NHERF associations with sodium-hydrogen exchanger isoform 3 (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3

The sodium-hydrogen exchanger regulatory factor (NHERF) is an essential cofactor for cAMP-mediated inhibition of the Na(+)/H(+) exchanger isoform, NHE3, in renal brush border membranes. NHERF is also an ezrin-binding protein. To define the functional importance of ezrin binding for NHERF's function as a NHE3 regulator, we transfected stable PS120 cells expressing NHE3 with plasmids encoding WT and truncated mouse NHERF proteins. Co-immunoprecipitation established that in PS120 cells, NHE3 bound to full-length NHERF(1-355), the C-terminal domain, NHERF(147-355), and NHERF(1-325), which lacks the proposed ezrin-binding domain. The N-terminal domain, NHERF(1-146), failed to bind the antiporter. Ezrin was also co-immunoprecipitated with NHERF(1-355) but not with NHERF(1-325). 8Br-cAMP inhibited NHE3 activity in cells that expressed NHERF(1-355) or NHERF(147-355) but had no effect on the formation of NHE3-NHERF or NHERF-ezrin complexes. Na(+)/H(+) exchange was unaffected by 8Br-cAMP in cells that expressed NHERF(1-146) or NHERF(1-325). NHE3 phosphorylation in vivo was enhanced by 8Br-cAMP only in cells where NHERF bound to both NHE3 and ezrin. The data suggest that NHERF functions as a scaffold to link NHE3 with ezrin and that this multiprotein complex is essential for cAMP-mediated phosphorylation of NHE3 and the inhibition of Na(+)/H(+) exchange.     

Ca(2+)-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis

Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na(+)/H(+) exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca(2+)-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca(2+) ([Ca(2+)](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca(2+)-dependent inhibition requires Ca(2+)-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca(2+)-dependent interaction between alpha-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of alpha-actinin-4. Elevation of [Ca(2+)](i) results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of alpha-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca(2+) inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-alpha-actinin-4 complex.     

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.     

Differential regulation of Na+/H+ exchange isoform activities by enteropathogenic E. coli in human intestinal epithelial cells

Enteropathogenic Escherichia coli (EPEC) is an important human intestinal foodborne pathogen associated with diarrhea, especially in infants and young children. Although EPEC produces characteristic attaching and effacing lesions and loss of microvilli, the pathophysiology of EPEC-associated diarrhea, particularly during early infection, remains elusive. The present studies were designed to examine the direct effects of EPEC infection on intestinal absorption via Na(+)/H(+) exchanger (NHE) isoforms. Caco-2 cells were infected with EPEC strain E2348/69 or nonpathogenic E. coli HB101 for a period of 60 to 120 min. Total NHE activity was significantly increased at 60 min, reaching approximately threefold increase after 90 min of EPEC infection. Similar findings were seen in HT-29 cells and T84 cells indicating that the response was not cell-line specific. Most surprising was the differential regulation of NHE2 and NHE3 by EPEC. Marked activation of NHE2 (300%) occurred, whereas significant inhibition ( approximately 50%) of NHE3 activity was induced. The activity of basolateral isoform NHE1 was also significantly increased in response to EPEC infection. Mutations that disrupted the type III secretion system (TTSS) ablated the effect of EPEC on the activity of both NHE2 and NHE3. These results suggest that EPEC, through a TTSS-dependent mechanism, exerts differential effects on NHE isoform activity in intestinal epithelial cells. Additionally, NHEs do not appear to play any role in EPEC-mediated inflammation, because the NHE inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride did not prevent EPEC-mediated IkappaBalpha degradation.     

cAMP-induced phosphorylation and inhibition of Na(+)/H(+) exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor.

