Na+/H+ Exchanger Regulatory Factor 2 Research Articles

Nephrin–Ephrin-B1–Na+/H+ Exchanger Regulatory Factor 2–Ezrin–Actin Axis Is Critical in Podocyte Injury

Ephrin-B1 is one of the critical components of the slit diaphragm of kidney glomerular podocyte. However, the precise function of ephrin-B1 is unclear. To clarify the function of ephrin-B1, ephrin-B1-associated molecules were studied. RNA-sequencing analysis suggested that Na+/H+ exchanger regulatory factor 2 (NHERF2), a scaffolding protein, is associated with ephrin-B1. NHERF2 was expressed at the apical area and the slit diaphragm, and interacted with the nephrin-ephrin-B1 complex at the slit diaphragm. The nephrin-ephrin-B1-NHERF2 complex interacted with ezrin bound to F-actin. NHERF2 bound ephrin-B1 via its first postsynaptic density protein-95/disks large/zonula occludens-1 domain, and podocalyxin via its second postsynaptic density protein-95/disks large/zonula occludens-1 domain. Both invitro analyses with human embryonic kidney 293 cells and invivo study with rat nephrotic model showed that stimulaiton of the slit diaphragm, phosphorylation of nephrin and ephrin-B1, and dephosphorylation of NHERF2 and ezrin, disrupted the linkages of ephrin-B1-NHERF2 and NHERF2-ezrin. It is conceivable that the linkage of nephrin-ephrin-B1-NHERF2-ezrin-actin is a novel critical axis in thepodocytes. Ephrin-B1 phosphorylation also disrupted the linkage of an apical transmembrane protein, podocalyxin, with NHERF2-ezrin-actin. The phosphorylation of ephrin-B1 and the consequent dephosphorylation of NHERF2 are critical initiation events leading to podocyte injury.

Autophagy regulates testosterone synthesis by facilitating cholesterol uptake in Leydig cells.

Testosterone is indispensable for sexual development and maintaining male characteristics, and deficiency of this hormone results in primary or late-onset hypogonadism (LOH). Testosterone is primarily produced in Leydig cells, where autophagy has been reported to be extremely active. However, the functional role of autophagy in testosterone synthesis remains unknown. In this study, we show that steroidogenic cell-specific disruption of autophagy influenced the sexual behavior of aging male mice because of a reduction in serum testosterone, which is similar to the symptoms of LOH. The decline in testosterone was caused mainly by a defect in cholesterol uptake in autophagy-deficient Leydig cells. Further studies revealed that once autophagic flux was disrupted, Na+/H+ exchanger regulatory factor 2 (NHERF2) accumulated in Leydig cells, resulting in the down-regulation of scavenger receptor class B, type I (SR-BI) and eventually leading to insufficient cholesterol supply. Collectively, these results reveal that autophagy promotes cholesterol uptake into Leydig cells by eliminating NHERF2, suggesting that dysfunction of autophagy might be causal in the loss of testosterone production in some patients.

Loss of ezrin expression reduced the susceptibility to the glomerular injury in mice

Ezrin is highly expressed in glomerular podocytes and is reported to form a multi-protein complex with scaffold protein Na+/H+ exchanger regulatory factor 2 (NHERF2) and podocalyxin, a major sialoprotein. Podocalyxin-knockout mice died within 24 h of birth with anuric renal failure, whereas NHERF2-knockout mice show no apparent changes in the glomerular functions. However, the physiological roles of ezrin in glomerular podocytes remain unclear. Here, we investigated the importance of ezrin in the regulation of glomerular podocyte function using ezrin-knockdown mice (Vil2kd/kd). The Vil2kd/kd mice did not exhibit apparent glomerular dysfunction, morphological defects or abnormal localisation of podocalyxin and NHERF2 in podocytes. Thus, we investigated the influence of ezrin defects on Rho-GTPase activity, as ezrin interacts with the Rho-GTPase dissociation inhibitor (Rho-GDI), which plays a key role in the regulation of podocyte actin organisation. In Vil2kd/kd glomeruli, Rac1 activity was significantly reduced compared to wildtype (WT) glomeruli at baseline. Furthermore, Vil2kd/kd mice showed reduced susceptibility to glomerular injury. In WT glomeruli, Rac1 activity was enhanced in nephrotic conditions, but remained at baseline levels in Vil2kd/kd glomeruli, suggesting that loss of ezrin protects podocytes from injury-induced morphological changes by suppressing Rac1 activation.

