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Endocrine Unit (M.J.M., G.V.S.), Massachusetts General Hospital, Boston, Massachusetts 02114; Department of Pharmacology (J.A.C.), University of Missouri School of Medicine, Columbia, Missouri 65212; Kidney Disease Program (E.D.L.), University of Louisville, Louisville, Kentucky 40202
Address all correspondence and requests for reprints to: Gino V. Segre, M.D., Endocrine Unit, Wellman 501, Massachusetts General Hospital, 50 Blossom Street, Boston, Massachusetts 02114. E-mail: segre{at}helix.mgh.harvard.edu.
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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60%) and the inhibition mediated by phorbol 12-myristate 13-acetate (30%) and the ability of these molecules to activate the protein kinase C-responsive reporter gene. Furthermore, we show that NHERF-1 directly interacts with type IIa cotransporter in OK cells. Although, PTH-mediated inhibition of Pi uptake in OK cells is largely NHERF-1 dependent, the signaling pathway(s) by which this occurs is still unclear. These pathways may involve cooperativity between cAMP- and protein kinase C-dependent pathways or activation/inhibition of an unrecognized NHERF-1-dependent pathway(s). |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Originally, NHERF-2 (13) and the closely related protein, NHERF-1 (14), were thought to regulate mainly the apical Na+/H+ exchanger 3. NHERFs are now recognized to mediate many cellular functions, as do other PDZ proteins, by assembling complexes of membrane-bound, cytosolic, and cytoskeletal proteins (15, 16, 17, 18). For example, NHERF-1 interacts with the type IIa sodium-phosphate cotransporter (Na-PiIIa) (19) and colocalizes with the type IIa cotransporter, Na-Pi4, expressed in opossum kidney (OK) cells (20). Furthermore, NHERF-1 interacts with the C terminus of the PTH1R in vitro and copurifies with the receptor in membrane extracts from the renal proximal tubule (PCT) (10). Combined, these data suggest that PTH induces phosphaturia in vivo by regulating functional links between PTH1R, NHERF-1, and Na-PiIIa.
To date, only the OK cell line displays a robust PTH-mediated inhibition of Pi uptake. Mechanistically, PTH induces internalization and degradation of Na-Pi4, the type-IIa sodium-phosphate cotransporter expressed in OK cells (21, 22, 23). A subclone of wild-type (wt) OK cells, OKH, displays even more vigorous PTH-mediated accumulation of cAMP and also specifically binds radioiodinated PTH(1–34), thus demonstrating expression of a functional PTH1R (24). However, PTH fails to increase total inositol phosphates or release of intracellular calcium in OKH cells (25) and, most importantly, OKH cells lack PTH-mediated inhibition of Pi uptake (24). Here we show that OKH cells are NHERF-1 deficient and that stable expression of NHERF-1 rescues the PTH-mediated inhibition of the sodium-phosphate cotransporter in the OKH subclone. Unexpectedly, stable expression of NHERF-1 also rescued the actions of cAMP and phorbol esters, demonstrating that NHERF-1 affects the inhibition of Pi at two or more cellular sites.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Clonal OKH cell lines that stably express NHERF-1 were generated; those selected for study expressed levels of NHERF-1 (OKH-N1–4, -5, -16, -18) comparable to the wt cells (Fig. 1). Consistent with reported findings (26, 27, 28, 29), PTH treatment (4 h) of the wt OK cells inhibits Pi uptake by 60%, as compared with vehicle-treated controls, with an EC50 of 50.5 pM (Fig. 2). PTH treatment of the OKH cell line maximally inhibits Pi uptake by only 20% with an EC50 of 0.68 nM, confirming the defective phenotype (Fig. 2). Stable expression of NHERF-1 in the OKH line (OKH-N1) rescues the defect in PTH action, resulting in a maximum inhibition of Pi uptake of 60% with an EC50 of 23.5 pM (Fig. 2). NHERF-1 lacking the 30 amino acids from the C terminus (NHERF-1
erm) does not interact with the ERM (ezrin, radixin, moesin) family of proteins (30). NHERF-1erm functions as a dominant negative with respect to the regulation of the Na+/H+-exchanger 3 in OK cells (31), suggesting that this biological function requires indirect interactions with the actin cytoskeleton via association with the ERM proteins. Suppression of PTH-mediated inhibition of Pi uptake by expression of NHERF-1erm indicates that the same or similar interactions of NHERF-1 with the actin cytoskeleton are required to modulate Pi uptake (Fig. 2). Among the four clonal cell lines developed, NHERF-1 expression reproducibly restores the PTH-mediated inhibition of Pi uptake in the OKH parental line (Fig. 3). Furthermore, analysis of the raw data demonstrates that overexpression of NHERF-1 does not significantly alter the basal Pi uptake in the OKH parental line (Fig. 3).