The members of the regulatory factor (RF) gene family, Na(+)/H(+) exchanger (NHE)-RF and NHE3 kinase A regulatory factor (E3KARP) are necessary for cAMP to inhibit the epithelial brush border NHE isoform 3 (NHE3). The mechanism of their action was studied using PS120 fibroblasts stably transfected with rabbit NHE3 and wild type rabbit NHE-RF or wild type human E3KARP. 8-Bromo-cAMP (8-Br-cAMP) had no effect on Na(+)/H(+) exchange activity in cells expressing NHE3 alone. In contrast, in cells co-expressing NHE-RF, 8-Br-cAMP inhibited NHE3 by 39%. In vivo phosphorylation of NHE3 demonstrated that cAMP increased phosphorylation in two chymotrypsin-generated phosphopeptides of NHE3 in cells containing NHE-RF or E3KARP but not in cells lacking these proteins. The requirement for phosphorylation of NHE-RF in this cAMP-induced inhibition of NHE3 was examined by studying a mutant NHE-RF in which serines 287, 289, and 290 were mutated to alanines. Wild type NHE-RF was a phosphorylated protein under basal conditions, but treatment with 8-Br-cAMP did not alter its phosphorylation. Mutant NHE-RF was not phosphorylated either under basal conditions or after 8-Br-cAMP. 8-Br-cAMP inhibited NHE3 similarly in PS120/NHE3 cells containing wild type or mutant NHE-RF. NHE-RF and NHE3 co-precipitated and did so similarly with and without cAMP. Mutant NHE-RF also similarly immunoprecipitated NHE3 in the presence and absence of 8-Br-cAMP. This study shows that members of the regulatory factor gene family, NHE-RF and E3KARP, are necessary for cAMP inhibition of NHE3 by allowing NHE3 to be phosphorylated. This inhibition is not dependent on the phosphorylation of NHE-RF.     

Calmodulin kinase II constitutively binds, phosphorylates, and inhibits brush border Na+/H+ exchanger 3 (NHE3) by a NHERF2 protein-dependent process

The epithelial brush border (BB) Na(+)/H(+) exchanger 3 (NHE3) accounts for most renal and intestinal Na(+) absorption. Ca(2+)/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 Ca(2+)-dependent manner, with less association when Ca(2+) 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 Ca(2+). 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.     

Serum- and glucocorticoid-induced kinase 3 in recycling endosomes mediates acute activation of Na+/H+ exchanger NHE3 by glucocorticoids

Na(+)/H(+) exchanger 3 (NHE3) is the major Na(+) transporter in the intestine. Serum- and glucocorticoid-induced kinase (SGK) 1 interacts with NHE regulatory factor 2 (NHERF2) and mediates activation of NHE3 by dexamethasone (Dex) in cultured epithelial cells. In this study, we compared short-term regulation of NHE3 by Dex in SGK1-null and NHERF2-null mice. In comparison to wild-type mice, loss of SGK1 or NHERF2 significantly attenuated regulation of NHE3 by Dex but did not completely obliterate the effect. We show that transfection of SGK2 or SGK3 in PS120 cells resulted in robust activation of NHE3 by Dex. However, unlike SGK1 or SGK2, SGK3 rapidly activated NHE3 within 15 min of Dex treatment in both PS120 and Caco-2bbe cells. Immunofluorescence analysis showed that SGK3 colocalized with NHE3 in recycling endosomes, whereas SGK1 and SGK2 were diffusely distributed. Mutation of Arg-90 of SGK3 disrupted the endosomal localization of SGK3 and delayed NHE3 activation. Activation of SGK3 and NHE3 by Dex was dependent on phosphoinositide 3-kinase (PI3K) and phosphoinositide-dependent kinase 1 (PDK1), and Dex induced translocation of PDK1 to endosomes. Our study identifies SGK3 as a novel endosomal kinase that acutely regulates NHE3 in a PI3K-dependent mechanism.     

