Departments of 1Physiology and 2Medicine, University of Maryland School of Medicine; and 3Department of Veterans Affairs Medical Center, Baltimore, Maryland, 21201; and 4Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710
Submitted 5 March 2003 ; accepted in final form 7 August 2003
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ABSTRACT |
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NHE3; Npt2; ezrin; PDZ domains; immunolocalization
The specificity of binding to PDZ sites usually resides in the COOH-terminal amino acids of the target proteins (21). Despite the similarities in the PDZ domains of NHERF-1 and NHERF-2, some target proteins are able to distinguish one protein from the other (7, 10, 12, 16, 29). We speculate that in addition to the requirement for unique amino acid sequences in the COOH termini, there may be another level of specificity related to differences in the cellular distribution of NHERF-1 compared with NHERF-2. The present experiments in wild-type and NHERF-1-null mice were designed, therefore, to study the subcellular localization of the NHERF isoforms and selected known target proteins in the mouse proximal tubule and to determine whether the absence of NHERF-1 results in mislocalization of NHERF-1-binding partners or loss of cell architecture.
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METHODS |
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All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
Antibodies. New antipeptide antibodies were made against NHERF-1 (amino acids 298-314): NH2-CSQDSPKKEDSTAPSSTS-COOH and NHERF-2 (amino acids 110-126) NH2-CRGLPPAHDPWEPKPDWA-COOH in both chickens and rabbits. Western immunoblots (22) used antiserum from these immunizations, whereas antibody prepared by affinity purification with immobilized peptide was used for immunocytochemistry (25). Commercially available anti-NHE3 monoclonal and polyclonal antibodies (Chemicon International) and an anti-Npt2 polyclonal (L697) antibody (11) provided by M. A. Knepper (National Heart, Lung, and Blood Institute, Bethesda, MD) were also used.
Immunocytochemistry. Mice were anesthetized with metofane and perfused through the left ventricle of the heart via a blunted 21-gauge needle. Perfusion was for 2 min in PBS to clear the kidneys of blood and 5 min in 2% paraformaldehyde. Kidneys were sliced and further fixed for 60 min in 2% paraformaldehyde followed by 60 min in a cryoprotectant of 10% EDTA in 0.1 M Tris. Tissue was then wrapped in aluminum foil and frozen on dry ice. Cryostat sections 8 µm thick were made and picked up on coverslips coated with HistoGrip (Zymed, San Francisco, CA). Sections were then treated with either 1% SDS (6) or 6 M guanidine (18) for 10 min to unmask antigenic sites. Sections were washed three times with high-salt buffer (50 ml PBS, 0.5 g BSA, 1.13 g NaCl) and incubated in blocking agent (50 ml PBS, 0.5 g BSA, 0.188 g glycine, pH 7.2) for 20 min, followed by incubation with primary antibody overnight at 4°C. Primary antibodies were diluted to 10 µg/ml with incubation medium (50 ml PBS, 0.05 g BSA, 200 µl 5% NaN3). After this incubation, sections were rinsed five times with high-salt buffer over the course of 1 h. Appropriate species-specific secondary antibodies coupled to Alexa 488 or 568 dyes (Molecular Probes, Eugene, OR) were diluted 1:200 with incubation medium and then incubated with the tissue sections for 2 h at 4°C. These samples were again washed five times with high-salt buffer over the course of 1 h and then in PBS to remove the excess salt before mounting and confocal microscopy.
