Lysophosphatidic acid stimulation of NHE3 exocytosis in polarized epithelial cells occurs with release from NHERF2 via ERK-PLC-PKCδ signaling.
The Na(+)/H(+) exchanger 3 (NHE3) is a brush border (BB) Na(+)/H(+) antiporter that accounts for the majority of physiologic small intestinal and renal Na(+) absorption. It is regulated physiologically and in disease via changes in endocytosis/exocytosis. Paradoxically, NHE3 is fixed to the microvillar (MV) actin cytoskeleton and has little basal mobility. This fixation requires NHE3 binding to the multi-PDZ domain scaffold proteins Na(+)/H(+) exchanger regulatory factor (NHERF)1 and NHERF2 and to ezrin. Coordinated release of NHE3 from the MV cytoskeleton has been demonstrated during both stimulation and inhibition of NHE3. However, the signaling molecules involved in coordinating NHE3 trafficking and cytoskeletal association have not been identified. This question was addressed by studying lysophosphatidic acid (LPA) stimulation of NHE3 in polarized renal proximal tubule opossum kidney (OK) cells that occurs via apical LPA5 receptors and is NHERF2 dependent and mediated by epidermal growth factor receptor (EGFR), Rho/Rho-associated kinase (ROCK), and ERK. NHE3 activity was determined by BCECF/fluorometry and NHE3 microvillar mobility by FRAP/confocal microscopy using NHE3-EGFP. Apical LPA (3 μM)/LPA5R stimulated NHE3 activity, increased NHE3 mobility, and decreased the NHE3/NHERF2 association. The LPA stimulation of NHE3 was also PKCδ dependent. PKCδ was necessary for LPA stimulation of NHE3 mobility and NHE3/NHERF2 association. Moreover, the LPA-induced translocation to the membrane of PKCδ was both ERK and phospholipase C dependent with ERK acting upstream of PLC. We conclude that LPA stimulation of NHE3 exocytosis includes a signaling pathway that regulates fixation of NHE3 to the MV cytoskeleton. This involves a signaling module consisting of ERK-PLC-PKCδ, which dynamically and reversibly releases NHE3 from NHERF2 to contribute to the changes in NHE3 MV mobility.
Departments of Physiology and Medicine, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and.
Boyoung
B
Cha
Departments of Physiology and Medicine, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and.
Tiane
T
Chen
Departments of Physiology and Medicine, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and.
Rafiquel
R
Sarker
Departments of Physiology and Medicine, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and.
Jianbo
J
Yang
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland.
Daniel
D
Raben
Departments of Physiology and Medicine, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and.
C Ming
CM
Tse
Departments of Physiology and Medicine, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and.
Olga
O
Kovbasnjuk
Departments of Physiology and Medicine, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and mdonowit@jhmi.edu.
Mark
M
Donowitz
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects* / Q000201:enzymology; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000201:enzymology; D008246:Lysophospholipids / Q000494:pharmacology*; D008871:Microvilli / Q000187:drug effects / Q000201:enzymology; D009893:Opossums; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D034622:RNA Interference; D011817:Rabbits; D049368:Receptors, Lysophosphatidic Acid / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism
D008246:Lysophospholipids; D010726:Phosphodiesterase Inhibitors; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D049368:Receptors, Lysophosphatidic Acid; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D066246:ErbB Receptors; D054460:rho-Associated Kinases; D051745:Protein Kinase C-delta; D048049:Extracellular Signal-Regulated MAP Kinases; D010738:Type C Phospholipases; D020741:rho GTP-Binding Proteins; C032881:lysophosphatidic acid; D002118:Calcium
FRAP; NHE3; PDZ domain; microvillus; trafficking