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). Na-Pi4 is also localized to the apical surface of OKH-N1; importantly, the presence of distinct apical patches is evident (Fig. 4). Mechanistically, PTH regulates Pi uptake by inducing Na-Pi4 internalization and degradation (21, 22). As shown in Fig. 5A, PTH does not alter the membrane expression level of NaPi4 protein in OKH cells, whereas, PTH treatment of the OKH-N1 cells markedly reduces membrane-associated NaPi4. Furthermore, immunohistochemical analysis reveals that 1-h PTH treatment of OKH cells does not alter expression of Na-Pi4 on the apical surface (Fig. 5B). In contrast, 1-h PTH treatment significantly decreases the apical expression of Na-Pi4 in the OKH-N1 cells, with a concomitant increase within intracellular vesicles (Fig. 5B). After 2 h of PTH treatment, expression of Na-Pi4 is not detectable in the OKH-N1 cells but remains detectable in the OKH cell line (data not shown).
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). Treatment of the OKH-N1 stable cell line with the cAMP agonists, however, markedly increases inhibition of Pi uptake (60%) (Fig. 6). Inhibition of Pi uptake mediated by the PKC activators is also significantly increased in cells expressing NHERF-1, albeit to a lesser extent (30%) (Fig. 6).
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). Dose-response analysis reveals a poor correlation between PTH-mediated inhibition of Pi and hormone-induced generation of cAMP. As shown in Fig. 8A, the EC50 for PTH(1–34) inhibition of Pi uptake is approximately 2 orders of magnitude lower than the EC50 for the generation of cAMP. Furthermore, the cAMP antagonist, adenosine 3'-5' cyclic monophosphorothioate Rp (Rp-cAMPs), did not affect PTH-mediated inhibition of Pi uptake, but reversed the inhibition of Pi uptake mediated by 8-Br-cAMP by more than 50% (Fig. 8B).
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). NHERF-1 expression in OKH cells modestly increases PTH-stimulated accumulation of total inositol phosphates in multiple clones of this line, which is consistent with the NHERF-1-dependent activation of the AP1-luciferase reporter gene by PTH in the same cells (Fig. 9). Robust responses elicited by PMA demonstrate that PKC-dependent activation of this reporter gene is intact in both cell lines (Fig. 9B).
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60%), but much higher doses are required to activate the PKC-responsive reporter gene (Fig. 10). Conversely, PMA only modestly inhibits Pi uptake (30%), but very potently activates the PKC-responsive reporter gene (Fig. 10). Furthermore, pretreatment of OKH-N1 cells with the PKC inhibitors, calphostin C and bisindolylmaleimide, fails to reverse the PTH-mediated inhibition of Pi uptake (data not shown). As reported by Pfister et al. (23), we also observed that, unlike treatment with PTH, PMA treatment alters OK cell morphology, including rounding of cells and an apparent loss of cell-to-cell contacts (data not shown). The C terminus of the Na-PiIIa cotransporters, TRL, is consistent with a motif that interacts with PDZ domains. The Na-PiIIa cotransporter interacts with NHERF-1 in glutathione-S-transferase pull-down assays using kidney brush border extracts (19) and NHERF-1 colocalizes with transfected Na-PiIIa in wt OK cells (20). We extend these observations by demonstrating that NHERF-1 coimmunoprecipitates with Na-Pi4 in OKH-N1 cells (Fig. 11A), suggesting that NHERF-1 regulates Na-Pi4 via a direct interaction. Notably, NHERF-1 also coimmunoprecipitates with Na-Pi4 in OKH cells, albeit to a lesser extent (Fig. 11A), which is consistent with deficient, but not absent, expression of NHERF-1 in OKH cells (Fig. 1
). NHERF-1 is also detectable in OKH cells by immunohistochemical staining (Fig. 11B). However, when compared with NHERF-1 expression in the OKH-N1 cells, the scanning laser power and the gain function were doubled to detect endogenous NHERF-1 expression in OKH cells. As shown in Fig. 11B, Na-Pi4 colocalizes with NHERF-1 in both OKH and OKH-N1 cells on the apical surface. However, apical patches are present only on the OKH-N1 cells and not the OKH cells, which display a punctate staining pattern for both NHERF-1 and Na-Pi4 (Fig. 11B).