CFTR-NHERF2-LPA₂ Complex in the Airway and Gut Epithelia.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and cGMP-regulated chloride (Cl−) and bicarbonate (HCO3−) channel localized primarily at the apical plasma membrane of epithelial cells lining the airway, gut and exocrine glands, where it is responsible for transepithelial salt and water transport. Several human diseases are associated with altered CFTR channel function. Cystic fibrosis (CF) is caused by the absence or dysfunction of CFTR channel activity, resulting from mutations in the gene. Secretory diarrhea is caused by the hyperactivation of CFTR channel activity in the gastrointestinal tract. CFTR is a validated target for drug development to treat CF, and extensive research has been conducted to develop CFTR inhibitors for therapeutic interventions of secretory diarrhea. The intracellular processing, trafficking, apical membrane localization, and channel function of CFTR are regulated by dynamic protein–protein interactions in a complex network. In this paper, we review the current knowledge of a macromolecular complex of CFTR, Na+/H+ exchanger regulatory factor 2 (NHERF2), and lysophosphatidic acids (LPA) receptor 2 (LPA2) at the apical plasma membrane of airway and gut epithelial cells, and discuss its relevance in human physiology and diseases. We also explore the possibilities of targeting this complex to fine tune CFTR channel activity, with a hope to open up new avenues to develop novel therapies for CF and secretory diarrhea.

173 The Absence of NHERF2 Attenuates Colon Cancer Growth

Background and Aims: Identification of new molecular targets is needed for better clinical care of colorectal cancer (CRC) patients. Na+/H+ exchanger regulatory factor 2 (NHERF2) was initially identified as a scaffold protein of NHE3, but NHERF2 interacts with a larger number of proteins and overexpression of NHERF2 in CRC has been reported. NHERF2 functions as a modulator of phospholipase Cβ and interacts with lysophosphatidic acid receptor type 2 (LPA2) to enhance LPA-dependent colon cancer cell growth in culture. We have previously shown that loss of LPA2 attenuates colon cancer progress in vivo. However, the importance of NHERF2 in cancer has not been studied and it is unclear whether the role of NHERF2 is limited to LPA2-dependent effects. The aim of the present work was to examine the role of NHERF2 in colonic tumor growth. Methods: HCT116 cells with stable knockdown of NHERF2 (shNHERF2), LPA2 (shLPA2) or control (shCont) were generated. The effects of NHERE2 knockdown on HCT116 cell growth was determined in vitro and in a xenograft model. The effects NHERF2 knockdown was compared with LPA2 knockdown. Apcmin/+ mice were crossed with Nherf2-/mice and the growth and number of intestinal tumors were determined in Apcmin/+, Apcmin/+;Nherf2+/-, and Apcmin/+;Nherf2-/mice. Results: Knockdown of NHERF2 significantly decreased the rates of cell proliferation in the presence of 1-10% FBS. Similarly, the rates of xenograft tumor growth of HCT116/ shNHERF2 cells were constantly slower compared with HCT116/shCont cells. Ki-67 staining of the xenografts confirmed the difference in tumor growth between shNHERF2 and shCont cells. Similar difference was obtained between shLPA2 and shCont cells. Intestinal tumor size was significantly smaller in Apcmin/+;Nherf2-/mice compared to Apcmin/+ mice. In contrast, there was no significant difference in the number of tumors between Apcmin/ +;Nherf2-/and Apcmin/+ mice. As a comparison, we reported earlier that both tumor numbers and sizes were decreased in Apcmin/+;Lpa2-/mice. To understand the molecular basis of NHERF2 depletion on colon cancer, we compared transcriptional profiling of control, shNHERF2 and shLPA2 xenograft cells by RNAseq. We found that expression of 549 and 778 genes in the shNHERF2 and shLPA2 cells, respectively, were altered (p< 0.05) compared with control. Interestingly, only 190 genes were altered in both shNHERF2 and shLPA2 cells, with 359 genes in the shNHERF2 and 588 genes in the shLPA2 cells uniquely regulated. Conclusion: Loss of NHERF2 attenuated tumor growth in both Apcmin/+ and xenograft models of colon cancer. Profile of dysregulated genes in NHERF2 deficient tumors indicates that the role of NHERF2 in tumor progress is potentially diverse and not limited to LPA signaling. This study highlights NHERF2 as a novel oncogenic factor and potential target for CRC treatment.

