Tian-e Chen

Cyclic GMP kinase II (cGKII) inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at three required sites: identification of a multifunctional phosphorylation site.

Background: cGKII acutely inhibits brush-border NHE3, but the mechanism is unknown. Results: cGMP/cGKII phosphorylates NHE3 at three sites. All are necessary for NHE3 inhibition. One of these is also phosphorylated by SGK1 to stimulate NHE3. Conclusion: cGKII inhibits NHE3 by phosphorylating and reducing NHE3 surface amount. Significance: Phosphorylation of the same site in a protein can alter function differently based on phosphorylation of additional sites. The epithelial brush-border Na+/H+ exchanger NHE3 is acutely inhibited by cGKII/cGMP, but how cGKII inhibits NHE3 is unknown. This study tested the hypothesis that cGMP inhibits NHE3 by phosphorylating it and altering its membrane trafficking. Studies were carried out in PS120/NHERF2 and in Caco-2/Bbe cells overexpressing HA-NHE3 and cGKII, and in mouse ileum. NHE3 activity was measured with 2′,7′-bis(carboxyethyl)-S-(and 6)carboxyfluorescein acetoxy methylester/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by iTRAQ/LC-MS/MS with TiO2 enrichment and immunoblotting with specific anti-phospho-NHE3 antibodies. cGMP/cGKII rapidly inhibited NHE3, which was associated with reduced surface NHE3. cGMP/cGKII increased NHE3 phosphorylation at three sites (rabbit Ser554, Ser607, and Ser663, equivalent to mouse Ser552, Ser605, and Ser659), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser663 phosphorylation was not necessary for cAMP inhibition of NHE3. Dexamethasone (4 h) stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to either S663A or S663D. We conclude that 1) cGMP inhibition of NHE3 is associated with phosphorylation of NHE3 at Ser554, Ser607, and Ser663, all of which are necessary for cGMP/cGKII to inhibit NHE3. 2) Dexamethasone stimulates NHE3 by phosphorylation of a single site, Ser663. The requirement for three phosphorylation sites in NHE3 for cGKII inhibition, and for phosphorylation of one of these sites for dexamethasone stimulation of NHE3, is a unique example of regulation by phosphorylation.

Calmodulin Kinase II Constitutively Binds, Phosphorylates, and Inhibits Brush Border Na/H Exchanger 3 (NHE3) by a NHERF2 Protein-dependent Process *

Background: NHE3 is regulated by a signaling complex on its C terminus, only some of the components of which are known. Results: CaMKIIγ binds to the NHE3 C terminus and phosphorylates and inhibits basal NHE3 activity by altering turnover number. Conclusion: CaMKIIγ is part of an NHE3 signaling complex. Significance: Signaling complexes that form on transport proteins take part in regulation of the transporter The epithelial brush border (BB) Na+/H+ exchanger 3 (NHE3) accounts for most renal and intestinal Na+ absorption. Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibits NHE3 activity under basal conditions in intact intestine, acting in the BB, but the mechanism is unclear. We now demonstrate that in both PS120 fibroblasts and polarized Caco-2BBe cells expressing NHE3, CaMKII inhibits basal NHE3 activity, because the CaMKII-specific inhibitors KN-93 and KN-62 stimulate NHE3 activity. This inhibition requires NHERF2. CaMKIIγ associates with NHE3 between aa 586 and 605 in the NHE3 C terminus in a Ca2+-dependent manner, with less association when Ca2+ is increased. CaMKII inhibits NHE3 by an effect on its turnover number, not changing surface expression. Back phosphorylation demonstrated that NHE3 is phosphorylated by CaMKII under basal conditions. This overall phosphorylation of NHE3 is not affected by the presence of NHERF2. Amino acids downstream of NHE3 aa 690 are required for CaMKII to inhibit basal NHE3 activity, and mutations of the three putative CaMKII phosphorylation sites downstream of aa 690 each prevented KN-93 stimulation of NHE3 activity. These studies demonstrate that CaMKIIγ is a novel NHE3-binding protein, and this association is reduced by elevated Ca2+. CaMKII inhibits basal NHE3 activity associated with phosphorylation of NHE3 by effects requiring aa downstream of NHE3 aa 690 and of the CaMKII-binding site on NHE3. CaMKII binding to and phosphorylation of the NHE3 C terminus are parts of the physiologic regulation of NHE3 that occurs in fibroblasts as well as in the BB of an intestinal Na+-absorptive cell.