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DISCUSSION |
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The stable expression of NHERF-1 in OKH cells increases the maximal PTH-inhibited Pi uptake from 20% to 60% and lowers the EC50 from 685 pM to 23.6 pM. These data demonstrate that expression of NHERF-1 is required for maximal and sensitive PTH-inhibited Pi uptake and suggests that inhibition of Pi uptake by PTH is mediated, at least largely, by activation of NHERF-1-assembled PTH1R. NHERF-1 is expressed in or adjacent to apical membranes of the proximal tubule cells, as defined by immunohistochemistry (31). Other lines of evidence also support a vital role for NHERF-1 in PTH signaling in PCT. First, NHERF-1 is estimated to comprise up to 0.01% of the total protein in the kidney (33). Despite the high levels of NHERF-1 mRNA expressed in wt OK cells, the levels in proximal tubules from mouse kidney are substantially higher (20). Thus, to be consistent with the cellular environment in vivo, it is essential that PCT cell line models express abundant amounts of NHERF-1. Second, the renal phosphate wasting in NHERF-1 knock-out mice was associated with a 50% reduction in expression of Na-PiIIa on the brush border of proximal tubules (32). Notably, Na-PiIIa was readily visualized in intracellular vesicles, suggesting that NHERF-1 is essential for Na-PiIIa membrane targeting and/or trafficking (32). Third, Na-PiIIa binds to NHERF-1, also via a PDZ-specific interaction (19), and expression of a dominant negative NHERF-1, lacking the ERM protein interaction domain, markedly reduced the localization of Na-Pi4 to apical patches of wt OK cells (20). We extend these findings by showing that NHERF-1erm also suppressed the PTH-mediated inhibition of phosphate uptake in OKH cells (Fig. 2).
NHERF-1 restores PTH1R signaling via PLC in the OKH subclone in association with the rescue of PTH-inhibited Pi uptake. PTH-mediated accumulation of total inositol phosphates and activation of a PKC-responsive reporter gene in the OKH-N1 cells are consistent with activation of this pathway reported in the literature using wt OK cells (34, 35, 36). Several other reports suggest a role for PKC in PTH-mediated regulation of Na-Pi4: the inhibition of Pi uptake mediated by phorbol esters (23, 37), the blockade of PTH-mediated inhibition of Pi uptake by staurosporine (26), the inhibition of Pi uptake by the 3–34 fragment of PTH (23), and our current data that show NHERF-1-dependent restoration of both PTH-mediated activation of PKC and inhibition of Pi uptake in OKH-N1 cells.
However, although inhibition of Pi uptake by PTH may be due to activation of the PKC pathway, this seems unlikely to be the sole mechanism. First, phorbol ester-mediated inhibition of Pi uptake is relatively weak despite the vigorous activation of PKC. Conversely, PTH mediates robust inhibition of Pi uptake despite a relatively weak activation of a PKC reporter gene. Second, phorbol esters strikingly change the morphology, including cell rounding, of OK cells, which is not apparent with PTH treatment. This obvious morphological difference makes it difficult to conclude, with certainty, that PTH and phorbol esters affect Pi uptake through similar signaling pathways. Third, staurosporine is a relatively nonspecific protein kinase inhibitor, as demonstrated by its ability to reverse the inhibition of Pi uptake mediated by cAMP agonists (26). Last, activation of PKC by PTH(3–34) in OK cells has never been demonstrated (35).