174 The RNA-Binding Protein Tristetraprolin Controls Intestinal Cell Differentiation and Tumorigenesis Through the Notch Pathway

Background and Aims: Identification of new molecular targets is needed for better clinical care of colorectal cancer (CRC) patients. Na+/H+ exchanger regulatory factor 2 (NHERF2) was initially identified as a scaffold protein of NHE3, but NHERF2 interacts with a larger number of proteins and overexpression of NHERF2 in CRC has been reported. NHERF2 functions as a modulator of phospholipase Cβ and interacts with lysophosphatidic acid receptor type 2 (LPA2) to enhance LPA-dependent colon cancer cell growth in culture. We have previously shown that loss of LPA2 attenuates colon cancer progress in vivo. However, the importance of NHERF2 in cancer has not been studied and it is unclear whether the role of NHERF2 is limited to LPA2-dependent effects. The aim of the present work was to examine the role of NHERF2 in colonic tumor growth. Methods: HCT116 cells with stable knockdown of NHERF2 (shNHERF2), LPA2 (shLPA2) or control (shCont) were generated. The effects of NHERE2 knockdown on HCT116 cell growth was determined in vitro and in a xenograft model. The effects NHERF2 knockdown was compared with LPA2 knockdown. Apcmin/+ mice were crossed with Nherf2-/mice and the growth and number of intestinal tumors were determined in Apcmin/+, Apcmin/+;Nherf2+/-, and Apcmin/+;Nherf2-/mice. Results: Knockdown of NHERF2 significantly decreased the rates of cell proliferation in the presence of 1-10% FBS. Similarly, the rates of xenograft tumor growth of HCT116/ shNHERF2 cells were constantly slower compared with HCT116/shCont cells. Ki-67 staining of the xenografts confirmed the difference in tumor growth between shNHERF2 and shCont cells. Similar difference was obtained between shLPA2 and shCont cells. Intestinal tumor size was significantly smaller in Apcmin/+;Nherf2-/mice compared to Apcmin/+ mice. In contrast, there was no significant difference in the number of tumors between Apcmin/ +;Nherf2-/and Apcmin/+ mice. As a comparison, we reported earlier that both tumor numbers and sizes were decreased in Apcmin/+;Lpa2-/mice. To understand the molecular basis of NHERF2 depletion on colon cancer, we compared transcriptional profiling of control, shNHERF2 and shLPA2 xenograft cells by RNAseq. We found that expression of 549 and 778 genes in the shNHERF2 and shLPA2 cells, respectively, were altered (p< 0.05) compared with control. Interestingly, only 190 genes were altered in both shNHERF2 and shLPA2 cells, with 359 genes in the shNHERF2 and 588 genes in the shLPA2 cells uniquely regulated. Conclusion: Loss of NHERF2 attenuated tumor growth in both Apcmin/+ and xenograft models of colon cancer. Profile of dysregulated genes in NHERF2 deficient tumors indicates that the role of NHERF2 in tumor progress is potentially diverse and not limited to LPA signaling. This study highlights NHERF2 as a novel oncogenic factor and potential target for CRC treatment.

SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells.

The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors.