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.

Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells.

ABSTRACT The intestinal brush border Na+/H+ exchanger NHE3 is tightly regulated through changes in its endocytosis and exocytosis. Myosin VI, a minus-end-directed actin motor, has been implicated in endocytosis at the inter-microvillar cleft and during vesicle remodeling in the terminal web. Here, we asked whether myosin VI also regulates NHE3 movement down the microvillus. The basal NHE3 activity and its surface amount, determined by fluorometry of the ratiometric pH indicator BCECF and biotinylation assays, respectively, were increased in myosin-VI-knockdown (KD) Caco-2/Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immunoelectron microscopy results showed that NHE3 was preferentially localized in the basal half of control microvilli but in the distal half in myosin VI KD cells. Treatment with dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis. However, NHE3 had an intermediate distribution along the microvillus (between that in myosin VI KD and untreated cells) in dynasore-treated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells, and suggest that myosin VI also moves NHE3 down the microvillus.

Mo1747 NHERF2/NHERF3 Hetero-Dimerization Is Required for Carbachol Inhibition of NHE3 Activity That Involves Formation of NHE3 Macro-Complexes

Background: BB NHE3 (Na+/H+ exchanger 3) plus at least 7 other proteins are assembled into 600 kDa -1000 kDa macro-complexes, which serve as the signaling platforms required for the regulation of NHE3. NHERF proteins are a family of multi-PDZ-scaffolding proteins. NHERF1, NHERF2 and NHERF3 are localized to the areas of and below the BB and are involved in NHE3 regulation. Of these, both NHERF2 and NHERF3 but not NHERF1 were required for the carbachol inhibition of NHE3 activity, suggesting possible roles of NHERF dimerization in NHE3 regulation. The purpose of this study was 1) to compare the dimerization strength of all possible NHERF-NHERF interactions and 2) to test the hypothesis that NHERF2-NHERF3 hetero-dimerization contributes to NHE3 macro-complexes and is required for the acute carbachol inhibition of NHE3. Methods: FLAG-tagged NHERF proteins were used for Co-Immunoprecipitation (CO-IP) with anti-FLAG magnetic beads. Bacterially produced recombinant GST-fusion and MBP-fusion NHERF proteins were purified for pulldown (PD) assays to study direct protein-protein interactions in vitro. NHERF2 PDZ (GYGF to GAGA) mutants and NHERF3-4Ala (last 4 aa were Ala) mutant were used to define their interaction domains. Inhibition of NHE3 activity by carbachol was determined with BCECF/ fluorometry in Caco-2 cells expressing HA-NHE3. shRNAwas used to knock-down NHERF3. Adenovirus was used to express the FLAG-tagged rat wild type and mutant NHERF3. Results: NHERF1-NHERF3 and NHERF2-NHERF3 hetero-dimers were detected by both CO-IP and PD assays. NHERF1 and NHERF2 homo-dimers and NHERF1-NHERF2 hetero-dimers were detected minimally by PD and not by CO-IP. There was no significant NHERF3 homodimerization. Hetero-dimerization of NHERF2-NHERF3 was stronger than that of NHERF1NHERF3. The single-PDZ domain defective mutants of NHERF2 had reduced binding to NHERF3, whereas the double-PDZ domain mutant did not bind NHERF3 at all. NHERF34A did not bind NHERF2. This lack of interactions was confirmed by FRET in live OK (opossum kidney proximal tubule) cells. Macro-complexes could be assembled in vitro from NHERF2, NHERF3 and NHE3. NHE3 activity was inhibited by carbachol in wild-type Caco2 cells, but not in NHERF3 KD cells. This could be rescued by overexpression of shRNAresistant rat NHERF3 but not the rat NHERF3-4Amutant. Conclusions: 1) NHERF2-NHERF3 hetero-dimerization is strongest among all possible NHERF dimerizations. 2) NHERF2 and NHERF3 interact via the PDZ domains of NHERF2 and C-terminal PDZ recognition motif of NHERF3. 3) NHERF3 prefers binding to PDZ1 domain of NHERF2 while NHE3 prefers PDZ2 domain. 4) The NHERF2-NHERF3 hetero-dimerization mediated NHE3 macro-complex is necessary for the inhibition of NHE3 activity by carbachol. 5) Hetero-dimerization of NHER2-NHERF3 explains why both NHERF2 and NHERF3 are required for carbachol inhibition of NHE3.