At first glance, we found seemingly confounding results regarding the inhibition of Pi uptake by cAMP in the OKH-N1 cells. On one hand, the extent to which cAMP agonists mediate the inhibition of Pi uptake in OKH-N1 cells is in accordance with the inhibition mediated by PTH. These data could be interpreted to mean that the cAMP/PKA pathway mediates the effects of PTH. On the other hand, stable expression of NHERF-1 also reduces the PTH-elicited accumulation of cAMP by approximately 50%. Compared with wild type OK cells and a series of OK subclones, OKH cells displayed the highest level of PTH-mediated accumulation of cAMP (24), which is possibly due to the lack of NHERF-1. These data are consistent with the ability of NHERF-2 to markedly inhibit PTH1R signaling via AC in the PS120 fibroblast model (10).
Remarkably, NHERF-1 expression is required to inhibit the Pi uptake elicited by pharmacological agents that bypass the PTH1R to activate PKA (forskolin, 8-Br-cAMP) and PKC (PMA, DOG). As might be expected, the PKA inhibitor, Rp-cAMPs, blocks the effects of 8-Br-cAMP on Pi uptake. However, neither Rp-cAMPs, nor inhibitors of PKC, calphostin C and bisindolymaleimide, block PTH-mediated inhibition of Pi uptake. The major mechanism by which PTH inhibits renal Pi uptake thus appears to be more complex than previously recognized; it may require novel downstream cross-talk among PTH signaling pathways and/or signaling by the NHERF-1-assembled PTH1R through previously unrecognized pathway(s). Additionally, NHERF-1 markedly augments forskolin, 8-Br-cAMP, and PMA inhibition of phosphate uptake. These data strongly support the notion that NHERF-1 regulates one or more mechanisms, in addition to assembly of the PTH1R, that are critical for inhibition of Pi uptake by agents that bypass the receptor to activate PKA and PKC.
Comparisons among PCTs of NHERF-1-null mice, OKH, and OKH-N1 cells strongly support the notion that the level of NHERF-1 expression, not only its presence, critically determines the cellular phenotype. For example, the absence of NHERF-1 in vivo is associated with relatively lower levels of NaPiIIa in PCT apical membranes and in a partial redistribution of NaPiIIa from brush-border membranes to intracellular vesicles. In contrast, we found indistinguishable levels of NaPi4 in membranes from OKH and OKH-N1 cells and no detectable intracellular NaPi4 in untreated cells of either line. Robust and sensitive PTH-mediated inhibition of Pi uptake and PTH-induced internalization of NaPi4, however, are restored only by the expression of high levels of NHERF-1 in OKH-N1 cells, which also appears to be necessary for the formation of apical membrane patches. Hernando et al. (20) first demonstrated that NHERF-1, transfected NaPiIIa, and actin colocalize in these apical patches in parental OK cells. Our finding that NHERF-1 and endogenous NaPi4 are bound together and that high levels of NHERF-1 are associated with the formation of apical patches confirm and extend these observations. Taken together, these data lead us to postulate that levels of NHERF-1 expressed in OKH cells are sufficient to target and/or anchor NaPi4 to apical membranes, but that higher levels, in association with NaPi4, actin, and perhaps other cellular proteins, are required to form apical patches. The proteins assembled in these patches possibly constitute functional units required for the inhibition of Pi uptake by PTH and activators of PKA and PKC.
The current findings demonstrate that NHERF-1 is critical at several steps in the inhibition of Pi uptake by proximal renal cells. It rescues PTH-dependent regulation of the type IIa sodium-phosphate cotransporter in association with restoring activation of PLC and PKC and lowering of the maximal cAMP response to PTH. NHERF-1 is also critical for inhibition of Pi uptake mediated by cAMP and PKC. Finally, high levels of NHERF-1 expression localize Na-Pi4 into distinct apical patches, possibly representing organized regulatory complexes. Combined, these data suggest that NHERF-1 globally regulates sodium-phosphate cotransport in the brush-border membrane of the kidney. However, the direct pathways responsible for the PTH-mediated inhibition of Pi uptake are unclear because the mechanisms by which PTH induces internalization and degradation of the cotransporter are poorly defined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture and Transfection
The OK cell lines were cultured in 1:1 DMEM/F12 medium containing 10% fetal bovine serum and an antibiotic solution (Invitrogen, San Diego, CA) in 5% CO2 at 37 C. Full-length, hemagglutinin-tagged, human NHERF-1 (a gift from Dr. Vijaya Ramesh, Massachusetts General Hospital) or NHERF-1 lacking 30 amino acids from the C terminus (NHERF-1erm) were cloned into the pcDNA3.1/HYGRO vector (Invitrogen), transfected into OKH cells with FuGene 6 (Roche Clinical Laboratories, Indianapolis, IN), and selected with 1 mg/ml of hygromycin B (Calbiochem). Four clonal cell lines expressing the highest levels of NHERF-1 were examined.
