Abstract

Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was ∼17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-1. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis.

  • sodium-dependent phosphate transporter type 2a
  • fluorescence recovery after photobleaching
  • parathyroid hormone
  • OK cells
  • PDZ domains

recent experiments have indicated that a number of transporters, ion channels, and receptors form highly ordered protein complexes and that these complexes play an important role in the physiological regulation of cell function. Npt2a, one of the major sodium-dependent phosphate transporters in the proximal tubule of the kidney, for example, is known to associate with a large number of binding proteins, including adaptor proteins containing PDZ-protein interactive domains (10, 11, 18). To date, the association between Npt2a and sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) in renal proximal tubule cells has been best characterized. Npt2a binds to the first PDZ domain of NHERF-1. The C terminus of NHERF-1 binds ezrin, thereby providing linkage of the complex to the actin cytoskeleton (10, 11, 16). NHERF-1 appears to function as a membrane retention signal for Npt2a and is critical for the physiological adaptation to phosphate deprivation in intact animals and to exposure of proximal tubule cells to low-phosphate media (5, 6, 20). Recent experiments have also indicated that the Npt2a/NHERF-1 complex is uniquely the target of parathyroid hormone (PTH). PTH interaction with the PTH 1 receptor activates protein kinase C- and protein kinase A- mediated pathways, resulting in the phosphorylation of NHERF-1, the dissociation of Npt2a/NHERF-1 complexes, the endocytosis of Npt2a, and inhibition of phosphate transport (4, 6, 7, 21). The fate of Npt2a dissociated from the NHERF-1/ezrin/actin complex is not known, but we speculate that the mobility of Npt2a might be altered, at least transiently, before its association with other proteins that result in its endocytosis and degradation (21). In the present experiments, we studied the role of interactions between Npt2a and PDZ adaptor proteins and the interplay among these adaptor proteins in the effect of PTH on the mobility of Npt2a in a model renal proximal tubule cell line using fluorescence recovery after photobleaching (FRAP). The results indicate that Npt2a is relatively immobile in the apical membrane of these cells due, in part, to its association with PDZ proteins. PTH treatment results in a rapid but transient increase in the percent mobile fraction of the transporter, thereby highlighting the dynamic interaction between Npt2a and its adaptor proteins such as NHERF-1.

MATERIALS AND METHODS

Native opossum kidney (OK) cells and OK-H cells, a cell line with markedly decreased endogenous NHERF-1 (kindly provided by Dr. Judith A. Cole, University of Memphis, and Drs. Eleanor D. Lederer and Sayed Jalal Khundmiri, University of Louisville) were used in the current experiments. The OK-H cells were stably transfected using 4 μl Lipofectamine 2000 with an empty pcDNA6/His vector, wild-type NHERF-1, or NHERF-1 containing a serine-to-aspartic acid mutation at position 77 using 10 μg/ml blasticidin to maintain selection pressure (21). The levels of expression of wild-type and mutated NHERF-1 were similar to the NHERF-1 levels in native OK cells as determined by immunoblotting of whole cell lysates (data not shown). Native OK cells were cultured on sterile Nunc Lab-Tek II Chamber (configuration 1) German borosilicate coverglasses in DMEM/F12 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO2-95% air atmosphere. OK-H cells were handled in a similar manner except the DMEM/F12 media was supplemented with 5% fetal bovine serum to facilitate polarization and adherence to the coverslips. Cells were grown to 100% confluence and serum starved overnight. Full-length wild-type mouse Npt2a cDNA and Npt2a-AAA, in which AAA was substituted for TRL in the C terminus, was cloned into the pEGFP-C1 vector (kindly provided by N. Hernando, University of Zurich). Each well was transfected with 2 μg of one of the above pEGFP cDNA vectors using 4 μl Lipofectamine 2000 in Opti-MEM media for 18 h. The media was then changed to serum-free DMEM/F12 containing no phenol red or antibiotics, and the cells were allowed to grow for an additional 48 h before study.

Cells were imaged in a static bath containing DMEM/F12 containing no penicillin, streptomycin, serum, or phenol red. Experiments were conducted at 37°C using a Zeiss LSM 510 confocal microscope equipped with an environmental chamber containing a heated, covered stage continuously superfused with humidified 5% CO2. Images were collected using the 488-nm line of a 400-mW Kr/Ar laser in conjunction with a Zeiss 100 × 1.4 numerical aperture Plan-Apochromat oil-immersion objective with a pixel size of 512 × 512 nm.