EM immunolocalizations. Cryostat sections of 20 µm thickness were prepared from frozen renal tissue as described above and treated in suspension on coverslips. Antigen retrieval treatment was omitted to preserve morphology, but blocking and primary antibody incubation and washes followed the schedule described above. As a secondary antibody, Alexa 488-labeled goat anti-rabbit (1:100) was applied for 2 h. After washes, tissue sections were fixed in 2% glutaraldehyde and then in 1% OsO4 according to standard protocols and embedded in Epon. Thin sections (80 nm, mounted on bare 300-400 mesh Ni grids) were prepared from suitable cortical regions. To allow labeling of sections, Epon was etched with sodium ethoxide (5). A stock of sodium ethoxide was made by allowing a saturated solution of NaOH in ethanol to age several days in the dark until a burgundy color developed. Saturated sodium ethoxide was diluted to 2% of saturation with ethanol and was used fresh. Grids were treated for three different etching times (from 20 s to 10 min) to achieve optimal etching. Sections were then rinsed in ethanol and water and then blocked as above before the application of the third antibody, which was rabbit anti-Alexa 488 diluted 1:200 (Molecular Probes) for at least 2 h. Use of this antibody takes advantage of the fact that the Alexa 488 fluorophore withstands fixation and embedding in Epon. This labeling was detected by using goat anti-rabbit-gold diluted 1:25 and incubated overnight at 4°C. After being washed in PBS, grids were fixed in 2% glutaraldehyde and stained with uranyl acetate and lead citrate.
Coimmunoprecipitation procedures. Renal cortices from wild-type and NHERF-1 (-/-) mice (22) were hand dissected and homogenized in 1 ml of IP buffer, a solution containing 100 mM NaCl and 10 mM sodium phosphate buffer (pH 7.4) with Complete Protease Inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). After homogenization, Triton X-100 was added to a final concentration of 1% and the samples were lysed by being drawn through a 27-gauge needle and rocked at 4°C for 30-60 min, followed by centrifugation at 12,000 g for 30 min to remove insoluble cellular debris. The supernatants were precleared with protein GSepharose fast flow beads (prewashed in IP buffer containing 1% Triton X-100) by rocking for 1 h. The beads were then spun down, and the supernatants were aliquoted into 2 samples and incubated overnight with polyclonal antibodies to NHERF-1 or NHERF-2 in the case of wild-type kidneys and NHERF-2 and NHE3 in the kidneys from null animals. Protein A-Sepharose CL4B beads (prewashed in IP buffer containing 1% Triton X-100) were added and allowed to rock for an additional 2 h. Beads were washed several times in IP buffer, and proteins were eluted from beads by being boiled in 200 µl of Laemmli SDS-sample buffer.
Immunoblotting. Proteins and cell lysates (15 µg/lane) were resolved using 10% SDS-polyacrylamide gels electro-phoretically transferred to nitrocellulose and analyzed by Western immunoblotting. The immune complexes were detected by enhanced chemical luminescence (ECL) (Amersham, Arlington Heights, Il).
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RESULTS |
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To define the subcellular location of NHERF-1 and NHERF-2 in the proximal tubule, wild-type mouse kidney was stained using rabbit antibody to NHERF-1 and chicken antibody to NHERF-2 (Fig. 2). NHERF-1 was very abundant in the BBM (Fig. 2A), whereas labeling by the NHERF-2 antibody raised in chicken was strongest at the base of the BBM (Fig. 2B). Although NHERF-2 could be detected in the BBM with this NHERF-2 antibody, its abundance there was low compared with that of NHERF-1. When labeling by the two different antibodies to NHERF-2 were compared, the antibody raised in rabbit (Fig. 3A) labeled the BBM more strongly, whereas the antibody raised in chicken (Fig. 3B) labeled the region at the base of the BBM more strongly. Although these antibodies were raised to the same peptide and both recognize NHERF-2 only as shown above, these immunolocalizations suggest that the two antibodies may recognize different epitopes on NHERF-2 and that the major epitope recognized by the rabbit antibody is more exposed in the BBM, whereas the major epitope recognized by the chicken antibody is more exposed at the base of the BBM. When identical procedures were used to test these same antibodies on rat sections, labeling was seen in the glomeruli and in the vasculature of the kidney, including the descending vasa rectae, but no labeling was detected in the cells of the proximal convoluted tubule, which is consistent with previous reports that NHERF-2 is not expressed proximally in the rat (25). The absence of NHERF-2 in rat proximal tubule was confirmed by RT-PCR of proximal tubules and descending vasa recta hand dissected from collagenase-treated rat kidneys. With the use of nested primers for NHERF-2, an mRNA of predicted size was detected in descending vasa recta but not in proximal tubules. RT-PCR for GAPDH was used as an internal control and was detected in both the proximal tubule and in the descending vasa rectae (data not shown).
