Na+/H+ Exchanger Regulatory Factor Research Articles

CGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

Roles of Ezrin in regulation of ciliary beating in lung multiciliated cell

The mucociliary clearance is the first line of defense in the lungs. The function is given by the beating cilia on the surface of ciliated cells cooperating with the protective mucous layer. The beating cilia plays removing inhaled particles and pathogens (viruses, bacteria and fungi). Thus, the beating cilia are the key apparatus to conduct the mucociliary clearance. For ciliary beating to function properly, localization of ciliary proteins that regulate ciliary beating and formation of epithelial cell polarity are important. Ezrin/Radixin/Moesin (ERM) proteins function as general cross-linkers between apical membrane proteins and the actin cytoskeleton and are involved in the functional expression of apical membrane proteins. Especially, ezrin may play the important role of apical localization of β2-adrenergic receptor (β2AR) and cAMP-dependent protein kinase (PKA) in multiciliated cells. Here we examined roles of ezrin in the regulation of ciliary beating in lung multiciliated cells by using ezrin-knockdown (Vil2kd/kd) mice. To evaluate ciliary function, we analyzed ciliary beat frequency (CBF) and ciliary bend distance (CBD, and index of amplitude) in lung multiciliated cells of mice. The multiciliated cells were isolated by an elastase treatment. Ciliary beatings of lung multiciliated cells were observed by the videomicroscopy equipped with high speed camera (500 fps) and CBF and CBD were measured by an image analysis program using recorded images. We performed immunofluorescence microscopy analysis to evaluate the localization of β2AR, Na+/H+ exchanger regulatory factor (NHERF) 1, which is a PSD-95/Discs-large/ZO-1 (PDZ) scaffold protein, and PKA. In addition, we also performed cell surface protein biotinylation assay. In the results, stimulation with 1 nM procaterol, which is selective β2AR agonist, increased the ratios of CBF and CBD by about 80% in WT multiciliated cells. However, in Vil2kd/kd, stimulation with 1 nM procaterol increased the ratios of CBF and CBD by only 40%. While it is no significant difference that the increase of CBF and CBD by stimulation with 100 μM IBMX, which is a broad-spectrum phosphodiesterase inhibitor, in WT and Vil2kd/kd. In addition, the cell surface localization of NHERF1 and β2AR is disturbed in Vil2kd/kd multiciliated cells. There were no apparent changes in the subcellular localizations of PKA RIα and RIIα, which are PKA regulatory subunits, in Vil2kd/kd multiciliated cells. These results suggest that ezrin regulates the ciliary beating in lung multiciliated cells by promoting the apical localization of β2AR.

NHERF3 is necessary for Escherichia coli heat-stable enterotoxin-induced inhibition of NHE3: differences in signaling in mouse small intestine and Caco-2 cells.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler's diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+ concentration [Ca2+]i). The ST elevation of [Ca2+]i was from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms.

PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class I PDZ-binding motifs

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.

Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol

The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the Gq/11 protein-phospholipase C (PLC) signaling pathway has remained elusive so far. In contrast to all other TRPC channels, the PLC product diacylglycerol (DAG) is not sufficient for channel activation, whereas TRPC4/5 channel activity is potentiated by phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na+/H+ exchanger regulatory factor (NHERF) 1 and 2. PKC inhibition or the exchange of threonine for alanine in the C-terminal PDZ-binding motif conferred DAG sensitivity to the channel. Altogether, we present a DAG-mediated activation mechanism for TRPC4/5 channels tightly regulated by NHERF1/2 interaction. PIP2 depletion evokes a C-terminal conformational change of TRPC5 proteins leading to dynamic dissociation of NHERF1/2 from the C terminus of TRPC5 as a prerequisite for DAG sensitivity. We show that NHERF proteins are direct regulators of ion channel activity and that DAG sensitivity is a distinctive hallmark of TRPC channels.

