Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport and cause congenital hypophosphatemia.

The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg495His or Arg495Cys variants within the internal PDZ motif. The wild-type internal 494TRL496 PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a 494AAA496 substitution blocked hormone-inhibitable phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg495His or Arg495Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast with WT NPT2A, NPT2A Arg495His, or Arg495Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg495 changes the electrostatics, preventing phosphorylation of the upstream Thr494, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxy-terminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

The Intricacies of Renal Phosphate Reabsorption-An Overview.

To maintain an optimal body content of phosphorus throughout postnatal life, variable phosphate absorption from food must be finely matched with urinary excretion. This amazing feat is accomplished through synchronised phosphate transport by myriads of ciliated cells lining the renal proximal tubules. These respond in real time to changes in phosphate and composition of the renal filtrate and to hormonal instructions. How they do this has stimulated decades of research. New analytical techniques, coupled with incredible advances in computer technology, have opened new avenues for investigation at a sub-cellular level. There has been a surge of research into different aspects of the process. These have verified long-held beliefs and are also dramatically extending our vision of the intense, integrated, intracellular activity which mediates phosphate absorption. Already, some have indicated new approaches for pharmacological intervention to regulate phosphate in common conditions, including chronic renal failure and osteoporosis, as well as rare inherited biochemical disorders. It is a rapidly evolving field. The aim here is to provide an overview of our current knowledge, to show where it is leading, and where there are uncertainties. Hopefully, this will raise questions and stimulate new ideas for further research.

LRP2 contributes to planar cell polarity-dependent coordination of motile cilia function.

Motile cilia are protruding organelles on specialized epithelia that beat in a synchronous fashion to propel extracellular fluids. Coordination and orientation of cilia beating on individual cells and across tissues is a complex process dependent on planar cell polarity (PCP) signaling. Asymmetric sorting of PCP pathway components, essential to establish planar polarity, involves trafficking along the endocytic path, but the underlying regulatory processes remain incompletely understood. Here, we identified the endocytic receptor LRP2 as regulator of PCP component trafficking in ependyma, a multi-ciliated cell type that is involved in facilitating flow of the cerebrospinal fluid in the brain ventricular system. Lack of receptor expression in gene-targeted mice results in a failure to sort PCP core proteins to the anterior or posterior cell side and, consequently, in the inability to coordinate cilia arrangement and to aligned beating (loss of rotational and translational polarity). LRP2 deficiency coincides with a failure to sort NHERF1, a cytoplasmic LRP2 adaptor to the anterior cell side. As NHERF1 is essential to translocate PCP core protein Vangl2 to the plasma membrane, these data suggest a molecular mechanism whereby LRP2 interacts with PCP components through NHERF1 to control their asymmetric sorting along the endocytic path. Taken together, our findings identified the endocytic receptor LRP2 as a novel regulator of endosomal trafficking of PCP proteins, ensuring their asymmetric partition and establishment of translational and rotational planar cell polarity in the ependyma.

Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly.

The Na+/H+ exchange regulatory cofactor 1 (NHERF1) protein modulates the assembly and intracellular trafficking of several transmembrane G protein-coupled receptors (GPCRs) and ion transport proteins with the membrane-cytoskeleton adapter protein ezrin. Here, we applied solution NMR and small-angle neutron scattering (SANS) to structurally characterize full-length NHERF1 and disease-associated variants that are implicated in impaired phosphate homeostasis. Using NMR, we mapped the modular architecture of NHERF1, which is composed of two structurally-independent PDZ domains that are connected by a flexible, disordered linker. We observed that the ultra-long and disordered C-terminal tail of NHERF1 has a type 1 PDZ-binding motif that interacts weakly with the proximal, second PDZ domain to form a dynamically autoinhibited structure. Using ensemble-optimized analysis of SANS data, we extracted the molecular size distribution of structures from the extensive conformational space sampled by the flexible chain. Our results revealed that NHERF1 is a diffuse ensemble of variable PDZ domain configurations and a disordered C-terminal tail. The joint NMR/SANS data analyses of three disease variants (L110V, R153Q, and E225K) revealed significant differences in the local PDZ domain structures and in the global conformations compared with the WT protein. Furthermore, we show that the substitutions affect the affinity and kinetics of NHERF1 binding to ezrin and to a C-terminal peptide from G protein-coupled receptor kinase 6A (GRK6A). These findings provide important insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes.