E. coli secreted protein F promotes EPEC invasion of intestinal epithelial cells via an SNX9‐dependent mechanism

Enteropathogenic Escherichia coli (EPEC) infection requires the injection of effector proteins into intestinal epithelial cells (IECs) via type 3 secretion. Type 3‐secreted effectors can interfere with IEC signalling pathways via specific protein–protein interactions. For example, E. coli secreted protein F (EspF) binds sorting nexin 9 (SNX9), an endocytic regulator, resulting in tubulation of the plasma membrane. Our aim was to determine the mechanism of EspF/SNX9‐induced membrane tubulation. Point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains significantly inhibited tubulation, as did inhibition of clathrin coated pit (CCP) assembly. Although characterized as non‐invasive, EPEC are known to invade IECs in vitro and in vivo. Indeed, we found significant invasion of Caco‐2 cells by EPEC, which, like tubulation, was blocked by pharmacological inhibition of CCPs. Interestingly, however, inhibition of dynamin activity did not prevent tubulation or EPEC invasion, which is in contrast to Salmonella invasion, which requires dynamin activity. Our data also indicate that EPEC invasion is dependent on EspF and its interaction with SNX9. Together, these findings suggest that EspF promotes EPEC invasion of IECs by harnessing the membrane‐deforming activity of SNX9.     

Targeting of enteropathogenic Escherichia coli EspF to host mitochondria is essential for bacterial pathogenesis: critical role of the 16th leucine residue in EspF

The attachment of enteropathogenic Escherichia coli (EPEC) to host cells and the induction of attaching and effacing (A/E) lesions are prominent pathogenic features. EPEC infection also leads to host cell death and damage to the intestinal mucosa, which is partly dependent upon EspF, one of the effectors. In this study, we demonstrate that EspF is a mitochondrial import protein with a functional mitochondrial targeting signal (MTS), because EspF activity for importing into the mitochondria was abrogated by MTS deletion mutants. Substitution of the 16th leucine with glutamic acid (EspF(L16E)) completely abolished EspF activity. Infection of HeLa cells with wild type but not the espF mutant (DeltaespF) decreased mitochondrial membrane potential (DeltaPsi(m)), leading to cell death. The DeltaPsi(m) decrease and cell death were restored in cells infected with DeltaespF/pEspF but not DeltaespF/pEspF(L16E), suggesting that the 16th leucine in the MTS is a critical amino acid for EspF function. To demonstrate the impact of EspF in vivo, we exploited Citrobacter rodentium by infecting C3H/HeJ mice with DeltaespF(CR), DeltaespF(CR)/pEspF(CR), or DeltaespF(CR)/pEspF(L16E)(CR). These results indicate that EspF activity contributes to bacterial pathogenesis, as judged by murine lethality and intestinal histopathology, and promotion of bacterial colonization of the intestinal mucosa.     

A novel proline-rich protein, EspF, is secreted from enteropathogenic Escherichia coli via the type III export pathway

Enteropathogenic Escherichia coli (EPEC) cause a characteristic attaching and effacing (A/E) lesion in intestinal epithelial cells that is associated with the expression and export of specific bacterial proteins via a type III secretion pathway. These effector proteins and components of the type III export apparatus are encoded on a pathogenicity island known as the locus of enterocyte effacement (LEE). In this study, we describe a proline-rich protein, EspF, encoded by the LEE that is secreted by the EPEC type III secretion apparatus. Whereas an espF deletion mutant does not synthesize or secrete EspF, surprisingly it retains the ability to induce host signaling events, perform A/E activities, and invade host epithelial cells. Although these results do not indicate an obvious role for EspF in the formation of A/E lesions nor in the invasion of epithelial cells, they do not preclude a role played by EspF in other aspects of EPEC pathogenesis.     

cGMP inhibition of Na+/H+ antiporter 3 (NHE3) requires PDZ domain adapter NHERF2, a broad specificity protein kinase G-anchoring protein

