments of Physiology and Medicine, Gastroenterology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Electronic address: mdonowit@jhmi.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
mesh_terms: D020816:Amino Acid Motifs; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D006152:Cyclic GMP / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D054731:PDZ Domains; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D055550:Protein Stability; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D006152:Cyclic GMP; D002118:Calcium
keywords: NHE3; PDZ domain; calcium; epithelial cell; sodium transport; sodium-proton exchange