Inhibition of ezrin causes PKCα-mediated internalization of erbb2/HER2 tyrosine kinase in breast cancer cells.

Unlike other ErbB family members, HER2 levels are maintained on the cell surface when the receptor is activated, allowing prolonged signaling and contributing to its transforming ability. Interactions between HER2, HSP90, PMCA2, and NHERF1 within specialized plasma membrane domains contribute to the membrane retention of HER2. We hypothesized that the scaffolding protein ezrin, which has been shown to interact with NHERF1, might also help stabilize the HER2-PMCA2-NHERF1 complex at the plasma membrane. Therefore, we examined ezrin expression and its relationship with HER2, NHERF1, and PMCA2 levels in murine and human breast cancers. We also used genetic knockdown and/or pharmacologic inhibition of ezrin, HSP90, NHERF1, PMCA2, and HER2 to examine the functional relationships between these factors and membrane retention of HER2. We found ezrin to be expressed at low levels at the apical surface of normal mammary epithelial cells, but its expression is up-regulated and correlates with HER2 expression in hyperplasia and tumors in murine mammary tumor virus-Neu mice, in human HER2-positive breast cancer cell lines, and in ductal carcinoma in situ and invasive breast cancers from human patients. In breast cancer cells, ezrin co-localizes and interacts with HER2, NHERF1, PMCA2, and HSP90 in specialized membrane domains, and inhibiting ezrin disrupts interactions between HER2, PMCA2, NHERF1, and HSP90, inhibiting HER2 signaling and causing PKCα-mediated internalization and degradation of HER2. Inhibition of ezrin synergizes with lapatinib in a PKCα-dependent fashion to inhibit proliferation and promote apoptosis in HER2-positive breast cancer cells. We conclude that ezrin stabilizes a multiprotein complex that maintains active HER2 at the cell surface.

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport.

Na+-H+ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290 Mutating 257VPF259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ß-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.

Alteration in the sensitivity to crizotinib by Na+/H+ exchanger regulatory factor 1 is dependent to its subcellular localization in ALK-positive lung cancers.

BACKGROUND: Na+/H+ exchanger regulatory factor 1 (NHERF1) is an important scaffold protein participates in the modulation of a variety of intracellular signal pathways. NHERF1 was able to enhance the effects of chemo-drugs in breast and cervical cancer cells. Anaplastic lymphoma kinase (ALK) fusion mutations are validated molecules targeted therapy in lung cancers, where crizotinib can be used as the specific inhibitor to suppress tumor progression. However, due to the less frequent occurrence of ALK mutations and the complexity for factors to determine drug responses, the genes that could alter crizotinib sensitivity are unclear. METHODS: Both ALK-translocated and ALK-negative lung adenocarcinoma specimens in tissue sections were collected for immunohistochemistry. The possible mechanisms of NHERF1 and its role in the cell sensitivity to crizotinib were investigated using an ALK-positive and crizotinib-sensitive lung adenocarcinoma cell line H3122. Either a NHERF1 overexpression vector or agents for NHERF1 knockdown was used for crizotinib sensitivity measures, in association with cell viability and apoptosis assays. RESULTS: The expression level of NHERF1 in ALK-translocated NSCLC was significantly higher than that in other lung cancer tissues. NHERF1 expression in ALK positive lung cancer cells was regulated by ALK activities, and was in return able to alter the sensitivity to crizotinib. The function of NHERF1 to influence crizotinib sensitivity was depending on its subcellular distribution in cytosol instead of its nucleus localized form. CONCLUSION: Ectopically overexpressed NHERF1 could be a functional protein for consideration to suppress lung cancers. The determination of NHERF1 levels in ALK positive NSCLC tissues might be useful to predict crizotinib resistance, especially by distinguishing cytosolic or nuclear localized NHERF1 for the overexpressed molecules.