For quantitative measurement of the mobile fraction and diffusion coefficient, we examined FRAP of an area 2 μm wide and 2–4 μm long directed at regions of bright fluorescence near the cell periphery. Fluorescence was measured at low laser power (30% power, 1% transmission), and regions of interest (ROIs) were photobleached at higher intensity (10 iterations of 30% power, 100% transmission at a slower scan speed) to achieve 50–70% of the initial intensity. In preliminary experiments, recovery rates from a 100% reduction in initial intensity were found to be the same as a reduction of 50–70%. Recovery was monitored until the intensity reached a plateau, usually within 10 min.

The mobile fraction was determined by comparing the fluorescence intensity in the bleached region after full recovery (F) with the fluorescence intensity before bleaching (Fi) and just after bleaching (F0) using the equation Mf=[(FF0)/(FiF0)]×100(%)

To calculate the effective diffusion constant (Deff), the experimental data were fit to the empirical formula (2, 8) F(t)=F0+F{1[w2(w2+4πDefft)1]1/2} where F(t) = intensity as a function of time; F0 = intensity just after bleaching; F = final intensity reached after complete recovery; and w = strip width of 2 μm.

Fluorescence intensities were measured with the LSM 510 FRAP software. Intensities were normalized for loss of fluorescence in nonbleached regions. This loss was generally <10%, and examples of wild-type EGFP-Npt2a-TRL and EGFP-Npt2a-AAA are provided in Fig. 1, A and B. The mobile fraction, expressed as a percentage, was calculated using Microsoft Excel, and curve-fitting analysis was performed using Origin 6.0 (Microcal) software to calculate the effective diffusion coefficient.

Fig. 1.

Representative figures showing prebleach and postbleach fluorescence in native opossum kidney (OK) cells expressing wild-type sodium-dependent phosphate transporter 2a [enhanced green fluorescent protein (EGFP)-Npt2a-TRL; A] or C-terminal mutated EGFP-Npt2a-AAA (B) as a function of time. The figure also illustrates the relative levels of expression of EGFP-Npt2a-TRL and EGFP-Npt2a-AAA as well as the magnitude of loss of fluorescence intensity in non-photobleached regions of the cell.

Where studied, cells were treated with Bis(sulfosuccinimidyl) suberate (BS3), a water-soluble cross-linking reagent (10 mM) at 4°C for 30 min, after which the cells were washed once with PBS and the cross-linking reaction was quenched by incubating the cells for 20 min with a solution of 50 mM Tris·HCl in PBS. To examine the effect of PTH, cells were treated with PTH (10−7 M) and FRAP was performed within 10 min, the earliest time point possible, up to 50 min. For each cell, two or more ROIs were identified and the results were averaged to yield a single value per cell. All data are shown as means ± SE; n = the number of cells analyzed in each group. Statistical comparison was performed using ANOVA.

RESULTS AND DISCUSSION

A number of transporters, ion channels, and receptors including Npt2a, a major sodium-dependent phosphate transporter in renal proximal tubule cells, form multiprotein complexes in the plasma membranes of target cells. Npt2a binds to a number of PDZ adaptor proteins including NHERF-1 (10, 11). As determined in NHERF-1 null proximal tubule cells, NHERF-1 acts as a retention signal for Npt2a and the Npt2a/NHERF-1 complex is the downstream target of protein kinase cascades initiated by PTH occupation of the PTH1 receptor (19, 20). Recent evidence has been presented that PTH exposure results in the dissociation of Npt2a/NHERF-1 complexes in OK cells and in renal proximal tubule cells; a process necessary for the endocytosis of Npt2a and inhibition of phosphate transport (7, 21).