Recruitment and membrane interactions of host cell proteins during attachment of enteropathogenic and enterohaemorrhagic Escherichia coli

EPEC (enteropathogenic Escherichia coli) and EHEC (enterohaemorrhagic Escherichia coli) are attaching and effacing pathogens frequently associated with infectious diarrhoea. EPEC and EHEC use a T3SS (type III secretion system) to translocate effectors that subvert different cellular processes to sustain colonization and multiplication. The eukaryotic proteins NHERF2 (Na(+)/H(+) exchanger regulatory factor 2) and AnxA2 (annexin A2), which are involved in regulation of intestinal ion channels, are recruited to the bacterial attachment sites. Using a stable HeLa-NHERF2 cell line, we found partial co-localization of AnxA2 and NHERF2; in EPEC-infected cells, AnxA2 and NHERF2 were extensively recruited to the site of bacterial attachment. We confirmed that NHERF2 dimerizes and found that NHERF2 interacts with AnxA2. Moreover, we found that AnxA2 also binds both the N- and C-terminal domains of the bacterial effector Tir through its C-terminal domain. Immunofluorescence of HeLa cells infected with EPEC showed that AnxA2 is recruited to the site of bacterial attachment in a Tir-dependent manner, but independently of Tir-induced actin polymerization. Our results suggest that AnxA2 and NHERF2 form a scaffold complex that links adjacent Tir molecules at the plasma membrane forming a lattice that could be involved in retention and dissemination of other effectors at the bacterial attachment site.

NHERF-2 maintains endothelial homeostasis

AbstractThe Na+/H+ exchanger regulatory factor-2 (NHERF-2) is an integral component of almost all endothelial cells (ECs), yet its endothelial function is not known. Here, we found that NHERF-2, is a key regulator of endothelial homeostasis because NHERF-2–silenced ECs proliferate at a much higher rate even in the absence of mitogens such as VEGF compared with control ECs. We further show that the hyperproliferation phenotype of NHERF-2–silenced EC is because of an accelerated cell cycle that is probably caused by a combination of the following factors: increased cytoplasmic calcium, increased expression of c-Myc, increased expression of cyclin D1, and reduced expression of p27. Using an experimental mouse model of human hemangioma, we found that the endothelial neoplasms derived from NHERF-2–silenced cells were much larger in volume than those derived from control cells. Thus, NHERF-2 is a negative regulator of endothelial proliferation and may have important roles in endothelial homeostasis and vascular modeling.

Macromolecular Complex of CFTR, NHERF2 and iNOS at the Plasma Membrane Contributes to Diarrhea in IBD

Macromolecular complex formation is a common theme in regulation of protein function. The cystic fibrosis transmembrane conductance regulator (CFTR), a cGMP/cAMP‐regulated chloride (Cl−) channel, is a well‐known hub protein involved in multiple protein complexes. In this study, we find that a signaling macromolecular complex of CFTR, Na+/H+ exchanger regulatory factor 2 (NHERF2), and inducible Nitric Oxide Synthase (iNOS) is formed at the plasma membrane in Inflammatory Bowel disease (IBD). We demonstrated the protein interactions using biochemical methods (e.g., protein pairwise binding assay, macromolecular complex assembly assay, and co‐immunoprecipitation) and in vitro methods for studying protein interaction in live cell (e.g., FRET). We conclude based on our in vitro (polarized colonic epithelial monolayers) and in vivo (DSS‐induced colitis in mice) functional studies that the interaction is integrative to CFTR dependent diarrhea in IBD. To summarize our findings: (a) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain‐dependent manner. (b) Upregulation of iNOS increased CFTR channel function by at least two folds in in vivo and in vitro models of inflammation. (c). Diarrhea was attenuated in Nherf2−/− mice compared to Nherf2+/+mice in DSS‐induced colitis model which simulates IBD. Taken together, our results demonstrate that the macromolecular complex of CFTR, NHERF2 and iNOS play important roles in the pathogenic process of diarrhea in IBD. Our study will not only help understand the molecular mechanisms underlying the pathogenic process of diarrhea in IBD, but help to identify novel therapeutic targets and agents for IBD therapy.