Prognostic and therapeutic implications of NHERF1 expression and regulation in colorectal cancer.

BACKGROUND: Na+ /H+ exchanger regulatory factor 1 (NHERF1) has been implicated in the tumorigenesis of several cancer types and is a potential therapeutic target. The current study evaluated the relationship between NHERF1 expression and clinical outcome in colorectal cancer (CRC). METHODS: NHERF1 expression was evaluated by immunohistochemistry in 167 patients with CRC primary tumors, 37 patients with no disease, and 27 patients with metastatic CRC (mCRC); and in the orthotopically implanted tumors in mice. NHERF1 expression was manipulated in CRC cells using inducible short hairpin RNAs to determine its biological functions. RESULTS: High expression of NHERF1 correlated with CRC progression and metastasis, as well as significantly worse overall survival, recurrence-free survival, and disease-specific survival. Orthotopic implantation studies demonstrated increased NHERF1 expression in liver metastases. Treatment of CRC xenografts with insulin-like growth factor 1 receptor (IGF1R) inhibitors downregulated NHERF1 expression, indicating NHERF1 is downstream of IGF1R signaling. Knockdown of NHERF1 increased apoptosis and reduced X-linked inhibitor of apoptosis protein (XIAP) and survivin expression, indicating NHERF1 is critical for CRC cell survival. CONCLUSION: NHERF1 expression levels correlated with worse prognosis in patients with CRC and plays a critical role in CRC cell survival. Together, our findings establish NHERF1 as a novel potential marker for increased risk of CRC-specific mortality and identify NHERF1 as an attractive therapeutic target for mCRC treatment.

Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation.

The fetal inflammatory response, a key contributor of infection-associated preterm birth (PTB), is mediated by nuclear factor kappa B (NF-kB) activation. Na+/H+ exchanger regulatory factor-1 (NHERF1) is an adapter protein that can regulate intracellular signal transduction and thus influence NF-kB activation. Accordingly, NHERF1 has been reported to enhance proinflammatory cytokine release and amplify inflammation in a NF-kB-dependent fashion in different cell types. The objective of this study was to examine the role of NHERF1 in regulating fetal membrane inflammation during PTB. We evaluated the levels of NHERF1 in human fetal membranes from term labor (TL), term not in labor (TNIL), and PTB and in a CD1 mouse model of PTB induced by lipopolysaccharide (LPS). Additionally, primary cultures of fetal membrane cells were treated with LPS, and NHERF1 expression and cytokine production were evaluated. Gene silencing methods using small interfering RNA targeting NHERF1 were used to determine the functional relevance of NHERF1 in primary cultures. NHERF1 expression was significantly (p < 0.001) higher in TL and PTB membranes compared to TNIL membranes, and this coincided with enhanced (p < 0.01) interleukin (IL)-6 and IL-8 expression levels. LPS-treated animals delivering PTB had increased levels of NHERF1, IL-6, and IL-8 compared to phosphate-buffered saline (PBS; control) animals. Silencing of NHERF1 expression resulted in a significant reduction in NF-kB activation and IL-6 and IL-8 production as well as increased IL-10 production. In conclusion, downregulation of NHERF1 increased anti-inflammatory IL-10, and reducing NHERF1 expression could be a potential therapeutic strategy to reduce the risk of infection/inflammation associated with PTB.

Repressing of NHERF1 inhibits liver cancer progression by promoting the production of ROS.