Sodium-Phosphate Cotransport Assay
OK cells, grown to confluence, were cultured in serum-free medium plus 0.2% BSA for 16 h and treated either with vehicle (10 mM acetic acid) or various concentrations of PTH(1–34) for 4 h before analysis. All inhibitors were added 20 min before PTH. Analysis of Pi is as described (24). Briefly, cells were washed with Pi uptake buffer (in mM: 150 NaCl, 5 KCl, 0.1 K2HPO4, 1 CaCl2, 1.8 MgSO4, 20 glucose, and 10 HEPES, pH 7.4), followed by treatment with Pi uptake buffer containing 1 µCi/ml monopotassium phosphate [32P] for 5 min at room temperature in Centigrade degrees. Phosphate uptake was stopped by washing cells with 3 x 1 ml of ice-cold stop buffer (same as uptake except 150 mM tetramethylammonium chloride instead of NaCl plus 5 mM sodium arsenate). Phosphate uptake in the absence of Na+ was less than 5% of the uptake in the presence of Na+ ions. Cells were solubilized in 1% Triton X-100 and counted in a liquid scintillation counter (Beckman Instruments, Inc./Hybritech, Fullerton, CA). All studies of Pi uptake are presented as a mean percent of vehicle-treated cells ± SEM (n = 6) and statistical analysis using the Student’s t test for at least three independent experiments.
Determination of cAMP and Inositol Phosphates
OK cells, grown to confluence, were treated with serum-free media supplemented with 0.2% BSA 16 h before the assay for cAMP determination or for 24 h before the determination of total inositol phosphates by these cells cultured in the same media supplemented with 2 µCi/ml of [3H]myoinositol. For determination of cAMP, cells were treated either with vehicle (10 mM acetic acid) or PTH(1–34) in the presence of 1 mM isobutyl-methylxanthine for 20 min at 37 C and cAMP determined by RIA (NEN Life Science Products, Boston, MA). For determination of total inositol phosphates, cells were treated with 30 mM LiCl for 20 min followed by treatment either with vehicle (10 mM acetic acid) or 100 nM PTH(1–34) and analyzed as previously described (38).
AP1-Luciferase Reporter Gene Assays
OK cells, grown to 80% confluence, were transiently transfected with the 100 ng AP1-luciferase reporter gene (CLONTECH, Palo Alto, CA) and 100 ng of the pTKGH human-GH control reporter gene per well using FuGene 6 (Roche Clinical Laboratories). Twenty-four hours after transfection, cells were treated with either vehicle, PTH(1–34), or 100 nM phorbol-12-myristate-13-acetate (PMA) for 16 h, followed by the analysis of luciferase activity using the Bright-Glo luciferase assay system (Promega Corp., Madison, WI). The relative luminescence was normalized among wells by the amount of human GH secreted into the media, which was calculated using a RIA (Nichols Institute Diagnostics, San Clemente, CA).
Protein Analysis and Coimmunoprecipitation
For the determination of Na-Pi4 expression, OK cells were treated either with vehicle or PTH(1–34) for 4 h before membrane extraction. Cells were pelleted and incubated with a hypotonic buffer (25 mM HEPES, pH 7.4, and 20% glycerol) containing a protease inhibitor cocktail (Sigma, St. Louis, MO) and 1 mM phenylmethylsulfonylfluoride for 15 min on ice. Cells were passed through a homogenizer (HGM Lab Equipment, Heidelberg, Germany), and nuclei were pelleted by low-speed (300 x g) centrifugation. Membrane pellets were collected by high-speed (15,000 x g) centrifugation of the supernatants and extracted in RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 25 mM HEPES, pH 7.4) plus protease inhibitors for 15 min on ice. Membrane extracts were analyzed by SDS-PAGE under nonreducing conditions and immunoblotted with a rabbit, polyclonal Na-Pi4 antibody, as described (39). Bands were visualized using enhanced chemiluminescence (NEN Life Sciences).