The interaction between megalin and ClC-5 is scaffolded by the Na+–H+ exchanger regulatory factor 2 (NHERF2) in proximal tubule cells

Albumin endocytosis in the proximal tubule is mediated by a number of proteins, including the scavenger receptor megalin/cubilin and the PSD-95/Dlg/ZO-1 (PDZ) scaffolds NHERF1 and NHERF2. In addition, in a number of in vitro and in vivo models, the loss of ClC-5 results in a decreased cell surface expression and whole cell level of megalin, suggesting an interaction between these two proteins in vivo. We investigated if ClC-5 and megalin interact directly, and as ClC-5 binds to NHERF2, we investigated if this PDZ scaffold was required for a megalin/ClC-5 complex. GST-pulldown and immunoprecipitation experiments using rat kidney lysate demonstrated an interaction between ClC-5 and megalin, which was mediated by their C-termini. As this interaction may be controlled by a scaffold protein, we characterised any interaction between megalin and NHERF2. Immunoprecipitation experiments indicated that megalin interacts with NHERF2 in vivo, and that this interaction was via an internal NHERF binding domain in the C-terminus of megalin and PDZ2 and the C-terminus of NHERF2. Silencing NHERF2 had no effect on megalin protein levels in the whole cell or plasma membrane. Using siRNA against NHERF2, we demonstrated that NHERF2 was required to facilitate the interaction between megalin and ClC-5. Using fusion proteins, we characterised a protein complex containing ClC-5 and megalin, which is scaffolded by NHERF2, in the absence of any other proteins. Importantly, these observations are the first to describe an interaction between megalin and ClC-5, which is scaffolded by NHERF2 in proximal tubule cells.

Phosphorylation of Podocalyxin (Ser415) Prevents RhoA and Ezrin Activation and Disrupts Its Interaction with the Actin Cytoskeleton

Podocalyxin (PC) is a polysialylated, anti-adhesin that is essential for maintaining foot process architecture and the integrity of the glomerular filtration barrier. We showed previously that PC is firmly attached to the actin cytoskeleton through ezrin, that in puromycin aminonucleoside (PAN)-mediated nephrosis the PC-ezrin-actin complex is disrupted, and that PC is uncoupled from actin. However, the precise mechanisms involved remained unknown. Here we show that detachment of PC from actin is regulated by phosphorylation of PC. PC is hyperphosphorylated at serines in PAN- and protamine sulfate (PS)-treated rat glomeruli. We determined that PC is a substrate of PKC and that the site of phosphorylation is Ser415, located within the juxtamembrane, ezrin-binding domain of the cytoplasmic tail of PC. Mutation of Ser415 to the phosphomimetic residues Glu (S415E) or Asp (S415D) interfered with direct binding of the PC cytoplasmic tail to ezrin in vitro. Moreover, stable expression of a phosphomimetic (S415E) PC mutant but not the WT or the phosphorylation-deficient (S415A) PC mutant, disrupted PC-ezrin-actin interaction, failed to activate RhoA, and the cytoskeletal linker, ezrin, remained inactive. Our data indicate that phosphorylation of PC at Ser415 prevents attachment of PC and ezrin to actin and highlights the strategic position of Ser415 and direct binding of PC to ezrin in regulating podocyte foot process architecture.

Alterations in the proteome of the NHERF2 knockout mouse jejunal brush border membrane vesicles

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.

Functional regulation of cystic fibrosis transmembrane conductance regulator-containing macromolecular complexes: a small-molecule inhibitor approach

CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein-protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)-NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR-NHERF2-LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2-NHERF2 interaction. We show that this compound can disrupt the LPA2-NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl- channel function can be finely tuned by modulating PDZ domain-based protein-protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl- channels.

MAGI-3 Competes With NHERF-2 to Negatively Regulate LPA2 Receptor Signaling in Colon Cancer Cells