NHERF1/EBP50 is a PDZ-scaffold protein initially identified as an organizer and modulator of transporters and channels at the apical side of epithelia via actin-binding ezrin-moesin-radixin proteins. Presently, hepatocellular carcinoma (HCC) is one of the most deadly cancers in the world and has no effective therapeutic strategies. In the present study, we attempted to explore the role of NHERF1 in regulating liver cancer progression. The results indicated that NHERF1 was significantly expressed in liver tumor samples compared to the corresponding adjacent normal tissues. HCC patients with low NHERF1 exhibited better survival rate. Additionally, repressing NHERF1 expression markedly down-regulated the cell proliferation. G0/G1 transition was highly induced by NHERF1 knockdown, accompanied with reduced expressions of Cyclin D1 and cyclin-dependent kinase 4 (CDK4), as well as the enhanced expression of p27, phosphatase and tensin homolog (PTEN) and p53. Moreover, NHERF1 suppression significantly induced apoptosis in liver cancer cells by promoting the activation of Caspase-3 and poly (ADP-ribose) polymerase (PARP). We also observed a remarkable increase of reactive oxygen species (ROS) production in NHERF1-knockdown cells, along with c-Jun-N-terminal kinase (JNK) phosphorylation. Importantly, suppressing ROS production abolished NHERF1 knockdown-induced JNK activation. Moreover, cell cycle-regulatory proteins meditated by NHERF1 knockdown in liver cancer cells were abrogated by the pre-treatment of ROS scavenger. Further, restraining ROS generation also diminished NHERF1 knockdown-induced apoptosis. In vivo, we also found that NHERF1 knockdown markedly reduced the tumor growth. In conclusion, the results suggested that NHERF1 played an essential role in regulating liver cancer progression, and repressing NHERF1 expression exhibited significant anticancer effects via the induction of G0/G1 phase arrest, apoptosis and ROS generation.

Loss of NHERF-1 expression prevents dopamine-mediated Na-K-ATPase regulation in renal proximal tubule cells from rat models of hypertension: aged F344 rats and spontaneously hypertensive rats.

Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.

Systematic Proteogenomic Approach To Exploring a Novel Function for NHERF1 in Human Reproductive Disorder: Lessons for Exploring Missing Proteins.

One of the major goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to fill the knowledge gaps between human genomic information and the corresponding proteomic information. These gaps are due to "missing" proteins (MPs)-predicted proteins with insufficient evidence from mass spectrometry (MS), biochemical, structural, or antibody analyses-that currently account for 2579 of the 19587 predicted human proteins (neXtProt, 2017-01). We address some of the lessons learned from the inconsistent annotations of missing proteins in databases (DB) and demonstrate a systematic proteogenomic approach designed to explore a potential new function of a known protein. To illustrate a cautious and strategic approach for characterization of novel function in vitro and in vivo, we present the case of Na(+)/H(+) exchange regulatory cofactor 1 (NHERF1/SLC9A3R1, located at chromosome 17q25.1; hereafter NHERF1), which was mistakenly labeled as an MP in one DB (Global Proteome Machine Database; GPMDB, 2011-09 release) but was well known in another public DB and in the literature. As a first step, NHERF1 was determined by MS and immunoblotting for its molecular identity. We next investigated the potential new function of NHERF1 by carrying out the quantitative MS profiling of placental trophoblasts (PXD004723) and functional study of cytotrophoblast JEG-3 cells. We found that NHERF1 was associated with trophoblast differentiation and motility. To validate this newly found cellular function of NHERF1, we used the Caenorhabditis elegans mutant of nrfl-1 (a nematode ortholog of NHERF1), which exhibits a protruding vulva (Pvl) and egg-laying-defective phenotype, and performed genetic complementation work. The nrfl-1 mutant was almost fully rescued by the transfection of the recombinant transgenic construct that contained human NHERF1. These results suggest that NHERF1 could have a previously unknown function in pregnancy and in the development of human embryos. Our study outlines a stepwise experimental platform to explore new functions of ambiguously denoted candidate proteins and scrutinizes the mandated DB search for the selection of MPs to study in the future.

Convergent Signaling Pathways Regulate Parathyroid Hormone and Fibroblast Growth Factor-23 Action on NPT2A-mediated Phosphate Transport.