Abstract P330: Role of Na-H Exchanger Regulatory Factor 1 (NHERF1) In Salt-sensitive Hypertension and Inflammation in Aging

Aging animals develop salt-sensitive hypertension due, in part, to desensitization of dopamine receptors and sensitization of AT1R in renal proximal tubules. We demonstrated old FBN rats develop salt-dependent hypertension. Preliminary data showed loss of NHERF1 expression in 22m old F344 rats. We hypothesized that loss of NHERF1 may contribute to salt reabsorption in aging. To address this hypothesis, Fischer Brown Norway (FBN) rats (1m, 4m, 12m, and 24m old) were fed 1% or 8% NaCl diet for one week and, urinary volume, sodium, potassium, and chloride and expression of Na-K ATPase α1 subunit (NKA), NHERF1, and D1R were measured. Feeding 8% NaCl increased 24 h urine volume compared to rats fed 1% NaCl diet in 1m (13.6±0.98 vs 29.2±3.8 mL) and 4m (15.3±1.5 vs 39.2±2.4) but not in 24 m rats (20.7±4.7 vs 15.9±2.4). 24 h urine Na increased 7 fold in 4 m old rats fed an 8% NaCl diet but only 3 fold in 24m old rats. To our surprise, NHERF1 expression increased with age in FBN rats (0.21±.03 (4m) vs 0.71±0.07 (24m) AU ratio NHERF1:actin). In F344 rats NHERF1 expression decreased with age (0.86±0.14 vs 0.39±0.05). Increased NHERF1 expression with age in FBN rats was associated with decreased D1R expression (0.8±0.16 vs 0.4±0.04) while in F344 rats D1R expression increased with age (0.6±0.1 vs 0.9±0.1). FBN rats are salt-sensitive while F344 are salt-resistant. F344 rats develop kidney inflammation with aging. We determined TNFα in kidney homogenates from FBN and F344 rats. TNFα increased with age in both F344 (4.04±0.15 vs 6.9±0.42) and FBN rats (2.1±0.3 vs 4.05±0.31 ng/mg protein). To determine if lack of NHERF1 is responsible for salt wasting, we measured urine volume and Na in 18 m old WT and NHERF1 KO mice. The urine volume in WT mice (2.7±0.8 (1% NaCl) vs 5.6±1.2 mL/24 h (8% NaCl)) were significantly lower than KO mice (3.6±0.57(1% NaCl) vs 7.5±0.88 mL/24 h (8% NaCl)). Similar results were observed in Na excretion. D1R expression increased in NHERF1 KO mice as compared to WT mice. TNFα increased significantly in NHERF1 KO mice kidneys as compared to WT mice. We conclude that increased NHERF1 expression as seen in FBN rats results in salt retention while lack of NHERF1 as seen F344 rats and NHERF1 KO mice increases renal inflammation. Further studies are required to delineate the two roles of NHERF1.

Abstract P244: Role of the Na-H Exchanger Regulatory Factor 1 (NHERF1) in Hypertension of Aging Animals

Aging animals develop hypertension when challenged with high salt diet due, in part, to desensitization of dopamine receptors (DR) in renal proximal tubules (RPT). We have demonstrated that NHERF1 associates with DR1 and Na-K ATPase (NKA) and is important for regulation of NKA in RPT. Preliminary data showed loss of NHERF1 expression in 22m old F344 rats. We hypothesized that loss of NHERF1 results in increased blood pressure (BP) and lack of natriuretic response to dopamine (DA) in aging animals. To address this hypothesis, Fischer Brown Norway (FBN) rats (1m, 4m, 12m, and 24m old) were fed diet containing 1% or 8% NaCl for one week and, BP was measured in anesthetized animals using an indwelling left femoral artery catheter. 8% NaCl did not increase BP in 1m or 4 month old rats. By contrast, 8% NaCl diet increased BP in 12m (84.3±3.5 vs 90.8±2.36) and 24m (73.5±7.58 vs 104±1.6) old animals. To determine if lack of NHERF1 is responsible for the increase in BP, we measured BP in 12 m old WT and NHERF1 KO mice. By contrast to WT mice, 8% NaCl diet did not increase BP in NHERF1 KO mice (84±4.9 vs 96.5±3.56 (WT) and 78.2±3.89 vs 81.8±9.2 (NHERF1 KO mice)). To confirm that NHERF1 is required for DA-mediated inhibition of NKA, NKA activity in primary proximal tubule cells (PTC) from young and old mice in culture was measured in the presence or absence of DA. DA decreased NKA activity in PTC from young animals (67.2±3.8 vs 32.7±5.3) but not in PTC from old animals. Transfection of NHERF1 restored NKA regulation by DA in PTC from old rats (58.4±4.2 vs 64.4±4.3 (in untransfected cells) 54.2±3.8 vs 31.1±3.4 (in NHERF1 transfected cells)). We conclude that NHERF1 regulates DA-mediated proximal tubule sodium handling; however, other factors modulate BP response to dietary sodium intake in young and old animals. The contribution of NHERF1 and dopamine signaling to sodium homeostasis requires further study.

