Defective jejunal and colonic salt absorption and alteredNa(+)/H (+) exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice.

We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting.

Na+/H+ exchanger regulatory factor (NHERF)-1 and NHERF-2, two structurally related protein adapters containing tandem PSD-95/Discs large/ZO-1 (PDZ) domains, were identified as essential factors for protein kinase A-mediated inhibition of the sodium-hydrogen exchanger, NHE3. NHERF-1 and NHERF-2 also bound other cellular targets including the sodium-phosphate cotransporter type IIa encoded by the NPT2 gene. Targeted disruption of the mouse NHERF-1 gene eliminated NHERF-1 expression in kidney and other tissues of the mutant mice without altering NHERF-2 levels in these tissues. NHERF-1 (+/-) and (-/-) male mice maintained normal blood electrolytes but showed increased urinary excretion of phosphate when compared with wild-type (+/+) animals. Although the overall levels of renal NHERF-1 targets, NHE3 and Npt2, were unchanged in the mutant mice, immunocytochemistry showed that the Npt2 protein was aberrantly localized at internal sites in the renal proximal tubule cells. The mislocalization of Npt2 paralleled a reduction in the transporter protein in renal brush-border membranes isolated from the mutant mice. In contrast, NHE3 was appropriately localized at the apical surface of proximal tubules in both wild-type and mutant mice. These data suggested that NHERF-1 played a unique role in the apical targeting and/or trafficking of Npt2 in the mammalian kidney, a function not shared by NHERF-2 or other renal PDZ proteins. Phosphate wasting seen in the NHERF-1(-/-) null mice provided a new experimental system for defining the role of PDZ adapters in the hormonal control of ion transport and renal disease.

Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3.

1. Rabbit ileal Na+-absorbing cell Na+-H+ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50% of BB NHE3 was associated with Triton X-100-soluble fractions and the other approximately 50% with Triton X-100-insoluble fractions; approximately 33% of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts). 2. The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na+-H+ exchange activity. 3. Disrupting rafts by removal of cholesterol with methyl-beta-cyclodextrin (MbetaCD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and MbetaCD treatment. 4. We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF- and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3.

Expanding the role of NHERF, a PDZ-domain containing protein adapter, to growth regulation.

NHERF (Na+/H+ exchanger regulatory factor or NHERF-1) and E3KARP (NHE3 kinase A regulatory protein or NHERF-2) are structurally related protein adapters that are highly expressed in epithelial tissues. NHERF proteins contain two tandem PDZ domains and a C-terminal sequence that binds several members of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal adapters. Although identified as a regulator of NHE3, recent evidence points to a broadening role for NHERF in the function, localization and/or turnover of G-protein coupled receptors, platelet-derived growth factor receptor and ion transporters such as CFTR, Na/Pi cotransporter, Na/HCO3 cotransporter and Trp (calcium) channels. NHERF also recruits non-membrane proteins such as the c-Yes/YAP-65 complex, members of the phospholipase Cbeta family and the GRK6A protein kinase to apical surface of polarized epithelial cells where they regulate or respond to membrane signals. While two distinct models have been proposed for NHERF's role in signal transduction, the common theme is NHERF's ability to bring together membrane and non-membrane proteins to regulate cell metabolism and growth. NHERF overexpression in human breast cancers and mutations in NHERF targets, such as CFTR and merlin, the product of Neurofibromatosis NF2 tumor suppressor gene, that impair NHERF binding suggest that aberrant NHERF function contributes to human disease.

Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO3- co-transporter in BSC-1 cells.

Prior studies have indicated a requirement for the PDZ domain-containing protein, Na(+)/H(+) Exchanger Regulatory Factor (NHERF), for protein kinase A (PKA)-mediated inhibition of the renal basolateral Na(+)-HCO(3)(-) co-transporter (NBC). The present studies explore the potential mechanisms by which NHERF transduces cAMP signals to inhibit NBC. In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 microm for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was expressed. mNHERF-(116-355) containing PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylated form of rabbit NHERF effectively transduced the cAMP signals that inhibited NBC. By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNHERF-(1-325), which lacks ezrin-binding, failed to support cAMP inhibition of NBC activity. NBC and NHERF did not associate with each other in yeast two-hybrid or co-immunoprecipitation assays, and confocal microscopy indicated distinct subcellular localization of the two proteins. NBC was phosphorylated in BSC-1 cells, but its phosphorylation was not increased by cAMP nor was immunoprecipitated NBC phosphorylated by PKA in vitro. Acute exposure of mNHERF-(1-355)-expressing BSC-1 cells to cAMP did not change cell surface expression of NBC. Although these results established an essential role for NHERF in cAMP-mediated inhibition of NBC in BSC-1 cells, they also suggest a novel mechanism for NHERF-mediated signal transduction distinct from that previously characterized from studies of other NHERF targets.