Electroneutral NaCl absorption mediated by Na+/H+ exchanger 3 (NHE3) is important in intestinal and renal functions related to water/Na+ homeostasis. cGMP inhibits NHE3 in intact epithelia. However, unexpectedly it failed to inhibit NHE3 stably transfected in PS120 cells, even upon co-expression of cGMP-dependent protein kinase type II (cGKII). Additional co-expression of NHERF2, the tandem PDZ domain adapter protein involved in cAMP inhibition of NHE3, restored cGMP as well as cAMP inhibition, whereas NHERF1 solely restored cAMP inhibition. In vitro conditions were identified in which NHERF2 but not NHERF1 bound cGKII. The NHERF2 PDZ2 C terminus, which binds NHE3, also bound cGKII. A non-myristoylated mutant of cGKII did not support cGMP inhibition of NHE3. Although cGKI also bound NHERF2 in vitro, it did not evoke inhibition of NHE3 unless a myristoylation site was added. These results show that NHERF2, acting as a novel protein kinase G-anchoring protein, is required for cGMP inhibition of NHE3 and that cGKII must be bound both to the plasma membrane by its myristoyl anchor and to NHERF2 to inhibit NHE3.     

NHERF2 is necessary for basal activity, second messenger inhibition, and LPA stimulation of NHE3 in mouse distal ileum

To test the hypothesis that Na(+)/H(+) exchanger (NHE) regulatory factor 2 (NHERF2) is necessary for multiple aspects of acute regulation of NHE3 in intact mouse small intestine, distal ileal NHE3 activity was determined using two-photon microscopy/SNARF-4F in a NHERF2-null mouse model. The NHERF2-null mouse ileum had shorter villi, deeper crypts, and decreased epithelial cell number. Basal rates of NHE3 activity were reduced in NHERF2-null mice, which was associated with a reduced percentage of NHE3 in the apical domain and an increase in intracellular NHE3 amount but no change in total level of NHE3 protein. cAMP, cGMP, and elevated Ca(2+) due to apical exposure to UTP all inhibited NHE3 activity in wild-type mouse ileum but not in NHERF2-null mice, while inhibition by hyperosmolarity occurred normally. The cAMP-increased phosphorylation of NHE3 at aa 552; levels of PKAIIα and cGMP-dependent protein kinase II (cGKII); and elevation of Ca(2+) were similar in wild-type and NHERF2-null mouse ileum. Luminal lysophosphatidic acid (LPA) stimulated NHE3 in wild-type but not in NHERF2-null ileum. In conclusion, 1) there are subtle structural abnormalities in the small intestine of NHERF2-null mouse which include fewer villus epithelial cells; 2) the decreased basal NHE3 activity and reduced brush border NHE3 amount in NHERF2-null mice show that NHERF2 is necessary for normal basal trafficking or retention of NHE3 in the apical domain; 3) hyperosmolar inhibition of NHE3 occurs similarly in wild-type and NHERF2-null ileum, demonstrating that some inhibitory mechanisms of NHE3 are not NHERF2 dependent; 4) cAMP inhibition of NHE3 is NHERF2 dependent at a step downstream of cAMP/PKAII phosphorylation of NHE3 at aa 552; 5) cGMP- and UTP-induced inhibition of NHE3 are NHERF2 dependent at steps beyond cGKII and the UTP-induced increase of intracellular Ca(2+); and 6) LPA stimulation of NHE3 is also NHERF2 dependent.     

Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2

Enteropathogenic Escherichia coli (EPEC) is a major causative agent of infant diarrhoea in developing countries. The EspF effector protein is injected from EPEC into host cells via a type III secretion system and is involved in the disruption of host intestinal barrier function. In addition, EspF is sorted to mitochondria and has a role in initiating the mitochondrial death pathway. To clarify the manner in which EspF affects host cells, we sought to identify eukaryotic EspF-binding proteins using affinity purification. Abcf2, a protein of unknown function and member of the ABC-transporter family, bound EspF in this assay. An interaction between EspF and Abcf2 was confirmed in a yeast two-hybrid system, by colocalization and by co-immunoprecipitation from EPEC-infected cells. Levels of Abcf2 were decreased in cells infected with EPEC in an EspF dose-dependent manner. Knock-down of Abcf2 expression by RNA interference increased EspF-induced caspase 9 and caspase 3 cleavage. In addition, Abcf2-knocked down cells showed increased caspase 3 cleavage upon treatment with the apoptosis inducing agent staurosporine. These results indicate that EspF induces or facilitates host cell death by targeting and interfering with the putative protective function of Abcf2.     

