NHERF1 and NHERF2 are necessary for multiple but usually separate aspects of basal and acute regulation of NHE3 activity
Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na(+)/H(+) exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na(+) absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca(2+) and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca(2+)-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca(2+)-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent.
[Show abstract] [Hide abstract] ABSTRACT: Sorting Nexin 27 (SNX27) contains a PDZ domain which is phylogenetically related to the PDZ domain of the NHERF proteins. Studies on non-epithelial cells have shown that this protein is located in endosomes where it regulates trafficking of cargo proteins in a PDZ domain dependent manner. However the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) which is primarily through SNX27 PDZ domain. A combination of knock down and reconstitution experiments with wild type or a PDZ domain mutant (GYGF→GAGA) of SNX27 demonstrated that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation based recycling and degradation studies in intestinal epithelial cells showed that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells. © 2015 by The American Society for Cell Biology.0Comments 2Citations
- "SK-CO15 cells were grown on Transwell filters (Corning) until 6–7 d postconfluence. Triple-HA-tagged rabbit NHE3 in replication-deficient adenovirus (Sarker et al., 2011) was transiently infected into SK-CO15 cells for endocytosis-based colocalization analysis. Transiently transfected SK-CO15 cells were incubated with 5 μg/ml HA or GFP antibody with 10% FBS for 1 h at 4°C. "
[Show abstract] [Hide abstract] ABSTRACT: The intestinal brush border (BB) Na(+)/H(+) exchanger, NHE3, is acutely regulated through changes in its endocytosis/exocytosis. Myosin VI, a minus-end directed motor, has been implicated in endocytosis at the inter-microvillar (MV) cleft and vesicle remodeling in the terminal web. We asked if myosin VI also regulates NHE3 movement down MV. Basal NHE3 activity and surface amount, determined by BCECF/fluorometry and biotinylation, respectively, were increased in myosin VI knock-down (KD) Caco-2/Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immuno-EM results showed NHE3 preferentially localized in the basal half of MV in control but in the distal half of myosin VI KD cells. Dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis; but NHE3's distribution along the MV was intermediate in dynasore-treated as compared to either myosin VI KD or untreated 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 MV.0Comments 3Citations
- "Cellular Na + /H + exchange activity in cells expressing HA–NHE3 was determined fluorometrically using the intracellular pH-sensitive dye BCECF-AM (Molecular Probes, Eugene, OR). Filter-grown cells (polycarbonate) were infected with Ad-HA-NHE3 at day 12 after reaching confluence, and were then serum-starved 48 h later for at least 4 h before Na + /H + exchange activity (NHE3) was determined as described previously (Janecki et al., 1998; Li et al., 2004; Murtazina et al., 2007; Sarker et al., 2011). HOE-694 (50 mM) was included to inhibit the contributions of NHE1, NHE2 and NHE8. "
[Show abstract] [Hide abstract] ABSTRACT: The anaphylatoxin C3a binds to the G protein coupled receptor (GPCR, C3aR) and activates divergent signaling pathways to induce degranulation and cytokine production in human mast cells. Adapter proteins such as the Na(+)/H(+) exchange regulatory factor (NHERF1 and NHERF2) have been implicated in regulating functions of certain GPCRs by binding to the class I PDZ (PSD-95/Dlg/Zo1) motifs present on their cytoplasmic tails. Although C3aR possesses a class I PDZ motif, the possibility that it interacts with NHERF proteins to modulate signaling in human mast cells has not been determined. Using reverse transcription PCR and Western blotting, we found that NHERF1 and NHERF2 are expressed in human mast cell lines (HMC-1, LAD2) and CD34(+)-derived primary human mast cells. Surprisingly, however, C3aR did not associate with these adapter proteins. To assess the roles of NHERFs on signaling downstream of C3aR, we used lentiviral shRNA to stably knockdown the expression of these proteins in human mast cells. Silencing the expression of NHERF1 and NHERF2 had no effect on C3aR desensitization, agonist-induced receptor internalization, ERK/Akt phosphorylation or chemotaxis. However, loss of NHERF1 and NHERF2 resulted in significant inhibition of C3a-induced mast cell degranulation, NF-κB activation and chemokine production. This study demonstrates that although C3aR possesses a class I PDZ motif, it does not associate with NHERF1 and NHERF2. Surprisingly, these proteins provide stimulatory signals for C3a-induced degranulation, NF-κB activation and chemokine generation in human mast cells. These findings reveal a new level of complexity for the functional regulation of C3aR by NHERFs in human mast cells.0Comments 4Citations
- "However, the molecular mechanism via which NHERF proteins promote chemokine gene expression is unknown. Na+/H+ exchanger 3 (NHE3) is a member of a group of integral transmembrane proteins that is regulated by NHERF . It has been proposed that in monocytes and macrophages, NHEs are rapidly activated by inflammatory stimulus such as IL-1, TNF-α and LPS which leads to the production of a variety of cytokines –. "