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To further characterize the site of NHERF-2 labeling, we carried out colabeling localizations with antibody to the coated pit/coated vesicle protein clathrin (Fig. 4). Whereas there was no significant overlap of NHERF-1 labeling with clathrin at the base of the BBM (Fig. 4, A-C), there was significant overlap of NHERF-2 labeling with that of clathrin using both the rabbit (Fig. 4, D and F) and the chicken (Fig. 4, G-I) antibodies. Thus both antibodies indicate that NHERF-2 occurs not only in the BBM with NHERF-1 but also at an adjacent site below the BBM, where NHERF-2 but not NHERF-1 colabels with clathrin. This distribution was confirmed by electron microscopic localizations that showed that NHERF-1 was exclusively detected in the brush border microvilli (Fig. 5A). NHERF-2, on the other hand, was largely localized to an apical band of small vesicles and minimally associated with microvilli (Fig. 5B).
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We next sought to define the cellular distribution of NHE3, Npt2, and ezrin because these three proteins have been identified as targets of both NHERF isoforms. NHE3, Npt2, and ezrin all localized strongly to the BBM where NHERF-1 labeling was strong. Modest labeling of NHE3, Npt2, and ezrin was also detectable at the base of the BBM where NHERF-2 is strong (arrows, Fig. 6). We also determined that the distribution of NHERF-2 with respect to NHE3 is unaltered in NHERF-1 null mice (Fig. 7).
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To extend these findings, NHERF-1 or NHERF-2 was immunoprecipitated from renal cortical lysates of wild-type mice and NHERF-1-null animals. As shown in Fig. 8, NHERF-1 coimmunoprecipitated NHE3, Npt2, and ezrin from lysates of wild-type mice. NHERF-1 was detected in these immunoprecipitates, as expected. In addition, NHERF-2 was recovered in the NHERF-1 immunoprecipitates. With the use of the rabbit antibody, NHERF-2 coimmunoprecipitated the same proteins from lysates of wild-type mice. In NHERF-1-null animals, however, immunoprecipitation of NHERF-2 coimmunoprecipitated NHE3 and ezrin but Npt2 could not be detected. Immunoprecipitation of NHE3 coimmunoprecipitated ezrin and NHERF-2 but also failed to bring down Npt2.
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The NHERF proteins, by binding to ezrin, link the actin cytoskeleton of the cell to their integral membrane targets. The absence of the NHERF proteins, therefore, has the potential to disrupt this connection and the organization of proximal tubular microvilli. However, evaluation of fluorescent phalloidin labeling in NHERF-1-null animals showed that the amount and cellular location of actin was the same in wild-type and NHERF-1-null animals (Fig. 9). The ezrin localization was also unaltered (data not shown). Electron microscopy was used to examine the fine structure of the BBM. As shown in Fig. 10, the substructure of the BBM appears the same in wild-type and NHERF-1-null mice.
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DISCUSSION |
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With the use of confocal microscopy, NHERF-1 was detected strongly in the BBM of the mouse proximal tubule as previously described in rat (4, 25) and mouse (9). Although previous studies had indicated that NHERF-2 does not occur in proximal tubule cells of rat (25) and mouse (9), our current work shows that NHERF-2 does occur in the proximal tubule of mouse. Other work indicates that NHERF-2 is also expressed in the human proximal tubule (26). With the use of an antibody raised in chicken, NHERF-2 intensely localized to the base of the microvilli where it colabeled with clathrin. Immunogold electron microscopy confirmed the presence of NHERF-1 in the microvilli and the greater abundance of NHERF-2 in the region at the base of the microvilli. Distinct microdomains of the renal brush border and associated clathrin labeling have been characterized at the base of the microvilli with a coated-pit intermicrovillar region (1-3, 19). Our finding that NHERF-2 but not NHERF-1 is strongly localized to this site suggests that the latter may be important in vesicular traffic.