The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.

In the brush border of intestinal and kidney epithelial cells, scaffolding proteins ezrin, Na(+)-H(+) exchanger regulatory factor (NHERF)1 and NHERF2 play important roles in linking transmembrane proteins to the cytoskeleton and assembling signalling regulatory complexes. The last 30 carboxyl residues of NHERF1 and NHERF2 form the EBDs [ezrin, radixin and moesin (ERM)-binding domain]. The current study found that NHERF1/2 contain an ERM-binding regulatory sequence (EBRS), which facilitates the interaction between the EBD and ezrin. The EBRSs are located within 24 and 19 residues immediately upstream of EBDs for NHERF1 and NHERF2 respectively. In OK (opossum kidney) epithelial cells, EBRSs are necessary along with the EBD to distribute NHERF1 and NHERF2 exclusively to the apical domain. Furthermore, phosphorylation of Ser(303) located in the EBRS of NHERF2, decreases the binding affinity for ezrin, dislocates apical NHERF2 into the cytosol and increases the NHERF2 microvillar mobility rate. Moreover, increased phosphorylation of Ser(303) was functionally significant preventing acute stimulation of NHE3 (Na(+)-H(+) exchanger 3) activity by dexamethasone.

Mo1763 Sorting Nexin 27 (SNX27) Is a Member of NHERF (Na+/H+ Exchanger Regulatory Factor) Family of PDZ Adaptor Proteins

Mo1763 Sorting Nexin 27 (SNX27) Is a Member of NHERF (Na+/H+ Exchanger Regulatory Factor) Family of PDZ Adaptor Proteins

CGMP-dependent kinase 2, Na+/H+ regulatory factor 2, and Na+/H+ exchanger isoform 3 assemble within lipid rafts in murine small intestinal brush border membrane

Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of the very homologous NHERFs. We studied the association of NHE3, as well as NHERF1 (EBP50), NHERF2 (E3KARP) and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM and their possible association with signaling molecules. NHE3 was found to partially associate with glycosphingolipid-enriched microdomains in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity as well as on second-messenger-dependent regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. A search for other signalling molecules that are implicated in regulating NHE3 through a NHERF interaction, cGMP-dependent kinase II, which together with NHERF2 is essential for guanylin/heat stable enterotoxin of E.coli (STa)-mediated NHE3 inhibition in the intestine, was found exclusively lipid-raft associated. In conclusion, the differential association of the NHERFs, as well as kinases, with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is likely one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

NHERF2 Protein Mobility Rate Is Determined by a Unique C-terminal Domain That Is Also Necessary for Its Regulation of NHE3 Protein in OK Cells

Na(+)/H(+) exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.

The membrane cytoskeletal crosslinker ezrin is essential for the regulation of phosphate and calcium homeostasis

Ezrin is a cross‐linker between the plasma membrane proteins and actin cytoskeleton. In the kidney, ezrin is mainly localized at brush border membrane of proximal tubules with a scaffolding protein, Na+/H+ exchanger regulatory factor (NHERF) 1. NHERF1 interacts with Na+/phosphate (Pi) cotransporter Npt2a. We focused on the physiological role of ezrin in the renal Pi reabsorption by using ezrin knockdown mice (Vil2kd/kd). The Vil2kd/kd mice exhibited hypophosphatemia, hypocalcemia, and osteomalacia. The reduced plasma concentration of Pi was attributed to the defective urinary Pi reabsorption. Immunofluorescence staining and immunoblotting revealed that the Npt2a and NHERF1 expressions at apical membrane of proximal tubules were markedly reduced in the kidney of Vil2kd/kd mice. On the other hand, urinary loss of Ca2+ was not observed in Vil2kd/kd mice. Plasma concentration of 1,25‐dihydroxyvitamin D was elevated following reduced plasma Pi level. Furthermore, bone mineral density in tibiae was significantly lowered in the adult and young Vil2kd/kd mice. These results suggest that ezrin is required for the regulation of systemic Pi homeostasis in vivo.

Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft- and the Non-Raft Brush Border Membrane Fractions of NHE3

Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na⁺/H⁺ exchanger NHE3 is regulated by the Na⁺/H⁺ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes (“lipid rafts”) were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO₂/HCO₃^- mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

NRFL-1, the C. elegans NHERF orthologue, interacts with amino acid transporter 6 (AAT-6) for age-dependent maintenance of AAT-6 on the membrane.

The NHERF (Na+/H+ exchanger regulatory factor) family has been proposed to play a key role in regulating transmembrane protein localization and retention at the plasma membrane. Due to the high homology between the family members, potential functional compensations have been a concern in sorting out the function of individual NHERF numbers. Here, we studied C. elegans NRFL-1 (C01F6.6) (nherf-like protein 1), the sole C. elegans orthologue of the NHERF family, which makes worm a model with low genetic redundancy of NHERF homologues. Integrating bioinformatic knowledge of C. elegans proteins into yeast two-hybrid scheme, we identified NRFL-1 as an interactor of AAT-6, a member of the C. elegans AAT (amino acid transporter) family. A combination of GST pull-down assay, localization study, and co-immunoprecipitation confirmed the binding and characterized the PDZ interaction. AAT-6 localizes to the luminal membrane even in the absence of NRFL-1 when the worm is up to four-day old. A fluorescence recovery after photobleaching (FRAP) analysis suggested that NRFL-1 immobilizes AAT-6 at the luminal membrane. When the nrfl-1 deficient worm is six-day or older, in contrast, the membranous localization of AAT-6 is not observed, whereas AAT-6 tightly localizes to the membrane in worms with NRFL-1. Sorting out the in vivo functions of the C. elegans NHERF protein, we found that NRFL-1, a PDZ-interactor of AAT-6, is responsible for the immobilization and the age-dependent maintenance of AAT-6 on the intestinal luminal membrane.

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.

Biochemical and Biophysical Characterizations of the Interaction between Two PDZ Adapter Proteins NHERF and E3KARP in vitro

NHERF (Na + /H + exchanger regulatory factor) and E3KARP (NHE3 kinase A regulatory protein) play important roles in membrane targeting, trafficking and sorting of ion channels, transmembrane receptors and signaling proteins in many tissues. Each of these proteins contains two PDZ (PSD-95/Dlg-1/ZO-1) domains, which mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. The interaction between NHERF and E3KARP was investigated by surface plasmon resonance spectroscopy (BIAcore), fluorescence measurement, His-tagged pull-down experiment, and size-exclusion column (SEC) chromatography. BIAcore experiments revealed that NHERF bound to E3KARP with an apparent K D of 7 nM. Fluorescence emission spectra of the NHERF-E3KARP complex suggested that the tight interaction between these proteins was accompanied by significant conformational changes in one or both. The CD spectra ofNHERF and E3KARP show that the conformational changes of these proteins were dependent on pH and temperature. These results implicate that the NHERF-E3KARP complex allows intracellular signaling complexes to form through PDZ-PDZ interactions.

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

Elevated intracellular Ca(2+) ([Ca(2+)](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 [Ca(2+)](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 Ca(2+) ionophore, 4-bromo-A23187 (0.5 mum): 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 [Ca(2+)](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 [Ca(2+)](i) inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca(2+)](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 Ca(2+).

