title: NHERF2 protein mobility rate is determined by a unique C-terminal domain that is also necessary for its regulation of NHE3 protein in OK cells.
abstract: Na(+)/H(+) exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Varsha
authors.initials: V
authors.lastname: Singh
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Boyoung
authors.initials: B
authors.lastname: Cha
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Tian-E
authors.initials: TE
authors.lastname: Chen
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Rafiquel
authors.initials: R
authors.lastname: Sarker
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Rakhilya
authors.initials: R
authors.lastname: Murtazina
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Shi
authors.initials: S
authors.lastname: Jin
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Zachos
authors.affiliation: Biophotonics Section, National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, Maryland 20892.
authors.forename: George H
authors.initials: GH
authors.lastname: Patterson
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: C Ming
authors.initials: CM
authors.lastname: Tse
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Olga
authors.initials: O
authors.lastname: Kovbasnjuk
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205.
authors.forename: Xuhang
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Medicine, Division of Gastroenterology, Baltimore, Maryland 21205; Department of Physiology, 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: D000818:Animals; D018938:Caco-2 Cells; D000001:Calcimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D061207:Calcium Ionophores / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008246:Lysophospholipids / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011817:Rabbits; D051381:Rats; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D061207:Calcium Ionophores; D003598:Cytoskeletal Proteins; D008246:Lysophospholipids; D046912:Multiprotein Complexes; D010750:Phosphoproteins; C000613385:SLC9A3 protein, human; C000613387:Slc9a3 protein, rat; C498832:Slc9a3r2 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D000001:Calcimycin; C033170:4-bromo-A-23187; C032881:lysophosphatidic acid
keywords: Epithelial Cell; Exocytosis; Mobility; Scaffold Proteins; Sodium Proton Exchange; Trafficking