Modest labeling of NHERF-2 could also be detected in the microvilli with the chicken antibody. To extend these findings, we also employed an anti-NHERF-2 antibody raised in rabbit to the same peptide used for the chicken antibody. The rabbit antibody showed more labeling of the microvillar region but confirmed the presence of NHERF-2 in the clathrin-rich microdomain. Both antibodies indicated that the abundance of NHERF-2 in the microvilli is significantly less than that of NHERF-1. It is of interest, however, that two antibodies to the same protein provided different localizations. The best explanation for the different staining patterns is that antibodies recognizing largely different epitopes were generated in the different animals and that these antigenic sites were differentially occluded in the different microvillar regions. Biemesderfer and colleagues (1) reached a similar conclusion based on experience with multiple antibodies to NHE3.
Given the localization of NHERF-1 in the BBM and NHERF-2 at the base, additional studies were performed to determine their association with some known target proteins. NHE3, Npt2, and ezrin strongly colocalize with NHERF-1 in the microvilli. Lesser amounts of these proteins were also identified at the base of the microvilli where NHERF-2 predominates. In wild-type mice, NHERF-1 coimmunoprecipitated NHE3, Npt2, and ezrin. The immunoprecipitates also contained NHERF-2, indicating the formation of heterodimers of the two isoforms. Evidence for such a direct interaction between the two NHERFs has recently been advanced by Lau and Hall (14). The current results, however, do not exclude the possibility that the interaction may be indirect via other proteins present in the complex. With the use of the rabbit antibody to NHERF-2, the antibody that appeared to detect NHERF-2 most broadly by confocal microscopy, the same proteins were coimmunoprecipitated from the wild-type kidney. In the NHERF-1-null kidneys, however, NHERF-2 antibody coimmunoprecipitated NHE3 and ezrin but not Npt2. This would suggest that the association of NHERF-2 and Npt2 seen in wild-type animals derives from the association between NHERF-1 and NHERF-2 and that NHERF-1 is the major isoform interacting with Npt2. This conclusion would be consistent with the physiological defects in the NHERF-1-null animals (22). It should also be noted that in the rat, in contrast to the mouse, only NHERF-1 is detectable in the renal proximal tubule (25), and in this species, Npt2 is normally targeted and regulated (15, 17) even in the absence of NHERF-2. When NHE3 antibody was used to immunoprecipitate proteins from the kidney of NHERF-1 (-/-) mice, ezrin and NHERF-2 were brought down but not Npt2. This indicates that NHE3 interacts with both NHERF-1 and NHERF-2 and, given that NHERF-1 is solely responsible for cAMP regulation, suggests that NHERF-1 and NHERF-2 subserve unique roles in the regulation of NHE3. A working model of the association among these proteins is shown in Fig. 11.
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By linking to the scaffold protein ezrin, NHERF-1 forms a part of the actin cytoskeleton of the cell. Sabolic and colleagues (20) have recently reported that disruption of the microtubule network with colchicine resulted in the trafficking of NHERF-1 and at least one of its targets, NHE3, to the basolateral surface of rat renal proximal tubules. By routine histology, the kidneys of NHERF-1 (-/-) mice appeared normal (22). To refine these initial observations, we used confocal and electron microscopy to define the role of NHERF-1 in maintaining the structure of renal cells. The abundance and distribution of actin, ezrin, as well as NHERF-2, appear to be the same in wild-type and NHERF-1 (-/-) mice. By electron microscopy, the structure of the BBM appeared normal in the null mice. Thus, although there is a potential for NHERF-1 to play a critical role in maintenance of the cytoskeleton, these observations suggest that its role is not essential or that other proteins can compensate for the absence of NHERF-1. Thus it seems unlikely that the mislocalization of Npt2 from the apical membrane of the proximal tubule seen in NHERF-1-null animals is due to a gross disruption of the cellular architecture. A more likely explanation for the defect is the absence of NHERF-1 and, as shown in the present experiments, the apparent absence of direct interaction between Npt2 with NHERF-2 in vivo.
