NHERF1 and NHERF2 are necessary for multiple but usually separate aspects of basal and acute regulation of NHE3 activity

Gastroenterology and Hepatology Division, Department of Medicine, Johns Hopkins Univ. School of Medicine, Baltimore, MD 21205-2195, USA.
AJP Cell Physiology (Impact Factor: 3.78). 12/2010; 300(4):C771-82. DOI: 10.1152/ajpcell.00119.2010
Source: PubMed


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.

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    • "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. "
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    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.
    Molecular biology of the cell 04/2015; 26(11). DOI:10.1091/mbc.E14-12-1597 · 4.47 Impact Factor
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    • "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 [47]. 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 [48]–[50]. "
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    PLoS ONE 12/2012; 7(12):e51355. DOI:10.1371/journal.pone.0051355 · 3.23 Impact Factor
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    • "EBVtransformed human lymphocytes (Blymphocytes ) were generated from a patient with Crohn's disease at Johns Hopkins Tissue Culture Facility, under IRB protocols approved by at the Johns Hopkins University. Caco-2/BBE epithelial cells were described previously [35]. "
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