422회 Brush Border (BB) Mobility of NHE3 is Dynamic as Part of Acute St…

422회 (2008. 11. 24.)

연사: 차 보 영, The Johns Hopkins University

제목: Brush Border (BB) Mobility of NHE3 is Dynamic as Part of Acute Stimulation by Lyophosphatidic Acid  

Abstract 

 The epithelial BB Na+/H+exchanger NHE3 is associated with the actin cytoskeleton by binding directly to ezrin and indirectly to ezrin via attachment to NHERF family proteins. NHE3 mobility in polarized epithelial cell BB is restricted by actin cytoskeleton binding such that only 30% of NHE3 in the renal proximal tubule OK cell line is mobile as judged by fluorescence recovery after photobleaching (FRAP) using confocal microscopy to examine the BB. Given that acute NHE3 regulation is partially by changes in trafficking, the hypothesis tested in this study was that the cytoskeleton association of NHE3 was dynamic and changed as part of acute regulation.  OK cells stably expressing NHERF2 (to mimic the small intestinal and proximal tubule distribution of NHERF family proteins) had NHE3 acutely stimulated by exposure to LPA (100 mM). With LPA, the BB NHE3 mobile fraction dramatically increased in a time dependent manner, with maximum mobility at 30 min after ligand exposure and returned to basal mobile fraction by 60 min. LPA stimulatory effect on NHE3 mobility requires NHERF2. NHERF2 null OK cells or NHERF2 binding deletion mutant NHE3_585 abolished LPA stimulatory effect on NHE3 mobility.  PI3-kinase inhibitor, LY294002 which blocks LPA stimulation effect on NHE3 activity did not block LPA stimulation on NHE3 mobility completely and water soluble surface cross linker, BS3 which blocks surface mobility of NHE3 did not block LPA stimulation on NHE3 mobility completely either.  These results shows that LPA stimulation on the NHE3 mobility contains two parts 1) PI3-kinase dependent exocytic insertion to the BB and 2) increase in surface mobility of NHE3.   To explain this LPA transient stimulation on the surface NHE3 mobility, the interaction of NHE3 with NHERF2 was studied. NHE3 dynamically interacts with NHERF2 by LPA. NHERF2 failed to co-precipitate NHE3 at 30 min but the physical association has been re-established at 60 min. This dynamic interaction between NHERF2 and NHE3 by LPA was confirmed by Fluorescence Resonance energy Transfer (FRET) as well. In conclusion: 1) Restricted BB mobility of NHE3 due to cytoskeleton association is dynamic and is increased as part of acute stimulation which change the amount of BB NHE3. 2) We suggest that acute stimulation of NHE3 by LPA occurs via addition of NHE3 to the BB and that this addition of NHE3 precedes binding of NHE3 to the actin cytoskeleton via NHERF/ezrin. These results identify dynamic steps in cytoskeleton involvement with NHE3 regulation which could be manipulated to stimulate or inhibit Na+ absorption.