Parathyroid hormone (PTH) and FGF23 are the primary hormones regulating acute phosphate homeostasis. Human renal proximal tubule cells (RPTECs) were used to characterize the mechanism and signaling pathways of PTH and FGF23 on phosphate transport and the role of the PDZ protein NHERF1 in mediating PTH and FGF23 effects. RPTECs express the NPT2A phosphate transporter, αKlotho, FGFR1, FGFR3, FGFR4, and the PTH receptor. FGFR1 isoforms are formed from alternate splicing of exon 3 and of exon 8 or 9 in Ir-like loop 3. Exon 3 was absent, but mRNA containing both exons 8 and 9 is present in cytoplasm. Using an FGFR1c-specific antibody together with mass spectrometry analysis, we show that RPTECs express FGFR-ß1C. The data are consistent with regulated FGFR1 splicing involving a novel cytoplasmic mechanism. PTH and FGF23 inhibited phosphate transport in a concentration-dependent manner. At maximally effective concentrations, PTH and FGF23 equivalently decreased phosphate uptake and were not additive, suggesting a shared mechanism of action. Protein kinase A or C blockade prevented PTH but not FGF23 actions. Conversely, inhibiting SGK1, blocking FGFR dimerization, or knocking down Klotho expression disrupted FGF23 actions but did not interfere with PTH effects. C-terminal FGF23(180-251) competitively and selectively blocked FGF23 action without disrupting PTH effects. However, both PTH and FGF23-sensitive phosphate transport were abolished by NHERF1 shRNA knockdown. Extended treatment with PTH or FGF23 down-regulated NPT2A without affecting NHERF1. We conclude that FGFR1c and PTHR signaling pathways converge on NHERF1 to inhibit PTH- and FGF23-sensitive phosphate transport and down-regulate NPT2A.

The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9.

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.

NHERF1 and NHERF2 regulation of SR-B1 stability via ubiquitination and proteasome degradation.

Scavenger receptor class B type 1 (SR-B1), an HDL receptor plays a crucial role in cholesterol metabolism in the liver, steroidogenic tissues, and vascular cells including macrophages. SR-B1 is subject to regulation at the transcription, posttranscription and posttranslational levels. We previously provided evidence that PDZ domain containing NHERF1 and NHERF2 regulate SR-B1 protein levels post-transcriptionally, although the underlying mechanism(s) by which NHERF1 and NHERF2 regulate SR-B1 protein levels is not well understood. In this study, we demonstrate that SR-B1 is degraded intracellularly via ubiquitin-proteasome pathway and that SR-B1 can be ubiquitinated at K500 and K508 residues. Overexpression of NHERF1 or NHERF2 enhanced SR-B1 ubiquitination and degradation. NHERF1 and NHERF2 promote SR-B1 ubiquitination at sites K508 and K500, respectively. These results suggest that NHERF1 and NHERF2 down-regulated SR-B1 at least in part via the ubiquitin/proteasome pathway.

Na+/H+ exchange regulatory factor 1 is required for ROMK1 K+ channel expression in the surface membrane of cultured M-1 cortical collecting duct cells.

The ROMK1 K+ channel, a member of the ROMK channel family, is the major candidate for the K+ secretion pathway in the renal cortical collecting duct (CCD). ROMK1 possesses a PDZ domain-binding motif at its C-terminus that is considered a modulator of ROMK1 expression via interaction with Na+/H+ exchange regulatory factor (NHERF) 1 and NHERF2 scaffold protein. Although NHERF1 is a potential binding partner of the ROMK1 K+ channel, the interaction between NHERF1 and K+ channel activity remains unclear. Therefore, in this study, we knocked down NHERF1 in cultured M-1 cells derived from mouse CCD and investigated the surface expression and K+ channel current in these cells after exogenous transfection with EGFP-ROMK1. NHERF1 knockdown resulted in reduced surface expression of ROMK1 as indicated by a cell biotinylation assay. Using the patch-clamp technique, we further found that the number of active channels per patched membrane and the Ba2+-sensitive whole-cell K+ current were decreased in the knockdown cells, suggesting that reduced K+ current was accompanied by decreased surface expression of ROMK1 in the NHERF1 knockdown cells. Our results provide evidence that NHERF1 mediates K+ current activity through acceleration of the surface expression of ROMK1 K+ channels in M-1 cells.