Lysophosphatidic acid (LPA) is a potent inducer of colon cancer and LPA receptor type 2 (LPA(2)) is overexpressed in colon tumors. LPA(2) interacts with membrane-associated guanylate kinase with inverted orientation-3 (MAGI-3) and the Na+/H+ exchanger regulatory factor 2 (NHERF-2), but the biological effects of these interactions are unknown. We investigated the roles of MAGI-3 and NHERF-2 in LPA(2)-mediated signaling in human colon cancer cells. We overexpressed or knocked down MAGI-3 in HCT116 and SW480 cells. The effects of MAGI-3 and NHERF-2 in LPA-induced cell migration, invasion, inositol phosphate generation, and nuclear factor-κB activation were determined. Expression of MAGI-3 and NHERF-2 in human colon tumor tissues was analyzed using tissue microarray analysis. NHERF-2 promoted migration and invasion of colon cancer cells, whereas MAGI-3 inhibited these processes. MAGI-3 competed with NHERF-2 for binding to LPA(2) and phospholipase C-β3. However, NHERF-2 and MAGI-3 reciprocally regulated LPA(2)-induced phospholipase C activity. MAGI-3 increased the interaction of LPA(2) with Gα(12), whereas NHERF-2 preferentially promoted interaction between LPA(2) and Gα(q). MAGI-3 decreased the tumorigenic capacity of LPA(2) by attenuating the activities of nuclear factor-κB and c-Jun N-terminal kinase. MAGI-3 and NHERF-2 were expressed differentially in colon adenocarcinomas, consistent with their opposing effects. LPA(2) is dynamically regulated by 2 distinct PDZ proteins via modulation of G-protein coupling and receptor signaling. MAGI-3 is a negative regulator of LPA(2) signaling.

Binding to Na(+) /H(+) exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map, EspI and NleH.

Enteropathogenic Escherichia coli (EPEC) strains are diarrhoeal pathogens that use a type III secretion system to translocate effector proteins into host cells in order to colonize and multiply in the human gut. Map, EspI and NleH1 are conserved EPEC effectors that possess a C-terminal class I PSD-95/Disc Large/ZO-1 (PDZ)-binding motif. Using a PDZ array screen we identified Na+/H+ exchanger regulatory factor 2 (NHERF2), a scaffold protein involved in tethering and recycling ion channels in polarized epithelia that contains two PDZ domains, as a common target of Map, EspI and NleH1. Using recombinant proteins and co-immunoprecipitation we confirmed that NHERF2 binds each of the effectors. We generated a HeLa cell line stably expressing HA-tagged NHERF2 and found that Map, EspI and NleH1 colocalize and interact with intracellular NHERF2 via their C-terminal PDZ-binding motif. Overexpression of NHERF2 enhanced the formation and persistence of Map-induced filopodia, accelerated the trafficking of EspI to the Golgi and diminished the anti-apoptotic activity of NleH1. The binding of multiple T3SS effectors to a single scaffold protein is unique. Our data suggest that NHERF2 may act as a plasma membrane sorting site, providing a novel regulatory mechanism to control the intracellular spatial and temporal effector protein activity.

Reduced 22Na+ Uptake and Dysregulation of NHERF2/Akt/NOS Pathway in Rat Renal Proximal Tubule Cells Stably Overexpressing p22phox

We showed that superoxide (O2−) reduced renal proximal tubule (PT) reabsorption (Hypertension. 2009 Oct.), but, the molecular mechanisms are unknown. We tested the hypothesis that O2− impairs Na+‐H+ exchanger 3 (NHE3) activity. A stably p22phox transfected rat PT cell line (s‐p22phox), compared to empty vector transfected wild type cells (Wt), had increased p22phox mRNA and protein by 4.1‐ and 1.59‐fold, and O2− by 2.2‐ fold but reduced 22Na+uptake by 36% (all P&lt;0.05). Immunoblot assay of s‐p22phox cells showed no change in NHE3 but increased expression of inhibitory Na+–H+ exchanger regulatory factor 2 (NHERF2), neuronal and inducible nitric oxide synthase (nNOS &amp; iNOS) by 183%, 293% and 372%, respectively (all P&lt;0.05). Since activated Akt regulates NOS and NHERF2, we compared the Akt in both cell types. Total Akt (t‐Akt) protein abundance was similar but the phosphorylated Akt at ser473 (p‐Aktser473) was markedly increased leading to a 207% higher p‐Akt/t‐Akt ratio in s‐p22phox cells. Knockdown of NHEFR2 by specific siRNA in s‐p22phox cells increased significantly (25±4.9%; P&lt;0.05) 22Na+uptake. In conclusion, overexpression of p22phox in PT cells inhibits NHE3 transport activity and upregulates NHERF2, nNOS and iNOS, and phosphorylated Akt. This dysregulation of NHERF2/Akt/NOS signaling pathways by O2− impairs PT function. This provides a novel insight into the molecular mechanisms underlying the effects of oxidative stress on ion and fluid homeostasis in the kidney.