Acute regulation of NHE3 by protein kinase A requires a multiprotein signal complex.

Biochemical and cellular experiments in fibroblasts have established the requirement for a member of the PDZ motif Na(+)/H(+) exchanger regulatory factor family of proteins (NHERF and NHERF2) in cAMP-mediated phosphorylation and inhibition of NHE3 activity. NHERF interacts with the actin cytoskeleton through the scaffolding protein ezrin to target a multiprotein signal complex to the plasma membrane. Recent experiments have focused on elements of this model. First, using specific antibodies, NHERF was identified in the renal proximal tubule, where it colocalized with ezrin and NHE3. NHERF2 was seen in glomeruli, the renal vasculature, and collecting duct cells, where it colocalized with ROMK. This distinct nephron localization suggests different physiologic roles for NHERF and NHERF2. Second, the signal-complex model of protein kinase A regulation of NHE3 developed in fibroblasts has been extended to epithelial cells by the development of a dominant-negative opossum kidney cell line expressing an ezrin binding domain-deficient truncation of NHERF. Preliminary studies indicate that these cells have normal basal Na+/H+ exchanger activity but a blunted inhibitory response to cAMP. Third, biochemical, biophysical, and cell experiments have indicated that NHERF binds to itself in a head-to-head configuration, raising the possibility that dimerization may alter the availability of active NHERF. The potential role of the NHERF proteins in the kidney has been expanded by recent studies indicating their involvement in the membrane targeting, trafficking, sorting, and regulation of a range of other transporters, receptors, and signaling proteins. NHERF and related PDZ-containing proteins may serve as adapters for regulation of renal transporters.

Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition of Na(+)/H(+) exchange in OK cells.

Na(+)/H(+) exchanger regulatory factor (NHERF), an essential protein cofactor in cAMP-mediated inhibition of Na(+)/H(+) exchange transporter 3 (NHE3), facilitates the formation of a signal complex of proteins that includes NHE3, NHERF, and ezrin. This model for NHE3 regulation was developed in fibroblasts and its applicability to epithelial cells remains to be established. Opossum kidney (OK) cells were transfected with either empty vector (control), full-length mouse (m) NHERF(1-355), or a truncated mNHERF(1-325) that lacked ezrin binding and had been demonstrated in fibroblasts to bind NHE3 but not mediate its cAMP-associated inhibition. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) at 10(-4) M inhibited Na(+)/H(+) exchange activity in control and OK cells expressing wild-type mNHERF(1-355) by >60% but by <10% in cells expressing mNHERF(1-325). NHE3 coimmunoprecipitated with mNHERF(1-325), but cAMP phosphorylation of NHE3 was impaired in cells expressing mNHERF(1-325). The inhibitory effect of hyperosmolality on NHE3 activity and the uptake of 3-O-methyl-D-glucose was the same in all three cell lines. Cell surface expression of NHE3 was not changed by cAMP in any of the cells lines. These data indicate that disruption of the NHERF-ezrin signal complex attenuates the inhibitory effect of cAMP on NHE3 activity in OK cells and provides evidence supporting the proposed model of protein kinase A regulation of NHE3 in epithelial cells.

NHERF: targeting and trafficking membrane proteins.

Vectorial ion transport initiated by Na+/H+ exchanger isoform 3 (NHE3) mediates the reabsorption of NaCl and NaHCO(3) in renal proximal tubule cells. NHE3 activity is modulated by numerous physiological stimuli. Biochemical and cellular experiments identified Na+/H+ exchanger regulatory factor (NHERF) as a protein cofactor essential for cAMP-mediated inhibition of NHE3 activity. Identification of numerous NHERF targets, including several transmembrane receptors and ion transporters, has broadened the role of this PSD-95/Dlg-1, Drososphila disk large/ZO-1 domain-containing adapter protein in membrane physiology. NHERF also associates with members of the ezrin/radixin/moesin family of actin-binding proteins and thus links NHE3 to the actin cytoskeleton. Formation of this multiprotein complex facilitates NHE3 phosphorylation and hormonal control of Na+/H+ exchange. NHERF also plays a critical role in targeting transport proteins to apical membranes. Moreover, the NHERF signaling complex functions as a regulatory unit to control endocytosis and internal trafficking of membrane proteins. This article reviews the new evidence that implicates NHERF in wider aspects of epithelial membrane biology.

N-terminal PDZ domain is required for NHERF dimerization.

NHERF, a 55 kDa PDZ-containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time- and concentration-dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N-terminal PDZ-I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling.

Differential renal distribution of NHERF isoforms and their colocalization with NHE3, ezrin, and ROMK.