886 Do Mutations of NHE3 At the Site of Poorly Functioning NHE3 Polymorphisms Contribute to Diarrhea of Congenital Na Diarrhea

of NHE3 from the membrane and diarrhea. Aim: We investigated the expression, brush border membrane (BBM) localization, and transport function of the major intestinal sodium absorptive transporter, the Na+/H+ exchanger NHE3, as well as the expression of its regulatory PDZ-adapter proteins NHERF1 and PDZK1, TNF-alpha, actin, and a variety of housekeeping genes, in the small and large intestine of mice after induction of a CD45RBhigh transfer colitis in the acute phase of diarrhea. Results: After colitis induction, TNF-alpha levels were increased both in the inflamed colon and microscopically normal-appearing small intestine. NHE3 mRNA expression was up regulated in the colon and unaltered in the small intestine, NHE3 protein expression and localization in the brush border membrane (BBM) was not altered, but acid-activated NHE3 transport rates in small intestinal villous and colonic surface cells were severely decreased, and fluid absorption In Vivo was decreased in the small intestine. PDZK1 mRNA expression was down regulated, whereas that of NHERF1 was not altered. Glucocorticoid treatment of colitic mice for 4 days decreased colonic mucosal cytokine expression and increased PDZK1 expression as well as fluid absorption. In order to investigate whether the down regulation of PDZK1 and the disturbed NHE3 transport activity is causally related, we studied PDZK1 protein expression and NHE3 transport activity in colonic surface enterocytes in the intestine of PDZK1 +/mice, and found PDZK1 content reduced by 60% and NHE3 transport activity significantly decreased. Conclusion: We suggest that during immune-mediated intestinal inflammation, NHE3 BBM protein abundance is normal, but the regulation of its transport activity is disturbed. PDZK1 down regulation during inflammation may be one factor responsible for inflammation-associated NHE3 dysfunction.

T1727 Myosin Vi Is Necessary for Setting Brush Border NHE3 Activity: An Effect On NHE3 Trafficking/Endocytosis

Serotonin transporter (SERT) plays a critical role in regulating serotonin (5-HT) availability in the gut via reuptake of released serotonin. A decrease in SERT function and expression has been implicated in inflammatory bowel diseases and irritable colon. Hence, SERT is a potential pharmacological target in gastrointestinal disorders, however, very little is known regarding regulation of SERT in the human intestine. In this regard, EGF, a potent growth factor, is known to influence intestinal electrolyte and glucose transport processes and has protective effects on mucosa in models of colitis. Whether EGF regulates SERT function and expression in the human intestine is not known. The present studies were, therefore, designed to examine the regulation of SERT by EGF utilizing Caco2 cells grown on Transwell inserts as an In Vitro model. SERT activity was measured as Na+ and Cldependent 3[H] 5-HT uptake in fully differentiated cell monolayers. SERT mRNA abundance was determined by real time QRT-PCR. SERT promoter activity was assessed by luciferase assay in cells transiently transfected with promoter constructs. Treatment of Caco-2 cells with EGF from basolateral side (5-10ng/ml) for 24h significantly stimulated SERT activity (~50%, P<0.005). However, acute treatment with EGF (1-2 h) did not affect SERT function in Caco-2 cells. Corresponding with increased function at 24h, EGF treatment resulted in up-regulation of SERT mRNA levels (~1.8 fold) compared to control. For studying the regulation of SERT gene by EGF, a 873 bp (-871/+2) SERT promoter fragment was cloned upstream to the luciferase reporter gene and was found to be highly active when transfected in Caco2 cells. EGF treatment (10 ng/ml) for 8-24h resulted in significant stimulation in SERT promoter activity (~50-60%; P<0.05). Inhibition of EGF receptor (EGFR) tyrosine kinase activity by PD168393 (1nM) blocked the stimulatory effects of EGF on SERT promoter. Studies utilizing progressive deletion constructs of the SERT promoter indicated that the putative EGF responsive elements are in the -672/+472 region of the promoter. Conclusion: Our findings demonstrate transcriptional regulation of SERT by EGF via EGFR. These data define novel mechanisms of modulation of SERT function and expression in intestinal epithelium that may be relevant to therapeutic benefits of EGF in diarrheal diseases associated with altered 5-HT availability. [Supported by NIDDK/Dept of Veteran Affairs].