These studies, then, extend prior observations and provide new information pertinent to the possible specific functional roles of the two NHERF isoforms. Initial studies showed an overlapping specificity of NHERF-1 and NHERF-2 with respect to target proteins. A number of recent papers, however, have described NHERF-2-mediated interactions that cannot be duplicated by NHERF-1. Our observations indicate that there is not only biochemical specificity but also an organizational specificity with NHERF-2 localizing to a cell region where NHERF-1 is undetectable and the predominant expression of NHERF-1 is relative to NHERF-2 in the brush border microvillar membrane. The distinct cellular locations of the two NHERF isoforms provide another mechanism allowing for specific interaction of each protein with its targets. This is consistent with the hypothesis that cell types expressing both NHERFs utilize the two isoforms for distinct as well as overlapping functions. The presence of NHERF-2 in the mouse proximal tubule and the ability of NHERF-2 to bind and stabilize NHE3 may explain the rather mild phenotype of NHERF-1-null mice. The apparent inability of NHERF-2 to interact with Npt2 in the absence of NHERF-1 could contribute to the mislocalization of Npt2 and elevated phosphate excretion seen in these animals (22). Finally, recent studies have expanded the biological targets of the NHERF proteins to include not only transporters and channels but also receptors and signaling proteins. Similar specific localization studies are needed to determine whether differential localization of the NHERF proteins affects the function of these target proteins. NHERF-1- and NHERF-2-null mice should be valuable models allowing study of each NHERF in these diverse biological processes.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Biemesderfer D, Dekan G, Aronson PS, and Farquhar MG. Assembly of distinctive coated pit and microvillar microdomains in the renal brush border. Am J Physiol Renal Fluid Electrolyte Physiol 262: F55-F67, 1992.
3. Biemesderfer D, Mentone SA, Mooseker M, and Hasson T. Expression of myosin VI within the early endocytic pathway in adult and developing proximal tubules. Am J Physiol Renal Physiol 282: F785-F794, 2002.
4. Breton S, Wiederhold T, Marshansky V, Nsumu NN, Ramesh V, and Brown D. The 1 subunit of the H+ ATPase is a PDZ-domain binding protein: colocalization with NHE-RF in renal B-intercalated cells. J Biol Chem 275: 18219-18224, 2000.
5. Brorson SH. Deplasticizing or etching of epoxy sections with different concentrations of sodium ethoxide to enhance the immunogold labeling. Micron 32: 101-105, 2001.[ISI][Medline]
6. Brown D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, and Alper S. Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol 105: 261-267, 1996.[ISI][Medline]
7. Demarco SJ, Chicka MC, and Strehler EE. Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+/H+ exchanger regulatory factor 2 in apical plasma membranes. J Biol Chem 277: 10506-10511, 2002.
8. Gisler SM, Stagljar I, Traebert M, Bacic D, Biber J, and Murer H. Interaction of the type IIa Na/Pi cotransporter with PDZ proteins. J Biol Chem 276: 9206-9213, 2001.
9. Ingraffea J, Reczek D, and Bretscher A. Distinct cell type-specific expression of scaffolding proteins EBP50 and E3KARP: EBP50 is generally expressed with ezrin in specific epithelia, whereas E3KARP is not. Eur J Cell Biol 81: 61-68, 2002.[ISI][Medline]
10. Kanai F, Marignani PA, Sarbassova D, Yagi R, Hall RA, Donowitz M, Hisaminato A, Fujiwara T, Ito Y, Cantley LC, and Yaffe MB. TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins. EMBO J 19: 6778-6791, 2000.
11. Kim GH, Martin SW, Fernandez-Llama P, Masilamani S, Packer RK, and Knepper MA. Long-term regulation of renal Na-dependent cotransporters and ENaC: response to altered acid-base intake. Am J Physiol Renal Physiol 279: F459-F467, 2000.
12. Kim JH, Lee-Kwon W, Park JB, Ryu SH, Yun CH, and Donowitz M. Ca2+-dependent inhibition of Na+/H+ exchanger 3 requires a NHE3/E3KARP/alpha-actinin-4 complex for oligomerization and endocytosis. J Biol Chem 277: 23714-23724, 2002.
13. Lamprecht G, Weinman EJ, and Yun CH. The role of NHERF and E3KARP in the cAMP-mediated inhibition of NHE3. J Biol Chem 273: 29972-29978, 1998.