The percent mobile fraction of wild-type EGFP-Npt2a-TRL was 17 ± 2%, and treatment of the cells with the water-soluble cross-linker BS3 decreased the fractional mobility to near zero (Fig. 2, Table 1). This indicates that under the conditions of these experiments and over the time course examined, the recovery of Npt2a relates to the lateral diffusion of the fluorescent-labeled transporter rather than to the recruitment of unbleached transporters to the surface of the cell. To assess the contribution of the C-terminal PDZ binding domain to the mobility of Npt2a, we studied Npt2a in which the C-terminal TRL amino acid sequence was mutated to AAA. The percent mobile fraction of EGFP-Npt2a-AAA was significantly higher (38 ± 5%) than wild-type EGFP-Npt2a-TRL (Fig. 2, Table 1). Thus the association between Npt2a and its PDZ binding proteins plays a significant role in limiting the lateral mobility of Npt2a. In accordance with this finding, the lateral mobility of both NHE3 and CFTR, two other proteins that associate with a significant number of binding proteins including NHERF-1, were also increased when C-terminal mutations predicted to decrease binding to PDZ domains were examined (2, 9). While the percent mobile fraction of EGFP-Npt2a-AAA was significantly higher than wild-type EGFP-Npt2a-TRL, it was significantly lower than EGFP-glycosylphosphatidylinositol used here to reflect a putatively nonfixed protein with significant differences in the transmembrane domains compared with Npt2a. This finding may indicate the association of Npt2a with other non-PDZ domain-containing proteins or the association of Npt2a with PDZ domain-containing proteins using internal rather than C-terminal sequences of the transporter for binding. Indeed, in OK cells and in an osteoclast cell line, evidence has been advanced that the binding of Npt2a to NHERF-1 may also involve internal amino acid sequences (12, 14).

Fig. 2.

Representative fluorescence recovery after photobleaching (FRAP) curves for EGFP-fusion proteins representing glycosylphosphatidylinositol (GPI), wild-type Npt2a-TRL, and C-terminal mutated Npt2a-AAA expressed in native OK cells. Also shown is a representative recovery curve of EGFP-Npt2a-TRL expressed in cells treated with the water-soluble cross-linker Bis(sulfosuccinimidyl) suberate (BS3).

View this table:
Table 1.

Percent mobile fraction and effective diffusion constant of EGFP-Npt2a-TRL, EGFP-Npt2a-AAA, and EGFP-GPI expressed in native OK cells

Npt2a binds to NHERF-1. The C terminus of NHERF-1 binds ezrin, which, in turn, binds actin. Accordingly, this multiprotein complex links the transporter to the cytoskeleton of the cell. By virtue of the findings that PTH phosphorylates NHERF-1 and dissociates Npt2a/NHERF-1 complexes, we have speculated that there might be a phase where Npt2a is detached from NHERF-1 with a change in its mobility within the plane of the plasma membrane (7, 19). As shown in Fig. 3A and Table 2, we examined the time course of the change in mobility of EGFP-Npt2a-TRL in native OK cells following treatment of the cells with PTH. PTH treatment resulted in an increase in the percent mobile fraction of EGFP-Npt2a-TRL to 30% when studied in the first 10 min, a time in which changes in the cell surface abundance of Npt2a in the plasma membrane are not clearly evident. Over the ensuing 20–50 min after PTH treatment, however, there is a decrease in the cell surface abundance of Npt2a, the appearance of EGFP-Npt2a-TRL in subapical membrane vesicles, and a return of the percent mobile fraction to baseline and below. Prior studies by Bonjour and colleagues (1) have documented increases in cAMP accumulation and decreases in sodium-dependent phosphate transport in OK cells as early as 5 min after treatment with PTH.

Fig. 3.

Effect of parathyroid hormone (PTH) as a function of time (minutes) on the percent mobile fraction of EGFP-Npt2a-TRL (A) and EGFP-Npt2a-AAA (B). Results are expressed as means ± SE. *P < 0.05 vs. control.

View this table:
Table 2.

Effect of PTH on the percent mobile fraction and effective diffusion constant of EGFP-Npt2a-TRL and EGFP-Npt2a-AAA expressed in native OK cells

The early increase in the mobile fraction of wild-type EGFP-Npt2a-TRL in response to PTH would be consistent with PTH-mediated dissociation of Npt2a from NHERF-1, as we previously proposed (21). It is important to note, however, that this is not precisely equivalent to the results obtained with EGFP-Npt2a-AAA since the detached wild-type transporter would have the opportunity to associate with other PDZ binding proteins. In fact, we think it remarkable that a nearly twofold increase in the percent mobile fraction was observed, suggesting the important role played by PDZ adaptor proteins in the mobility of Npt2a. On the other hand, the reason for the decline in the mobile fraction with longer exposures to PTH is unknown. One consideration is that there is less EGFP-Npt2a-TRL to diffuse into the bleached area since endocytosis of Npt2a is well established at these longer times of PTH exposure. Moreover, labeled transporter diffusing into the bleached area would be rapidly removed. The net effect would be to yield a decrease in the apparent mobile fraction as measured by FRAP. We also considered the possibility that PTH may affect other cell components including the lipid composition of the plasma membranes (15, 17). To this end, we examined the effect of PTH on the mobility of EGFP-Npt2a-AAA expressed in native OK cells (Fig. 3B, Table 2). PTH had no effect on the percent mobile fraction of Npt2a-AAA in the early or longer times of exposure periods. These results highlight the importance of PDZ binding domains in the early increase in Npt2a mobility and would tend to exclude nonspecific effects of PTH on the cells in the longer term decrease in mobility.

To examine the role of NHERF-1 in particular, we determined the percent mobile fraction of Npt2a expressed in OK-H cells, an OK cell line that was originally isolated due to its inability to respond to PTH and subsequently shown to have a marked decrease in the abundance of NHERF-1 (3, 13). As shown in Table 3, the basal percent mobile fraction of EGFP-Npt2a-TRL in OK-H cells transfected with the empty pcDNA6/His vector was significantly higher than in native OK cells. In these cells, PTH had no effect on the percent mobile fraction of EGFP-Npt2a-TRL. Compared with OK-H cells transfected with the empty vector, OK-H cells stably expressing wild-type NHERF-1 had a significantly lower baseline mobility of EGFP-Npt2a-TRL (48 ± 4 vs. 39 ± 2%, P < 0.05). The percent mobile fraction of EGFP-Npt2a-TRL in OK-H cells expressing NHERF-1, however, was significantly higher than in native OK cells. This may suggest that the lack of NHERF-1 affects the assembly of protein complexes that determine the mobility of Npt2a or that OK-H cells have other alterations in protein expression in addition to decreased NHERF-1. By contrast to the OK-H cells expressing the empty vector, OK-H cells expressing NHERF-1 showed a transient increase in the percent mobile fraction of Npt2a in response to PTH (Table 3). Our prior studies have indicated that PTH results in the phosphorylation of serine 77 in the first PDZ domain of NHERF-1, thereby resulting in the dissociation of NHERF-1/Npt2a complexes and inhibition of phosphate transport (21). To extend these observations, we determined the mobility of EGFP-Npt2a-TRL in OK-H cells expressing NHERF-1 containing a phosphomimetic serine 77 aspartic acid mutation. In these cells, the basal percent mobile fraction of EGFP-Npt2a-TRL was higher than in OK-H cells expressing wild-type NHERF-1 and did not change in response to PTH (Table 3). Taken together, these findings indicate that NHERF-1 is likely one of the major factors affecting the mobility of Npt2a and that Npt2a not tethered to NHERF-1 is not responsive to PTH.

View this table:
Table 3.

Effect of PTH on the percent mobile fraction and effective diffusion constant of EGFP-Npt2a-TRL in OK-H cells expressing the empty pcDNA6/His vector, wild-type NHERF-1, or NHERF-1 containing a S77D mutation

In summary, these experiments were designed to determine the mobile fraction of Npt2a in the plasma membrane of proximal tubule-like OK cells. The relatively limited mobility of Npt2a is due, at least in part, to its association with PDZ proteins, in general, and with NHERF-1, specifically. PTH has a biphasic effect on the percent mobile fraction of Npt2a, resulting in an early increase followed by a time-dependent decrease. Based on these findings and in association with our prior studies, we would speculate that the PTH-mediated increase in the percent mobile fraction of Npt2a represents dissociation of the transporter from NHERF-1 and that this reaction may be critical for engagement of Npt2a with processes that mediate its removal from the plasma membrane and, subsequently, a decrease in the reabsorption of phosphate in renal proximal tubule cells (7, 21).

GRANTS

These studies were supported by National Institutes of Health (NIH) Grants DK55881 (E. J. Weinman and S. Shenolikar) and MH080046 (T. A. Blanpied), Research Service, Department of Veterans Affairs (E. J. Weinman), and the Kidney Foundation of Maryland (R. Cunningham). R. Cunningham is a recipient of a Harold Amos Faculty Development Award from the Robert Wood Johnson Foundation, and N. A. Frost was support by an Integrative Membrane Biology training grant (5T32-GM008181). Additional support was provided by NIH Grants DK26523, DK61765, and DK072084 (M. Donowitz) and DK64388 (The Hopkins Basic Disease Development Core Center) and the Hopkins Center for Epithelial Disorders.

DISCLOSURES

No conflicts of interest are declared by the authors.

REFERENCES

  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
  13. 13.
  14. 14.
  15. 15.
  16. 16.
  17. 17.
  18. 18.
  19. 19.
  20. 20.
  21. 21.
View Abstract