Constitutively active phosphatidylinositol 3-kinase and AKT are sufficient to stimulate the epithelial Na+/H+ exchanger 3

Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors and has been shown to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the protein kinase Akt. Previous studies have indicated that PI 3-kinase is necessary for basal Na(+)/H(+) exchanger 3 (NHE3) transport and for fibroblast growth factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 activity or whether Akt is involved in this PI 3-kinase effect. We used an adenoviral infection system to test the possibility that activation of PI 3-kinase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesis was investigated in PS120 fibroblasts stably expressing NHE3 after somatic gene transfer using a replication-deficient recombinant adenovirus containing constitutively active catalytic subunit of PI 3-kinase or constitutively active Akt. The adenovirus construct used was engineered with an upstream ecdysone promoter to allow time-regulated expression. Adenoviral infection was nearly 100% at 48 h after infection. Forty-eight hours after infection (24 h after activation of the ecdysone promoter), PI 3-kinase and Akt amount and activity were increased. Increases in both PI 3-kinase activity and Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also increased the percentage of NHE3 on the plasma membrane. The magnitudes of stimulation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, and constitutively active Akt were similar to each other. These results demonstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NHE3 transport activity in PS120/NHE3 cells.     

Overexpression, purification, and characterization of PDZ domain proteins NHERF and E3KARP in Escherichia coli

NHERF (Na(+)/H(+) exchanger regulatory factor) and E3KARP (NHE3 kinase A regulatory protein or NHERF2) are structurally related adapter proteins that contain two tandem PDZ (PSD-95/Dlg-1/ZO-1) domains. Recent studies suggest that these proteins play important roles in the membrane targeting, trafficking, and sorting of several ion channels, transmembrane receptors, and signaling proteins in many tissues. Both NHERF and E3KARP interact with NHE3 through their C-terminally extended second PDZ domain, and the last 30 amino acids of these PDZ domain proteins interact with ezrin. However, the structural bases of the full-length human NHERF and E3KARP, in their physiological roles on the regulation of NHE3 trafficking, are still unknown. To obtain pure and soluble proteins for crystallization and X-ray studies, NHERF and E3KARP were subcloned into pET-30b and pET-30a vectors, and overexpressed in Escherichia coli strains of BL21(DE3). The soluble NHERF and E3KARP proteins were purified using Ni-NTA, anion-exchange column and gel filtration chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high-performance liquid chromatography, matrix-assisted laser desorption-ionization-time-of-flight mass spectroscopy and circular dichroism studies, respectively.     

NHERF2/NHERF3 Protein Heterodimerization and Macrocomplex Formation Are Required for the Inhibition of NHE3 Activity by Carbachol

NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na⁺/H⁺ exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na⁺/H⁺ exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.     

Enteropathogenic E. coli-induced barrier function alteration is not a consequence of host cell apoptosis

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that perturbs intestinal epithelial function. Many of the alterations in the host cells are mediated by effector molecules that are secreted directly into epithelial cells by the EPEC type III secretion system. The secreted effector molecule EspF plays a key role in redistributing tight junction proteins and altering epithelial barrier function. EspF has also been shown to localize to mitochondria and trigger membrane depolarization and eventual host cell death. The relationship, if any, between EspF-induced host cell death and epithelial barrier disruption is presently not known. Site-directed mutation of leucine 16 (L16E) of EspF impairs both mitochondrial localization and consequent host cell death. Although the mutation lies within a region critical for type III secretion, EspF(L16E) is secreted efficiently from EPEC. Despite its inability to promote cell death, EspF(L16E) was not impaired for tight junction alteration or barrier disruption. Consistent with this, the pan-caspase inhibitor Q-VD-OPH, despite reducing EPEC-induced host cell death, had no effect on infection-mediated barrier function alteration. Thus EPEC alters the epithelial barrier independent of its ability to induce host cell death.     