Na(+)/H(+) exchanger regulatory factor (NHERF) and NHERF2 are PDZ motif proteins that mediate the inhibitory effect of cAMP on Na(+)/H(+) exchanger 3 (NHE3) by facilitating the formation of a multiprotein signaling complex. With the use of antibodies specific for NHERF and NHERF2, immunocytochemical analysis of rat kidney was undertaken to determine the nephron distribution of both proteins and their colocalization with other transporters and with ezrin. NHERF was most abundant in apical membrane of proximal tubule cells, where it colocalized with ezrin and NHE3. NHERF2 was detected in the glomerulus and in other renal vascular structures. In addition, NHERF2 was strongly expressed in collecting duct principal cells, where it colocalized with ROMK. These results indicate a striking difference in the nephron distribution of NHERF and NHERF2 and suggests NHERF is most likely to be the relevant biological regulator of NHE3 in the proximal tubule, while NHERF2 may interact with ROMK or other targets in the collecting duct. The finding that NHERF isoforms occur in different cell types suggests that NHERF and NHERF2 may subserve different functions in the kidney.

Signal complex regulation of renal transport proteins: NHERF and regulation of NHE3 by PKA.

The activity of the sodium/hydrogen exchanger 3 (NHE3) isoform of the sodium/hydrogen exchanger in the brush-border membrane of the renal proximal tubule is tightly regulated. Recent biochemical and cellular experiments have established the essential requirement for a new class of regulatory factors, sodium/hydrogen exchanger regulatory factor (NHERF) and NHERF-like proteins, in cAMP-mediated inhibition of NHE3 activity. NHERF is the first PSD-95/Dlg/ZO-1 (PDZ) motif-containing protein localized to apical membranes and appears to facilitate cAMP-dependent protein kinase A (PKA) phosphorylation of NHE3 by interacting with the cytoskeleton to target a multiprotein complex to the brush-border membrane. Other recent experiments have indicated that NHERF also regulates the activity of other renal transport proteins, suggesting that the signal complex model of signal transduction in the kidney may be more common than presently appreciated. This article reviews studies on the regulation of NHE3 by NHERF, PKA, and ezrin and introduces the concept of regulation of renal transporters by signal complexes. Although not the primary focus of this review, recent studies have indicated a role for NHERF in membrane targeting, trafficking, and sorting of transporters, receptors, and signaling proteins. Thus NHERF and related PDZ-containing proteins appear to be essential adapters for regulation of renal transporters in the mammalian kidney that maintain salt and water balance.

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

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

Assembly of signaling complexes by the sodium-hydrogen exchanger regulatory factor family of PDZ-containing proteins.

The sodium-hydrogen exchanger regulatory factor (NHERF) was first identified as an essential cofactor for cyclic AMP-mediated inhibition of the epithelial isoform of rabbit kidney sodium-hydrogen exchanger (NHE3). More recent work shows that NHERF constitutes a family of PSD-95/DIg/ZO-1 (PDZ) domain-containing adapter proteins, only some of which associate with the NHE3 antiporter. Other targets of the NHERF proteins include members of the ezrin-radixin-moesin family of cytoskeletal proteins. In the current model for NHE3 regulation, NHERF links NHE3 to the protein kinase A-anchoring protein, ezrin, and thereby facilitates its phosphorylation and inhibition by protein kinase A. Recent studies have also established the interaction of NHERF and its homologs with the beta2-adrenergic receptor and the platelet-derived growth factor receptor tyrosine kinase that facilitates signal transduction by these receptors. Association with NHERF may also regulate the cystic fibrosis transmembrane conductance regulator and the sodium-bicarbonate transporter. With the rapid increase in the intracellular targets identified for NHERF, the emerging data point to a broad role for these PDZ-containing proteins in the organization of signaling complexes and control of cell physiology.

Molecular cloning of the cDNA and promoter sequences for the mouse sodium-hydrogen exchanger regulatory factor.

The Na/H exchanger regulatory factor (NHE-RF) was first identified as a co-factor for cAMP dependent protein kinase regulation of the rabbit epithelial Na/H exchanger. Subsequently, this protein which contains two PDZ motifs, was shown to interact with multiple cellular targets. To understand more fully the function of NHE-RF and its regulation, we have cloned the full-length cDNA for mouse NHE-RF and a portion of the mouse gene containing the promoter elements. NHE-RF cDNA, isolated from a mouse kidney cDNA library, predicted a polypeptide of 356 amino acids that shares striking sequence conservation within the two PDZ domains and in-vitro phosphorylation sites with the human and rat homologs. The nucleotide sequence 5' of the transcription start site, identified by primer extension analysis, was highly 'GC' rich and lacked canonical TATA or CAAT sequences. Using a luciferase reporter construct, deletion analyses localized the critical segment for gene expression in mouse medullary thick ascending limb cells to 114 bp 5' of the transcription start site. Although NHE-RF has been recently identified as an estrogen-inducible gene, the lack of an estrogen-response element in the mouse NHE-RF 5'-non-coding-sequence and the inability to demonstrate estrogen stimulation of reporter gene expression in MCF-7 cells suggests a non-conventional or indirect mechanism for NHE-RF regulation by estrogen.

Regulation of the Na/H exchanger regulatory factor in OK cells.

The Na/H exchanger regulatory factor (NHE-RE), a recently cloned renal protein, is a necessary cofactor in protein kinase A-mediated inhibition of the renal brush border membrane Na/H exchanger. No studies to date, however, have examined the regulation of NHE-RF itself. The rabbit NHE-RF cDNA and an antibody to rabbit NHE-RF were used to study the effects of serum and cyclic adenosine monophosphate (cAMP) on the steady-state levels of NHE-RF mRNA and on the abundance and intracellular distribution of the protein in OK cells. Incubation of quiescent cells with serum was associated with a significant decrease in steady-state NHE-RF mRNA and protein abundance in the cytosolic and membrane fractions. Incubation of cells with cAMP for 6 h was associated with no change in NHE-mRNA at 24 h. There was, however, a 46% increase in protein abundance in the cytosolic fraction of the cell and a 43% decrease in the membrane fraction. Despite the decrease in membrane-associated NHE-RF in quiescent cells treated with serum of cAMP, there were no differences in either the basal rate of Na/H exchange transport or the inhibitory effect of the acute addition of cAMP on the transporter between experimental and control cells. These studies provide the first description of the regulation of NHE-RF. The results indicate that serum is associated with a decrease in NHE-RF mRNA and protein, while chronic exposure to cAMP is associated with an altered distribution of NHE-RF between the cytosolic and membrane fractions of OK cells.

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

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

Basolateral Na(+)/HCO(3)(-) cotransport activity is regulated by the dissociable Na(+)/H(+) exchanger regulatory factor.

In the renal proximal tubule, the activities of the basolateral Na(+)/HCO(3)(-) cotransporter (NBC) and the apical Na(+)/H(+) exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif-containing protein, the Na(+)/H(+) exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10(-8)M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-RF-expressing B-SC-1 cells (B-SC-RF) exhibited markedly lower basal levels of NBC activity than did wild-type controls. Inhibition of NBC activity in B-SC-RF cells was enhanced after 10 microM of forskolin treatment, consistent with a postulated role for NHE-RF in mediating the inhibition of NBC activity by PKA. These findings not only suggest NHE-RF involvement in PKA-regulated NBC activity, but also provide a unique molecular mechanism whereby basolateral NBC and apical NHE3 activities may be coordinately regulated in renal proximal tubule cells.

Estrogen receptor regulation of the Na+/H+ exchange regulatory factor.

To better understand the actions of estrogens and antiestrogens in estrogen target cells, we have searched for estrogen-regulated genes in human breast cancer cells, in which the number of genes known to be directly activated by estrogen is quite small. Using differential display RNA methods, we have identified the human homolog of the Na+ -H+ exchanger regulatory factor (NHE-RF), an approximately 50-kDa protein that is also an ezrin-radixin-moesin-binding phosphoprotein, as being under rapid and direct regulation by estrogen in estrogen receptor (ER)-containing breast cancer cells. Stimulation by estrogen of NHE-RF RNA is rapid, being near maximal (approximately 6-fold) by 1 h, and is not blocked by cycloheximide, indicating that it is a primary response. Stimulation is selective for estrogen ligands, with no stimulation by other classes of steroid hormones, and stimulation by estrogen is suppressed by the antiestrogens tamoxifen and ICI 182,780. Induction is shown to require an active ER through several approaches, including the use of ER-negative breast cancer cells containing a stably integrated ER. NHE-RF protein levels, monitored using antibodies specific for this protein, increase after estrogen and reach maximal levels at 24-48 h. Interestingly, NHE-RF is a PDZ domain-containing protein that is enriched in polarized epithelia, where it is known to be localized in microvilli. Among various human tissues we have examined, we found that NHE-RF is expressed at a fairly high level in mammary tissue. NHE-RF regulates protein kinase A inhibition of the Na+ -H+ exchanger and may serve as a scaffold adaptor protein that contributes to the specificity of signal transduction events. Our findings suggest that the early, known effects of estrogen on cell cytoarchitecture (e.g. increasing microvilli on breast cancer cells) and on some cell signaling pathways (e.g. those involving cAMP) may involve rapid estrogen-mediated changes in the production of NHE-RF.