NHERF1 in Microvilli of Vomeronasal Sensory Neurons.

In most mammals, the vomeronasal system detects a variety of (semio)chemicals that mediate olfactory-driven social and sexual behaviors. Vomeronasal chemosensation depends on G protein-coupled receptors (V1R, V2R, and FPR-rs) that operate at remarkably low stimulus concentrations, thus, indicating a highly sensitive and efficient signaling pathway. We identified the PDZ domain-containing protein, Na+/H+ exchanger regulatory factor-1 (NHERF1), as putative molecular organizer of signal transduction in vomeronasal neurons. NHERF1 is a protein that contains 2 PDZ domains and a carboxy-terminal ezrin-binding domain. It localizes to microvilli of vomeronasal sensory neurons and interacts with V1Rs. Furthermore, NHERF1 and Gαi2 are closely colocalized. These findings open up new aspects of the functional organization and regulation of vomeronasal signal transduction by PDZ scaffolding proteins.

NHERF1 Enhances Cisplatin Sensitivity in Human Cervical Cancer Cells.

Cervical cancer is one of the most common female malignancies, and cisplatin-based chemotherapy is routinely utilized in locally advanced cervical cancer patients. However, resistance has been the major limitation. In this study, we found that Na⁺/H⁺ Exchanger Regulatory Factor 1 (NHERF1) was downregulated in cisplatin-resistant cells. Analysis based on a cervical cancer dataset from The Cancer Genome Atlas (TCGA) showed association of NHERF1 expression with disease-free survival of patients received cisplatin treatment. NHERF1 overexpression inhibited proliferation and enhanced apoptosis in cisplatin-resistant HeLa cells, whereas NHERF1 knockdown had inverse effects. While parental HeLa cells were more resistant to cisplatin after NHERF1 knockdown, NHERF1 overexpression in CaSki cells promoted cisplatin sensitivity. Overexpression and knockdown studies also showed that NHERF1 significantly inhibited AKT and extracellular signal-regulated kinase (ERK) signaling pathways in cisplatin-resistant cells. Taken together, our results provide the first evidence that NHERF1 can sensitize cisplatin-refractory cervical cancer cells. This study may help to increase understanding of the molecular mechanisms underlying cisplatin resistance in tumors.

Phosphorylation of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) by Akt promotes stability and mitogenic function of S-phase kinase-associated protein-2 (Skp2).

The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.

Histone Deacetylase Inhibitors Target the Leukemic Microenvironment by Enhancing a Nherf1-Protein Phosphatase 1α-TAZ Signaling Pathway in Osteoblasts.

Disrupting the protective signals provided by the bone marrow microenvironment will be critical for more effective combination drug therapies for acute myeloid leukemia (AML). Cells of the osteoblast lineage that reside in the endosteal niche have been implicated in promoting survival of AML cells. Here, we investigated how to prevent this protective interaction. We previously showed that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis of AML cells, unless the leukemic cells receive protective signals provided by differentiating osteoblasts (8, 10). We now identify a novel signaling pathway in differentiating osteoblasts that can be manipulated to disrupt the osteoblast-mediated protection of AML cells. Treating differentiating osteoblasts with histone deacetylase inhibitors (HDACi) abrogated their ability to protect co-cultured AML cells from SDF-1-induced apoptosis. HDACi prominently up-regulated expression of the Nherf1 scaffold protein, which played a major role in preventing osteoblast-mediated protection of AML cells. Protein phosphatase-1α (PP1α) was identified as a novel Nherf1 interacting protein that acts as the downstream mediator of this response by promoting nuclear localization of the TAZ transcriptional modulator. Moreover, independent activation of either PP1α or TAZ was sufficient to prevent osteoblast-mediated protection of AML cells even in the absence of HDACi. Together, these results indicate that HDACi target the AML microenvironment by enhancing activation of the Nherf1-PP1α-TAZ pathway in osteoblasts. Selective drug targeting of this osteoblast signaling pathway may improve treatments of AML by rendering leukemic cells in the bone marrow more susceptible to apoptosis.