Identification Of The NHERF2 Binding Site For The Chloride/Proton Transporter ClC-5

The chloride/proton transporter ClC-5 mediates the re-absorption of filtered proteins in the kidney by promoting the formation of the macromolecular endocytic complex and by aiding in endosomal acidification upon complex internalization in proximal tubule cells. Mutations disrupting ClC-5 lead to proteinuria reflecting a severe impairment of renal receptor endocytosis and several disease-causing mutations in ClC-5 translate into truncations of the carboxy terminus (Ct), highlighting the significance of this region. The Ct interacts with the scaffold protein NHERF2 (Na+/H+ exchanger regulatory factor 2) and leads to enhanced endocytic uptake of albumin by kidney cells [Hryciw, D. et al. 2006]. This interaction occurs through the second PDZ domain of NHERF2 and an unknown binding site on the Ct of ClC-5 [Hryciw, D. et al. 2006]. In the present study NHERF2/ClC-5 Ct interactions were confirmed and a putative internal PDZ binding motif TSII (residues 657-60) was identified in the Ct. Mapping of this motif on the crystal structure of ClC-5 reveals it lies up-stream of a β-turn, a secondary structure element thought to be critical for PDZ domain recognition of an internal motif [Hillier, B. et al. 1999]. The strategic S658A mutation completely abolishes NHERF2 binding in vitro suggesting the PDZ binding motif was targeted. CD analysis confirmed similar spectra between wildtype and S658A mutant, indicating that this mutation does not lead to gross misfolding of the Ct. The disease mutant R648X which removes this critical S658 abolishes NHERF2 interactions providing further evidence the internal PDZ binding motif was targeted. Future work will focus on identifying proteins interacting with the ClC-5/NHERF2 complex and deciphering the role of this complex in renal endocytosis. This work was supported by the Kidney Foundation of Canada operating gant to C.B. and a NSERC studentship to L.W.

Muscarinic-induced Recruitment of Plasma Membrane Ca2+-ATPase Involves PSD-95/Dlg/Zo-1-mediated Interactions

Efflux of cytosolic Ca2+ mediated by plasma membrane Ca2+-ATPases (PMCA) plays a key role in fine tuning the magnitude and duration of Ca2+ signaling following activation of G-protein-coupled receptors. However, the molecular mechanisms that underpin the trafficking of PMCA to the membrane during Ca2+ signaling remain largely unexplored in native cell models. One potential mechanism for the recruitment of proteins to the plasma membrane involves PDZ interactions. In this context, we investigated the role of PMCA interactions with the Na+/H+ exchanger regulatory factor 2 (NHERF-2) during muscarinic-induced Ca2+ mobilization in the HT-29 epithelial cell line. GST pull-downs in HT-29 cell lysates showed that the PDZ2 module of NHERF-2 bound to the PDZ binding motif on the C terminus of PMCA. Co-immunoprecipitations confirmed that PMCA1b and NHERF-2 associated under normal conditions in HT-29 cells. Cell surface biotinylations revealed significant increases in membrane-associated NHERF-2 and PMCA within 60 s following muscarinic activation, accompanied by increased association of the two proteins as seen by confocal microscopy. The recruitment of NHERF-2 to the membrane preceded that of PMCA, suggesting that NHERF-2 was involved in nucleating an efflux complex at the membrane. The muscarinic-mediated translocation of PMCA was abolished when NHERF-2 was silenced, and the rate of relative Ca2+ efflux was also reduced. These experiments also uncovered a NHERF-2-independent PMCA retrieval mechanism. Our findings describe rapid agonist-induced translocation of PMCA in a native cell model and suggest that NHERF-2 plays a key role in scaffolding and maintaining PMCA at the cell membrane.

The PDZ Scaffold NHERF-2 Interacts with mGluR5 and Regulates Receptor Activity

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G(q) to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs1 and 5are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na(+)/H(+) exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.