14. Lau AG and Hall RA. Oligomerization of NHERF-1 and NHERF-2 PDZ domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation. Biochemistry 40: 8572-8580, 2002.[ISI]
15. Levi M, Lotscher M, Sorribas V, Custer M, Arar M, Kaissling B, Murer H, and Biber J. Cellular mechanisms of acute and chronic adaptation of rat renal Pi transporter to alterations in dietary Pi. Am J Physiol Renal Fluid Electrolyte Physiol 267: F900-F908, 1994.
16. Li Y, Li J, Straight SW, and Kershaw DB. PDZ domain-mediated interaction of rabbit podocalyxin and Na+/H+ exchange regulatory factor-2. Am J Physiol Renal Physiol 282: F1129-F1139, 2002.
17. Lotscher M, Wilson P, Nguyen S, Kaissling B, Biber J, Murer H, and Levi M. New aspects of adaptation of rat renal Na-Pi cotransporter to alterations in dietary phosphate. Kidney Int 49: 1012-1018, 1996.[ISI][Medline]
18. Peranen J, Rikkonen M, and Kaariainen L. A method for exposing hidden antigenic sites in paraformaldehyde-fixed cultured cells, applied to initially unreactive antibodies. J Histochem Cytochem 41: 447-454, 1993.
19. Rodman JS, Seidman L, and Farquhar MG. The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell. J Cell Biol 102: 77-87, 1986.[Abstract]
20. Sabolic I, Herak-Kramberger CM, Ljubojevic M, Biemesderfer D, and Brown D. NHE3 and NHERF are targeted to the basolateral membrane in proximal tubules of colchicine-treated rats. Kidney Int 61: 1351-1364, 2002.[ISI][Medline]
21. Sheng M and Sala C. PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24: 1-29, 2001.[ISI][Medline]
22. Shenolikar S, Voltz JW, Minkoff CM, Wade JB, and Weinman EJ. Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting. Proc Natl Acad Sci USA 99: 11470-11475, 2002.
23. Shenolikar S and Weinman EJ. NHERF: targeting and trafficking membrane proteins. Am J Physiol Renal Physiol 280: F389-F395, 2001.
24. Voltz JW, Weinman EJ, and Shenolikar S. Expanding the role of NHERF, a PDZ-domain containing protein adapter, to growth regulation. Oncogene 20: 6309-6314, 2001.[ISI][Medline]
25. Wade JB, Welling PA, Donowitz M, Shenolikar S, and Weinman EJ. Differential renal distribution of NHERF isoforms and their colocalization with NHE3, ezrin, and ROMK. Am J Physiol Cell Physiol 280: C192-C198, 2001.
26. Weinman EJ, Lakkis J, Akom M, Wali RK, Drachenberg CB, Coleman RA, and Wade JB. Expression of NHERF-1, NHERF-2, PDGFR-á, and PDGFR-â in normal human kidneys and in renal transplant rejection. Pathobiology 70: 314-323, 2003.[ISI]
27. Weinman EJ, Minkoff C, and Shenolikar S. Signal complex regulation of renal transport proteins: NHERF and regulation of NHE3 by PKA. Am J Physiol Renal Physiol 279: F393-F399, 2000.
28. Weinman EJ, Steplock D, and Shenolikar S. NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse renal apical membranes. FEBS Lett 536: 141-144, 2003.[ISI][Medline]
29. Yun CC, Chen Y, and Lang F. Glucocorticoid activation of Na+/H+ exchanger isoform 3 revisited. The roles of SGK1 and NHERF2. J Biol Chem 277: 7676-7683, 2002.
30. Yun CH, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman EJ, and Donowitz M. cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein. Proc Natl Acad Sci USA 94: 3010-3015, 1997.
31. Zizak M, Lamprecht G, Steplock D, Tariq N, Shenolikar S, Donowitz M, Yun CH, and Weinman EJ. cAMP-induced phosphorylation and inhibition of Na+/H+ exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor. J Biol Chem 274: 24753-24758, 1999.