title: NHERF2/NHERF3 protein heterodimerization and macrocomplex formation are required for the inhibition of NHE3 activity by carbachol.
abstract: NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na(+)/H(+) exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na(+)/H(+) exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yang
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: Varsha
authors.initials: V
authors.lastname: Singh
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: Tian-E
authors.initials: TE
authors.lastname: Chen
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: Rafiquel
authors.initials: R
authors.lastname: Sarker
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: Lishou
authors.initials: L
authors.lastname: Xiong
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: Boyoung
authors.initials: B
authors.lastname: Cha
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: Shi
authors.initials: S
authors.lastname: Jin
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: Xuhang
authors.initials: X
authors.lastname: Li
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: C Ming
authors.initials: CM
authors.lastname: Tse
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Zachos
authors.affiliation: From the Department of Medicine, Division of Gastroenterology and Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 mdonowit@jhmi.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D018938:Caco-2 Cells; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D006224:Cricetinae; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D054731:PDZ Domains; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C000613385:SLC9A3 protein, human; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D002217:Carbachol
keywords: Dimerization; Epithelial Cell; Fluorescence Resonance Energy Transfer (FRET); Macrocomplex; Microvillus; NHE3 (Na+/H+ Exchanger 3); NHERF Proteins; Protein-Protein Interaction; Scaffold Protein; Trafficking