For coimmunoprecipitations, membranes were resuspended in 25 mM HEPES containing the reversible cross-linker 0.25 mM dithiobis(succinimidylpropionate) (Pierce Chemical Co., Rockford, IL) and incubated for 2 h on ice. Membranes were repelleted and extracted with RIPA buffer. Membrane extracts were precleared with 1 µg of normal rabbit IgG and protein A/G plus agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 4 C, followed by an overnight incubation with a 1:200 dilution with polyclonal Na-Pi4 antibodies at 4 C. Immune complexes were pelleted with protein A/G plus agarose, washed with RIPA buffer, eluted with sodium dodecyl sulfate sample buffer supplemented with 50 mM dithiothreitol, and immunoblotted with monoclonal EBP50/NHERF-1 antibodies (Abcam Limited, Cambridge, UK; ab9526).
For immunohistochemistry, cells were cultured on microscope chamber slides (Nunc, Rochester, NY) and fixed for 30 min with 2% paraformaldehyde in PBS. Cells were then permeabilized and blocked with 5% nonfat dry milk in PBS containing 0.1% Triton X-100 for 30 min. Cells were then incubated with a 1:500 dilution of Na-Pi4 rabbit polyclonal antibodies and a 1:500 dilution of NHERF-1 monoclonal antibodies (Abcam Limited; ab9526) for 1 h, followed by incubations with appropriate, species-specific secondary antibodies conjugated with either Alexa Fluor 488 or Alexa Fluor 546 (Molecular Probes, Eugene, OR). Immunostained samples were analyzed with a Radiance 2000 (Bio-Rad Laboratories, Hercules, CA) laser confocal microscope and the associated software.
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FOOTNOTES |
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Abbreviations: AC, Adenylyl cyclase; 8-Br-cAMP, 8-bromo-cAMP; DOG, 1,2-di-octanoyl-sn-glycerol; ERM, ezrin, radixin, moesin; Na-Pi4, type IIa cotransporter; Na-PiIIa, type IIa sodium-phosphate cotransporter; NHERF-1, sodium-hydrogen exchanger regulatory factor; OK, opossum kidney; Pi, phosphate; PCT, renal proximal tubule; PDZ, PSD-95, Discs-large, and ZO1; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; PTH1R, PTH receptor; Rp-cAMPs, adenosine 3'-5' cyclic monophosphorothioate Rp; wt, wild-type.
Received for publication February 6, 2003. Accepted for publication July 15, 2003.
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J. Biber, S. M. Gisler, N. Hernando, C. A. Wagner, and H. Murer PDZ interactions and proximal tubular phosphate reabsorption Am J Physiol Renal Physiol, November 1, 2004; 287(5): F871 - F875. [Abstract] [Full Text] [PDF]
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R. Cunningham, D. Steplock, F. Wang, H. Huang, X. E, S. Shenolikar, and E. J. Weinman Defective Parathyroid Hormone Regulation of NHE3 Activity and Phosphate Adaptation in Cultured NHERF-1-/- Renal Proximal Tubule Cells J. Biol. Chem., September 3, 2004; 279(36): 37815 - 37821. [Abstract] [Full Text] [PDF]
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P. Huang, D. Steplock, E. J. Weinman, R. A. Hall, Z. Ding, J. Li, Y. Wang, and L.-Y. Liu-Chen {kappa} Opioid Receptor Interacts with Na+/H+-exchanger Regulatory Factor-1/Ezrin-Radixin-Moesin-binding Phosphoprotein-50 (NHERF-1/EBP50) to Stimulate Na+/H+ Exchange Independent of Gi/Go Proteins J. Biol. Chem., June 11, 2004; 279(24): 25002 - 25009. [Abstract] [Full Text] [PDF]
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M. J. Mahon and G. V. Segre Stimulation by Parathyroid Hormone of a NHERF-1-assembled Complex Consisting of the Parathyroid Hormone I Receptor, Phospholipase C{beta}, and Actin Increases Intracellular Calcium in Opossum Kidney Cells J. Biol. Chem., May 28, 2004; 279(22): 23550 - 23558. [Abstract] [Full Text] [PDF]
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