Regulation of the Intestinal Anion Exchanger DRA (Downregulated in Adenoma)

Barrier function and transepithelial transport are intimately linked and are sometimes disturbed in parallel. DRA (downregulated in adenoma) is an intestinal chloride/bicarbonate exchanger that is functionally coupled to CFTR (cystic fibrosis transmembrane regulator) in the upper gastrointestinal tract to mediate chloride and bicarbonate secretion and to NHE3 (Na/H exchanger- isoform 3) in the lower gastrointestinal tract to mediate electroneutral NaCl absorption. All three transport proteins possess PDZ domain binding motifs that facilitate binding to members of the NHERF (Na/H exchanger regulatory factor) family of adapter proteins [NHERF, E3KARP (NHE3 kinase A regulatory protein), PDZK1 (PDZ protein kidney 1) and IKEPP (intestinal and kidney enriched PDZ protein)]. Regulation of DRA appears to depend on the presence of a partner transport protein, and this may involve the assembly of different complexes of transporters, adapter proteins, and signaling molecules. We have established stable expression of DRA in HEK cells. In these cells, that do not express significant amounts of CFTR or NHE3, DRA is inhibited by intracellular calcium but not by protein kinase C or protein kinase A. At high calcium concentrations induced by 4Br-A23187 this inhibition is independent of the PDZ interaction of DRA. These data show that DRA can be individually regulated and may be confirmed in a more physiologically relevant expression system (i.e., Caco-2/BBE cells) using natural agonists of the intracellular calcium signal.

GABARAP deficiency modulates expression of NaPi-IIa in renal brush-border membranes.

Renal reabsorption of inorganic phosphate (P(i)) is mainly mediated by the Na(+)-dependent P(i)-cotransporter NaPi-IIa that is expressed in the brush-border membrane (BBM) of renal proximal tubules. Regulation and apical expression of NaPi-IIa are known to depend on a network of interacting proteins. Most of the interacting partners identified so far associate with the COOH-terminal PDZ-binding motif (TRL) of NaPi-IIa. In this study GABA(A) receptor-associated protein (GABARAP) was identified as a novel interacting partner of NaPi-IIa applying a membrane yeast-two-hybrid system (MYTH 2.0) to screen a mouse kidney library with the TRL-truncated cotransporter as bait. GABARAP mRNA and protein are present in renal tubules, and the interaction of NaPi-IIa and GABARAP was confirmed by using glutathione S-transferase pulldowns from BBM and coimmunoprecipitations from transfected HEK293 cells. Amino acids 36-68 of GABARAP were identified as the determinant for the described interaction. The in vivo effects of this interaction were studied in a murine model. GABARAP(-/-) mice have reduced urinary excretion of P(i), higher Na(+)-dependent (32)P(i) uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with GABARAP(+/+) mice. The expression of Na(+)/H(+) exchanger regulatory factor (NHERF)1, an important scaffold for the apical expression of NaPi-IIa, is also increased in GABARAP(-/-) mice. The absence of GABARAP does not interfere with the regulation of the cotransporter by either parathyroid hormone or acute changes of dietary P(i) content.

NHERF1/EBP50 Head-to-Tail Intramolecular Interaction Masks Association with PDZ Domain Ligands

Loss of cell polarity is one of the initial alterations in the development of human epithelial cancers. Na(+)/H(+) exchanger regulatory factor (NHERF) homologous adaptors 1 and 2 are membrane-associated proteins composed of two amino (N)-terminal PDZ domains and an ezrin-radixin-moesin (ERM)-binding (EB) carboxyl (C)-terminal region. We describe here an intramolecular conformation of NHERF1/EBP50 (ERM-binding phosphoprotein 50) in which the C-terminal EB region binds to the PDZ2 domain. This novel head-to-tail conformation masked the interaction of both PDZ domains with PDZ domain-specific ligands, such as PTEN and beta-catenin. An EB region composite structure comprising an alpha-helix ending in a PDZ-binding motif imparted opposite effects to NHERF1 associations, mediating binding to ERM proteins and inhibiting binding of PDZ domain ligands. The PDZ domain inhibition was released by prior association of ezrin with the EB region, a condition that occurs in vivo and likely disrupts NHERF1 head-to-tail interaction. In contrast, NHERF2 did not present a regulatory mechanism for protein complex formation. Functionally, NHERF1 is required to organize complexes at the apical membranes of polarized epithelial cells. The regulation of NHERF1 interactions at the apical membrane thus appears to be a dynamic process that is important for maintaining epithelial-tissue integrity.