NHERF3 (PDZK1) Contributes to Basal and Calcium Inhibition of NHE3 Activity in Caco-2BBe Cells

Elevated intracellular Ca²⁺ ([Ca²⁺]_i) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca²⁺]_i regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca²⁺ ionophore, 4-bromo-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (0.5 μm): 1) inhibited NHE3 V_max activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca²⁺]_i by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca²⁺]_i inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca²⁺]_i; 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca²⁺.In normal digestive physiology, the brush border (BB)² Na⁺/H⁺ exchanger, NHE3, mediates the majority of the NaCl and NaHCO₃ absorption in the ileum (1). Sequential inhibition and stimulation of NHE3 occur as part of digestive physiology. Short-term regulation of NHE3 activity is achieved through a variety of factors that affect NHE3 turnover number and/or surface expression and often involve a role for the cytoskeleton and accessory proteins, including the multi-PDZ domain containing proteins, NHERF1 and NHERF2 (1, 2). However, many details of this regulation are not understood.The NHERF (Na⁺/H⁺ exchanger regulatory factor) family of multi-PDZ domain containing proteins consists of four evolutionarily related members, all of which are expressed in epithelial cells of the mammalian small intestine (2). NHERF1 and NHERF2 have been previously shown to contribute to acute NHE3 stimulation and inhibition (3–10). Recently, two additional PDZ domain containing proteins, termed NHERF3/PDZK1 and NHERF4/PDZK2/IKEPP, have been demonstrated to possess sequence homology with NHERF1 and NHERF2 (11–14). However, unlike NHERF1 and NHERF2, which are comprised of two tandem PDZ domains flanked by a C-terminal ezrin/radixin/moesin binding domain, NHERF3 and NHERF4 consist of four PDZ domains but no other protein-protein interacting domains (12).NHERF3 was initially identified by a yeast two-hybrid screen from a human kidney cDNA library using the membrane-associated protein MAP17, as bait (12). NHERF3 is expressed in the brush border of epithelial cells of the kidney proximal tubule and the small intestine (12). NHERF3 associates with and, in a few cases, has been shown to regulate the activity of multiple apical membrane ion transporters including the cystic fibrosis transmembrane regulator (CFTR), urate anion exchanger 1 (URAT1), sodium-phosphate cotransporter type IIa (NaP_iIIa), proton-coupled peptide transporter (PEPT2), and organic cation/carnitine cotransporter (OCTN2) (15–19). Furthermore, NHERF3 directly binds the C terminus of NHE3 (20). Recent studies have begun evaluating the effect of NHERF3 on mouse intestinal Na⁺ and Cl⁻ transport. Basal electroneutral sodium absorption was decreased by >40% in the NHERF3 null mouse jejunum (21) and by >80% in the colon (22). In addition, Cinar et al. (22) demonstrated that cAMP and [Ca²⁺]_i inhibition of NHE3 activity was abolished in the NHERF3 null mouse colon. However, the mechanism by which NHERF3 regulates NHE3 activity was not resolved.Several physiological and pathophysiological agonists, acting through [Ca²⁺]_i-induced second messenger systems, are known to inhibit electroneutral NaCl absorption in the small intestine (1, 23). Elevation of [Ca²⁺]_i has previously been demonstrated to inhibit NHE3 activity in a NHERF2-, but not NHERF1-dependent manner (5). NHERF2 regulation of NHE3 involves the formation of multiprotein complexes at the plasma membrane that include NHE3, NHERF2, α-actinin-4, and PKCα, which induce endocytic removal of NHE3 from the plasma membrane by a PKC-dependent mechanism (5, 24). Because multiple PDZ proteins exist in the apical pole of epithelial cells (2), the current study was designed to determine whether NHERF3 could reconstitute Ca²⁺ regulation of NHE3 activity and to define how that occurred.     

EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins β1‐integrin and Na⁺/K⁺ ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na⁺/K⁺ ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na⁺/K⁺ ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical–basal polarity in an EspF‐dependent manner, which would contribute to EPEC‐associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes.