The role of NHERF-1 in the regulation of renal proximal tubule sodium–hydrogen exchanger 3 and sodium-dependent phosphate cotransporter 2a
1 Department of Medicine
2 Department of Physiology University of Maryland School of Medicine
3 Medical Service, Department of Veterans Affairs Medical Center, Baltimore, MD 21201, USA
4 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
Abstract
Adaptor proteins containing PDZ interactive domains have been recently identified to regulate the trafficking and activity of ion transporters and channels in epithelial tissue. In the renal proximal tubule, three PDZ adaptor proteins, namely NHERF-1, NHERF-2 and PDZK1, are expressed in the apical membrane, heterodimerize with one another, and, at least in vitro, are capable of binding to NHE3 and Npt2a, two major regulated renal proximal tubule apical membrane transporters. Studies using NHERF-1 null mice have begun to provide insights into the organization of these adaptor proteins and their specific interactions with NHE3 and Npt2a. Experiments using brush border membranes and cultured renal proximal tubule cells indicate a specific requirement for NHERF-1 for cAMP-mediated phosphorylation and inhibition of NHE3. NHERF-1 null mice demonstrate increased urinary excretion of phosphate associated with mistargeting of Npt2a to the apical membrane of renal proximal tubule cells. NHERF-1 null animals challenged with a low phosphate diet and proximal tubule cells from these animals cultured in a low phosphate media fail to adapt as well as wild-type mice. These studies indicate a unique requirement for NHERF-1 in cAMP regulation of NHE3 and in the trafficking of Npt2a.
, 百拇医药
Introduction
The abundance and activity of epithelial transporters and ion channels is tightly regulated and it is likely that these membrane proteins exist as multiprotein complexes. Our recent work has focused on the organization of three PSD-95/Drosophila Discs large/ZO-1 (PDZ) adaptor proteins, namely the Na+–H+ exchanger regulatory factor or NHERF-1, NHERF-2 and PDZK1 in the regulation and targeting of the sodium–hydrogen exchanger isoform 3 (NHE3) and the sodium-dependent phosphate transporter 2a (Npt2a), two important regulated transport proteins in renal proximal convoluted tubule cells.
, 百拇医药
In 1995 we isolated and cloned a protein that was a necessary cofactor for cAMP regulation of NHE3 activity initially called NHERF (also known as ezrin-binding phosphoprotein of 50 kDa (EBP50)), later renamed NHERF-1 (Weinman et al. 1993, 1995). A second member of the NHERF family was identified, NHERF-2 (also known as NHE-3 kinase A-regulatory protein (E3KARP), tyrosine kinase activator-1 (TKA1), and sex-determining region of the Y chromosome (SRY-1)-interacting protein-1 (SIP-1)) in a yeast 2-hybrid screen using the NHE3 C-terminus as bait (Yun et al. 1997). NHERF-1 and NHERF-2 are encoded by unique genes, contain no recognizable catalytic domains, and share approximately 52% over all amino acid homology. They are considered members of a protein family by virtue of their common modular structure with two tandem PDZ homology domains and a C-terminal domain that binds all members of the ezrin–radixin–moesin (ERM) family of structural proteins (Reczek et al. 1997). PDZK1 (PDZ containing 1, also known as NaPiCap1 or CAP70) was identified using differential display PCR in studies of phosphate-deprived rats. PDZK1 contains four PDZ domains but unlike the NHERF proteins, lacks the C-terminal ERM domain (Custer et al. 1997). NHERF-1, NHERF-2 and PDZK1 are expressed in the apical side of renal proximal tubule cells and in the small intestines (Wade et al. 2001, 2003). NHERF-1 and NHERF-2 form homodimers and all three proteins heterodimerize with one another (Fouassier et al. 2000; Lau et al. 2001; Shenolikar et al. 2001; Gisler et al. 2003). It has been proposed that they array a microvillar/submicrovillar mesh or scaffold that regulates its target proteins. NHE3 and Npt2a appear to bind to all three of these adaptor proteins (Lamprecht et al. 1998; Zizak et al. 1999; Gisler et al. 2001; Hernando et al. 2002). A vexing question relates to whether NHERF-1, NHERF-2 and PDZK1 represent a redundant physiological control mechanism, function independently of one another, or act cooperatively in the regulation of target proteins.
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In this review, we will trace the evolution of our thinking about the role of NHERF-1, NHERF-2 and PDZK1 in the regulation of NHE3 and Npt2a, and the renal tubular reabsorption of sodium and phosphate. We initially believed these adaptor proteins had overlapping functions based on results from model cell systems (Yun et al. 1997). Our more recent experiments, particularly studies using NHERF-1 null mice, have led us to consider that the organization of these adaptor proteins in native tissues such as the renal proximal tubule importantly influences the regulation of NHE3 and Npt2a (Shenolikar et al. 2002). These studies highlight the fact that model cell systems, while providing important information about biochemical interactions between proteins, must be supported by studies in native tissues where the interactions between these adaptor proteins and their targets may differ.
, 百拇医药
NHERF-1, NHERF-2 and the regulation of NHE3 in PS120 cell fibroblasts
Initial study of the cellular function of NHERF-1 and NHERF-2 was undertaken in PS120 cell fibroblasts, a cell line negatively selected to express no endogenous NHE activity (Yun et al. 1997). This cell line also does not express endogenous NHERF-1 or NHERF-2. Rabbit NHE3 was readily expressed in these cells but treatment with cAMP failed to regulate NHE3 activity. When NHERF-1 or NHERF-2 were coexpressed with NHE3, however, cAMP inhibition of NHE3 activity was observed. These studies established that NHERF-1 or NHERF-2 was required for protein kinase A (PKA) associated down-regulation of NHE3 activity. The biochemical bases for the effect of the NHERF proteins was elucidated by the demonstration that NHERF-1 and NHERF-2 bound to NHE3 and to ezrin. Ezrin, acting as a protein kinase A anchor protein, recruited PKA thereby facilitating the rapid phosphorylation of specific serine residues in the C-terminus of NHE3 with a consequent down-regulation of the activity of the transporter (Lamprecht et al. 1998; Zizak et al. 1999). The model that emerged was that the NHERF proteins were required for the formation of a multiprotein complex and that the binding of NHERF to both NHE3 and ezrin was critical for this type of hormonal regulation (Weinman et al. 2000). In PS120 cells, NHERF-1 and NHERF-2 were equally effective in mediating cAMP inhibition of NHE3 activity (Yun et al. 1997).
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Localization of NHERF-1 and NHERF-2 in the kidney of rodents and man
Highly specific antipeptide antibodies that differentiated NHERF-1 from NHERF-2 were used to determine the tissue distribution of the NHERF isoforms. Initial studies were performed using the rat kidney (Wade et al. 2001). Although both isoforms were detected in specific cells of the nephron, NHERF-1 but not NHERF-2 was readily detected in the apical membrane of renal proximal tubule cells and we suggested that NHERF-1 was the relevant isoform mediating hormonal regulation of NHE3 activity. Additional studies in man and mouse, however, indicated the presence of both NHERF isoforms in the renal proximal tubule (Wade et al. 2003; Weinman et al. 2003b). As shown in Fig. 1, the distribution of NHERF-1 and NHERF-2 in mice was not identical. NHERF-1 was expressed in the microvillar membrane and colocalized with NHE3 and Npt2a. NHERF-2, on the other hand, was expressed predominantly in the submicrovillar region of renal proximal tubule cells but only minimally in the microvillus itself. This distribution was confirmed using immunogold staining and electron microscopy. The factors that determine the unique distribution of the NHERF isoforms are unknown but when considered from a physiological perspective, the findings that both NHERF-1 and NHERF-2 were expressed in renal proximal tubule cells necessitated a reconsideration of the question of the roles of these proteins in control of NHE3 and Npt2a activity and abundance.
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A, representative confocal images of proximal convoluted tubules of the mouse kidney using antibodies specific for NHERF-1 (a) and NHERF-2 (b). c is the merged images showing the localization of NHERF-1 in the microvillar membranes and NHERF-2 predominantly in a region just below the microvilli. B, electron microscopical images of wild-type renal proximal tubules using immunogold showing the localization of NHERF-1 in the microvillus (a) and NHERF-2 in vesicle-rich region at the base of the microvilli (b). (Figures adapted from Wade et al. 2003.)
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Development of the NHERF-1–/– mouse
The potential complexity of the interactions between NHERF-1 and NHERF-2 with each other and with other PDZ scaffolding proteins in renal cells suggested that a genetic approach would be required to determine the individual role of each of these proteins on sodium and phosphate transport in the proximal tubule of the kidney. Using homologous recombination and a vector targeting exon 1 of the mouse NHERF-1 gene, we successfully abolished NHERF-1 expression in all mouse tissues (Shenolikar et al. 2002). Male NHERF-1–/– mice displayed no overt phenotype. Blood pressure, serum electrolytes, renal function and renal histology were normal. However, mutant male mice demonstrated mild hypophosphatemia and, as compared with wild-type mice, increased urinary excretion of phosphate. Some, but not all, NHERF-1 null female mice were runts, displayed severe osteoporosis and bone fractures, and died shortly after weaning. The more normal appearing females were used for breeding to establish an NHERF-1 null mouse colony. The availability of NHERF-1–/– mice permitted an assessment of the relative contribution of NHERF-1 and NHERF-2 to the regulation of NHE3 and Npt2a.
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NHERF-1 uniquely regulates cAMP-associated inhibition of NHE3 activity
As determined using tissue fractionation and confocal microscopy, the expression of NHE3 in the renal apical membrane did not differ between wild-type and NHERF-1–/– mice suggesting that NHERF-1 did not affect the trafficking of NHE3 (Shenolikar et al. 2002). Moreover, the abundance and cellular distribution of NHERF-2 was not affected by the absence of NHERF-1 (Shenolikar et al. 2002; Weinman et al. 2003a; Cunningham et al. 2004). To study the role of NHERF-1 in the regulation of NHE3 activity, brush border membrane vesicles (BBM) were harvested from wild-type and NHERF-1 null mice, PKA was activated ex vivo, and NHE3 activity was measured as the amiloride inhibitable component of pH gradient-stimulated uptake of sodium (Weinman et al. 2003c). Basal NHE3 activity did not differ between wild-type and knockout BBM consistent with the finding that the abundance of the transporter was not altered in NHERF-1 null mice. Activation of PKA resulted in a 50% decrease in NHE3 activity in wild-type BBM but failed to affect the activity of the transporter in NHERF-1–/– membranes. The defect in the regulation of NHE3 in NHERF-1 null renal BBM was associated with the lack of PKA-mediated phosphorylation of NHE3, the biochemical signature of this form of regulation, despite the presence of normal amounts and activity of BBM PKA. The abundance of NHERF-2 and PDZK1 also was not different. We concluded that NHERF-1 uniquely transduces the cAMP signals that inhibit NHE3 activity and that NHERF-2 and PDZK1 could not substitute for the absence of NHERF-1.
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These studies were extended to measurements of NHE3 activity in cultured renal proximal tubule cells from wild-type and NHERF-1 null animals (Cunningham et al. 2004). PTH stimulated cAMP accumulation and activated PKC to the same extent in both cell types. Basal NHE3 activity determined using fluorescence measurements did not differ between the cell types but while PTH and forskolin significantly inhibited NHE3 activity in wild-type cells, neither PTH nor forskolin inhibited NHE3 activity in NHERF-1 null cells. Infection of NHERF-1–/– proximal tubule cells with adenovirus-GFP-NHERF-1 completely restored the inhibitory effect of PTH and cAMP on NHE3 activity. Thus, these experiments established that in renal tissue, NHERF-1 was required for cAMP-mediated inhibition of NHE3 activity and that the effect of NHERF-1, NHERF-2 and PDZK1 were not redundant, as they appeared to be in transfected PS120 cells (Yun et al. 1997).
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NHERF-1 regulates renal brush border abundance of Npt2a
Male and female NHERF-1–/– mice exhibit a decrease in the serum concentration of phosphate, an increase in the urinary excretion of phosphate, and a decrease in the renal BBM expression of Npt2a, the major regulated sodium-dependent phosphate transporter in the proximal convoluted tubule (Shenolikar et al. 2002; Murer et al. 2003; Bacic et al. 2004; Biber et al. 2004). These results were consistent with expression studies in OK cells, a proximal tubule cell line, where disruption of binding of NHERF-1 to ezrin resulted in reduced membrane expression of Npt2a (Hernando et al. 2002). A physiological approach was undertaken to discern the involvement of NHERF-1 in the regulation of Npt2a. Wild-type mice rapidly decrease the urinary excretion of phosphate when fed a diet low in phosphate (Weinman et al. 2003a). This adaptive response is associated with recruitment of Npt2a to the apical membrane of renal proximal tubule cells. NHERF-1–/– mice also adapted rapidly to dietary limitation of phosphate intake but, as compared with wild-type mice, never adapted fully. This was associated with decreased abundance of Npt2a in the plasma membrane of the mutant mice and increased detection of Npt2a in submicrovillar vesicular structures.
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Wild-type proximal tubule cells in culture adapt to growth in low phosphate media in a manner analogous to restricting the dietary intake of phosphate and demonstrate increased sodium-dependent phosphate uptake and BBM Npt2a abundance (Cunningham et al. 2004). By contrast, proximal tubule cells from NHERF-1 null mice have lower rates of sodium-dependent phosphate transport compared with wild-type cells and fail to increase the rate of phosphate transport or Npt2a abundance in response to growth in a low phosphate media. The phosphate content of the media did not affect the abundance of NHERF-1 in the plasma membrane of wild-type cells and the abundance of NHERF-2 also was not affected by the phosphate content of the media in either wild-type or NHERF-1 null cells. Interestingly, the abundance of PDZK1 in the plasma membrane of cultured proximal tubule cells was higher in cells grown in low phosphate media but the increase in abundance was equivalent in wild-type and NHERF-1–/– cells.
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Preliminary studies suggest that NHERF-1 is required for the inhibition of sodium-dependent phosphate transport and the decrease in apical membrane Npt2a abundance in response to parathyroid hormone (R. Cunningham & E. J. Weinman, unpublished data). It is likely that independent processes mediate the plasma membrane recruitment of Npt2a in response to phosphate deprivation and the removal of Npt2a in response to PTH. While NHERF-1 may be involved in both processes, we have speculated that NHERF-1 functions as an Npt2a membrane retention signal and that the absence of NHERF-1 limits the amount of Npt2a capable of remaining on the BBM.
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Perspectives
The interaction between NHERF-1 and NHE3 differs significantly from its interactions with Npt2a. NHERF-1 forms a complex with NHE3 affecting its phosphorylation in response to stimuli that activate PKA. The association between NHE3 and NHERF-1 seems to occur within the plasma membrane, and the binding is constitutive and not affected by activation of PKA (Zizak et al. 1999). By contrast, NHERF-1 binds Npt2a and affects the trafficking and tissue distribution of the transporter (Shenolikar et al. 2002). There is no evidence that NHERF-1 affects the phosphorylation state of Npt2a although this possibility has not formally been excluded.
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The role of NHERF-2 and PDZK1 on NHE3 and Npt2a has been less well studied. NHERF-2 affects calcium-mediated changes in NHE3 trafficking in model cell systems (Lee-Kwon et al. 2003a,b). Npt2a also binds to NHERF-2 in yeast 2-hydrid screens (Gisler et al. 2001). In mice, we have observed that NHERF-2 and Npt2a can be coimmunopreciptiated only from lysates of the renal cortex from wild-type but not NHERF-1–/– mice suggesting that the association between NHERF-2 and Npt2a was indirect and required the presence of NHERF-1 (Wade et al. 2003). The potential role of PDZK1 in the regulation of NHE3 and Npt2a is not clear although PDZK1 binds to both transporters (Gisler et al. 2003). PDZK1 null mice manifest no overt defects in renal phosphate transport or the expression of Npt2a when fed a normal diet but exhibit differences from wild-type mice when challenged with a high phosphate diet (Kocher et al. 2003; Capuano et al. 2005). PDZK1 mRNA is increased in response to phosphate depletion, and we have observed a modest but significant increase in PDZK1 protein in proximal tubule cells grown in low phosphate media (Cunningham et al. 2004). It remains distinctly possible that the adaptive response to low phosphate requires a cooperative interaction between NHERF-1 and PDZK1.
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Since the initial discovery of NHERF-1 and the subsequent discoveries of NHERF-2, PDZK1, and other adaptor proteins, there has been an evolution in our thinking about how these proteins interact not only with one another but also with target proteins. We have reviewed our results and speculations with respect to NHE3 and Npt2a but have not considered the multiple other targets of these adaptor proteins that directly or indirectly may affect cell function (Voltz et al. 2001; Shenolikar et al. 2004). It is our view that future studies will need to combine cell expression experiments in model cell systems with studies in intact tissues of appropriate animals. Recognizing the cautions inherent in using genetically altered mice, the development of NHERF-1, NHERF-2 and PDZK1 null mice should provide the necessary models to decipher the unique as well as the common roles of these adaptor proteins.
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Footnotes
This report was presented at The Journal of Physiology Symposium on PDZ domain scaffolding proteins and their functions in polarized cells, San Diego, CA, USA, 4 April 2005. It was commissioned by the Editorial Board and reflects the views of the authors.
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2 Department of Physiology University of Maryland School of Medicine
3 Medical Service, Department of Veterans Affairs Medical Center, Baltimore, MD 21201, USA
4 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
Abstract
Adaptor proteins containing PDZ interactive domains have been recently identified to regulate the trafficking and activity of ion transporters and channels in epithelial tissue. In the renal proximal tubule, three PDZ adaptor proteins, namely NHERF-1, NHERF-2 and PDZK1, are expressed in the apical membrane, heterodimerize with one another, and, at least in vitro, are capable of binding to NHE3 and Npt2a, two major regulated renal proximal tubule apical membrane transporters. Studies using NHERF-1 null mice have begun to provide insights into the organization of these adaptor proteins and their specific interactions with NHE3 and Npt2a. Experiments using brush border membranes and cultured renal proximal tubule cells indicate a specific requirement for NHERF-1 for cAMP-mediated phosphorylation and inhibition of NHE3. NHERF-1 null mice demonstrate increased urinary excretion of phosphate associated with mistargeting of Npt2a to the apical membrane of renal proximal tubule cells. NHERF-1 null animals challenged with a low phosphate diet and proximal tubule cells from these animals cultured in a low phosphate media fail to adapt as well as wild-type mice. These studies indicate a unique requirement for NHERF-1 in cAMP regulation of NHE3 and in the trafficking of Npt2a.
, 百拇医药
Introduction
The abundance and activity of epithelial transporters and ion channels is tightly regulated and it is likely that these membrane proteins exist as multiprotein complexes. Our recent work has focused on the organization of three PSD-95/Drosophila Discs large/ZO-1 (PDZ) adaptor proteins, namely the Na+–H+ exchanger regulatory factor or NHERF-1, NHERF-2 and PDZK1 in the regulation and targeting of the sodium–hydrogen exchanger isoform 3 (NHE3) and the sodium-dependent phosphate transporter 2a (Npt2a), two important regulated transport proteins in renal proximal convoluted tubule cells.
, 百拇医药
In 1995 we isolated and cloned a protein that was a necessary cofactor for cAMP regulation of NHE3 activity initially called NHERF (also known as ezrin-binding phosphoprotein of 50 kDa (EBP50)), later renamed NHERF-1 (Weinman et al. 1993, 1995). A second member of the NHERF family was identified, NHERF-2 (also known as NHE-3 kinase A-regulatory protein (E3KARP), tyrosine kinase activator-1 (TKA1), and sex-determining region of the Y chromosome (SRY-1)-interacting protein-1 (SIP-1)) in a yeast 2-hybrid screen using the NHE3 C-terminus as bait (Yun et al. 1997). NHERF-1 and NHERF-2 are encoded by unique genes, contain no recognizable catalytic domains, and share approximately 52% over all amino acid homology. They are considered members of a protein family by virtue of their common modular structure with two tandem PDZ homology domains and a C-terminal domain that binds all members of the ezrin–radixin–moesin (ERM) family of structural proteins (Reczek et al. 1997). PDZK1 (PDZ containing 1, also known as NaPiCap1 or CAP70) was identified using differential display PCR in studies of phosphate-deprived rats. PDZK1 contains four PDZ domains but unlike the NHERF proteins, lacks the C-terminal ERM domain (Custer et al. 1997). NHERF-1, NHERF-2 and PDZK1 are expressed in the apical side of renal proximal tubule cells and in the small intestines (Wade et al. 2001, 2003). NHERF-1 and NHERF-2 form homodimers and all three proteins heterodimerize with one another (Fouassier et al. 2000; Lau et al. 2001; Shenolikar et al. 2001; Gisler et al. 2003). It has been proposed that they array a microvillar/submicrovillar mesh or scaffold that regulates its target proteins. NHE3 and Npt2a appear to bind to all three of these adaptor proteins (Lamprecht et al. 1998; Zizak et al. 1999; Gisler et al. 2001; Hernando et al. 2002). A vexing question relates to whether NHERF-1, NHERF-2 and PDZK1 represent a redundant physiological control mechanism, function independently of one another, or act cooperatively in the regulation of target proteins.
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In this review, we will trace the evolution of our thinking about the role of NHERF-1, NHERF-2 and PDZK1 in the regulation of NHE3 and Npt2a, and the renal tubular reabsorption of sodium and phosphate. We initially believed these adaptor proteins had overlapping functions based on results from model cell systems (Yun et al. 1997). Our more recent experiments, particularly studies using NHERF-1 null mice, have led us to consider that the organization of these adaptor proteins in native tissues such as the renal proximal tubule importantly influences the regulation of NHE3 and Npt2a (Shenolikar et al. 2002). These studies highlight the fact that model cell systems, while providing important information about biochemical interactions between proteins, must be supported by studies in native tissues where the interactions between these adaptor proteins and their targets may differ.
, 百拇医药
NHERF-1, NHERF-2 and the regulation of NHE3 in PS120 cell fibroblasts
Initial study of the cellular function of NHERF-1 and NHERF-2 was undertaken in PS120 cell fibroblasts, a cell line negatively selected to express no endogenous NHE activity (Yun et al. 1997). This cell line also does not express endogenous NHERF-1 or NHERF-2. Rabbit NHE3 was readily expressed in these cells but treatment with cAMP failed to regulate NHE3 activity. When NHERF-1 or NHERF-2 were coexpressed with NHE3, however, cAMP inhibition of NHE3 activity was observed. These studies established that NHERF-1 or NHERF-2 was required for protein kinase A (PKA) associated down-regulation of NHE3 activity. The biochemical bases for the effect of the NHERF proteins was elucidated by the demonstration that NHERF-1 and NHERF-2 bound to NHE3 and to ezrin. Ezrin, acting as a protein kinase A anchor protein, recruited PKA thereby facilitating the rapid phosphorylation of specific serine residues in the C-terminus of NHE3 with a consequent down-regulation of the activity of the transporter (Lamprecht et al. 1998; Zizak et al. 1999). The model that emerged was that the NHERF proteins were required for the formation of a multiprotein complex and that the binding of NHERF to both NHE3 and ezrin was critical for this type of hormonal regulation (Weinman et al. 2000). In PS120 cells, NHERF-1 and NHERF-2 were equally effective in mediating cAMP inhibition of NHE3 activity (Yun et al. 1997).
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Localization of NHERF-1 and NHERF-2 in the kidney of rodents and man
Highly specific antipeptide antibodies that differentiated NHERF-1 from NHERF-2 were used to determine the tissue distribution of the NHERF isoforms. Initial studies were performed using the rat kidney (Wade et al. 2001). Although both isoforms were detected in specific cells of the nephron, NHERF-1 but not NHERF-2 was readily detected in the apical membrane of renal proximal tubule cells and we suggested that NHERF-1 was the relevant isoform mediating hormonal regulation of NHE3 activity. Additional studies in man and mouse, however, indicated the presence of both NHERF isoforms in the renal proximal tubule (Wade et al. 2003; Weinman et al. 2003b). As shown in Fig. 1, the distribution of NHERF-1 and NHERF-2 in mice was not identical. NHERF-1 was expressed in the microvillar membrane and colocalized with NHE3 and Npt2a. NHERF-2, on the other hand, was expressed predominantly in the submicrovillar region of renal proximal tubule cells but only minimally in the microvillus itself. This distribution was confirmed using immunogold staining and electron microscopy. The factors that determine the unique distribution of the NHERF isoforms are unknown but when considered from a physiological perspective, the findings that both NHERF-1 and NHERF-2 were expressed in renal proximal tubule cells necessitated a reconsideration of the question of the roles of these proteins in control of NHE3 and Npt2a activity and abundance.
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A, representative confocal images of proximal convoluted tubules of the mouse kidney using antibodies specific for NHERF-1 (a) and NHERF-2 (b). c is the merged images showing the localization of NHERF-1 in the microvillar membranes and NHERF-2 predominantly in a region just below the microvilli. B, electron microscopical images of wild-type renal proximal tubules using immunogold showing the localization of NHERF-1 in the microvillus (a) and NHERF-2 in vesicle-rich region at the base of the microvilli (b). (Figures adapted from Wade et al. 2003.)
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Development of the NHERF-1–/– mouse
The potential complexity of the interactions between NHERF-1 and NHERF-2 with each other and with other PDZ scaffolding proteins in renal cells suggested that a genetic approach would be required to determine the individual role of each of these proteins on sodium and phosphate transport in the proximal tubule of the kidney. Using homologous recombination and a vector targeting exon 1 of the mouse NHERF-1 gene, we successfully abolished NHERF-1 expression in all mouse tissues (Shenolikar et al. 2002). Male NHERF-1–/– mice displayed no overt phenotype. Blood pressure, serum electrolytes, renal function and renal histology were normal. However, mutant male mice demonstrated mild hypophosphatemia and, as compared with wild-type mice, increased urinary excretion of phosphate. Some, but not all, NHERF-1 null female mice were runts, displayed severe osteoporosis and bone fractures, and died shortly after weaning. The more normal appearing females were used for breeding to establish an NHERF-1 null mouse colony. The availability of NHERF-1–/– mice permitted an assessment of the relative contribution of NHERF-1 and NHERF-2 to the regulation of NHE3 and Npt2a.
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NHERF-1 uniquely regulates cAMP-associated inhibition of NHE3 activity
As determined using tissue fractionation and confocal microscopy, the expression of NHE3 in the renal apical membrane did not differ between wild-type and NHERF-1–/– mice suggesting that NHERF-1 did not affect the trafficking of NHE3 (Shenolikar et al. 2002). Moreover, the abundance and cellular distribution of NHERF-2 was not affected by the absence of NHERF-1 (Shenolikar et al. 2002; Weinman et al. 2003a; Cunningham et al. 2004). To study the role of NHERF-1 in the regulation of NHE3 activity, brush border membrane vesicles (BBM) were harvested from wild-type and NHERF-1 null mice, PKA was activated ex vivo, and NHE3 activity was measured as the amiloride inhibitable component of pH gradient-stimulated uptake of sodium (Weinman et al. 2003c). Basal NHE3 activity did not differ between wild-type and knockout BBM consistent with the finding that the abundance of the transporter was not altered in NHERF-1 null mice. Activation of PKA resulted in a 50% decrease in NHE3 activity in wild-type BBM but failed to affect the activity of the transporter in NHERF-1–/– membranes. The defect in the regulation of NHE3 in NHERF-1 null renal BBM was associated with the lack of PKA-mediated phosphorylation of NHE3, the biochemical signature of this form of regulation, despite the presence of normal amounts and activity of BBM PKA. The abundance of NHERF-2 and PDZK1 also was not different. We concluded that NHERF-1 uniquely transduces the cAMP signals that inhibit NHE3 activity and that NHERF-2 and PDZK1 could not substitute for the absence of NHERF-1.
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These studies were extended to measurements of NHE3 activity in cultured renal proximal tubule cells from wild-type and NHERF-1 null animals (Cunningham et al. 2004). PTH stimulated cAMP accumulation and activated PKC to the same extent in both cell types. Basal NHE3 activity determined using fluorescence measurements did not differ between the cell types but while PTH and forskolin significantly inhibited NHE3 activity in wild-type cells, neither PTH nor forskolin inhibited NHE3 activity in NHERF-1 null cells. Infection of NHERF-1–/– proximal tubule cells with adenovirus-GFP-NHERF-1 completely restored the inhibitory effect of PTH and cAMP on NHE3 activity. Thus, these experiments established that in renal tissue, NHERF-1 was required for cAMP-mediated inhibition of NHE3 activity and that the effect of NHERF-1, NHERF-2 and PDZK1 were not redundant, as they appeared to be in transfected PS120 cells (Yun et al. 1997).
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NHERF-1 regulates renal brush border abundance of Npt2a
Male and female NHERF-1–/– mice exhibit a decrease in the serum concentration of phosphate, an increase in the urinary excretion of phosphate, and a decrease in the renal BBM expression of Npt2a, the major regulated sodium-dependent phosphate transporter in the proximal convoluted tubule (Shenolikar et al. 2002; Murer et al. 2003; Bacic et al. 2004; Biber et al. 2004). These results were consistent with expression studies in OK cells, a proximal tubule cell line, where disruption of binding of NHERF-1 to ezrin resulted in reduced membrane expression of Npt2a (Hernando et al. 2002). A physiological approach was undertaken to discern the involvement of NHERF-1 in the regulation of Npt2a. Wild-type mice rapidly decrease the urinary excretion of phosphate when fed a diet low in phosphate (Weinman et al. 2003a). This adaptive response is associated with recruitment of Npt2a to the apical membrane of renal proximal tubule cells. NHERF-1–/– mice also adapted rapidly to dietary limitation of phosphate intake but, as compared with wild-type mice, never adapted fully. This was associated with decreased abundance of Npt2a in the plasma membrane of the mutant mice and increased detection of Npt2a in submicrovillar vesicular structures.
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Wild-type proximal tubule cells in culture adapt to growth in low phosphate media in a manner analogous to restricting the dietary intake of phosphate and demonstrate increased sodium-dependent phosphate uptake and BBM Npt2a abundance (Cunningham et al. 2004). By contrast, proximal tubule cells from NHERF-1 null mice have lower rates of sodium-dependent phosphate transport compared with wild-type cells and fail to increase the rate of phosphate transport or Npt2a abundance in response to growth in a low phosphate media. The phosphate content of the media did not affect the abundance of NHERF-1 in the plasma membrane of wild-type cells and the abundance of NHERF-2 also was not affected by the phosphate content of the media in either wild-type or NHERF-1 null cells. Interestingly, the abundance of PDZK1 in the plasma membrane of cultured proximal tubule cells was higher in cells grown in low phosphate media but the increase in abundance was equivalent in wild-type and NHERF-1–/– cells.
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Preliminary studies suggest that NHERF-1 is required for the inhibition of sodium-dependent phosphate transport and the decrease in apical membrane Npt2a abundance in response to parathyroid hormone (R. Cunningham & E. J. Weinman, unpublished data). It is likely that independent processes mediate the plasma membrane recruitment of Npt2a in response to phosphate deprivation and the removal of Npt2a in response to PTH. While NHERF-1 may be involved in both processes, we have speculated that NHERF-1 functions as an Npt2a membrane retention signal and that the absence of NHERF-1 limits the amount of Npt2a capable of remaining on the BBM.
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Perspectives
The interaction between NHERF-1 and NHE3 differs significantly from its interactions with Npt2a. NHERF-1 forms a complex with NHE3 affecting its phosphorylation in response to stimuli that activate PKA. The association between NHE3 and NHERF-1 seems to occur within the plasma membrane, and the binding is constitutive and not affected by activation of PKA (Zizak et al. 1999). By contrast, NHERF-1 binds Npt2a and affects the trafficking and tissue distribution of the transporter (Shenolikar et al. 2002). There is no evidence that NHERF-1 affects the phosphorylation state of Npt2a although this possibility has not formally been excluded.
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The role of NHERF-2 and PDZK1 on NHE3 and Npt2a has been less well studied. NHERF-2 affects calcium-mediated changes in NHE3 trafficking in model cell systems (Lee-Kwon et al. 2003a,b). Npt2a also binds to NHERF-2 in yeast 2-hydrid screens (Gisler et al. 2001). In mice, we have observed that NHERF-2 and Npt2a can be coimmunopreciptiated only from lysates of the renal cortex from wild-type but not NHERF-1–/– mice suggesting that the association between NHERF-2 and Npt2a was indirect and required the presence of NHERF-1 (Wade et al. 2003). The potential role of PDZK1 in the regulation of NHE3 and Npt2a is not clear although PDZK1 binds to both transporters (Gisler et al. 2003). PDZK1 null mice manifest no overt defects in renal phosphate transport or the expression of Npt2a when fed a normal diet but exhibit differences from wild-type mice when challenged with a high phosphate diet (Kocher et al. 2003; Capuano et al. 2005). PDZK1 mRNA is increased in response to phosphate depletion, and we have observed a modest but significant increase in PDZK1 protein in proximal tubule cells grown in low phosphate media (Cunningham et al. 2004). It remains distinctly possible that the adaptive response to low phosphate requires a cooperative interaction between NHERF-1 and PDZK1.
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Since the initial discovery of NHERF-1 and the subsequent discoveries of NHERF-2, PDZK1, and other adaptor proteins, there has been an evolution in our thinking about how these proteins interact not only with one another but also with target proteins. We have reviewed our results and speculations with respect to NHE3 and Npt2a but have not considered the multiple other targets of these adaptor proteins that directly or indirectly may affect cell function (Voltz et al. 2001; Shenolikar et al. 2004). It is our view that future studies will need to combine cell expression experiments in model cell systems with studies in intact tissues of appropriate animals. Recognizing the cautions inherent in using genetically altered mice, the development of NHERF-1, NHERF-2 and PDZK1 null mice should provide the necessary models to decipher the unique as well as the common roles of these adaptor proteins.
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Footnotes
This report was presented at The Journal of Physiology Symposium on PDZ domain scaffolding proteins and their functions in polarized cells, San Diego, CA, USA, 4 April 2005. It was commissioned by the Editorial Board and reflects the views of the authors.
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