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Articles in PresS. Am J Physiol Renal Physiol (September 30, 2009). doi:10.1152/ajprenal.90657.2008 PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP. Edward J. Weinman1,4, Deborah Steplock1, Boyoung Cha2, Olga Kovbasnjuk2, Nicholas A. Frost1, Rochelle Cunningham1,4, Shirish Shenolikar3, Thomas A. Blanpied1, Mark Donowitz2 1 University of Maryland School of Medicine, 2Johns Hopkins University School of Medicine, Baltimore, Maryland; 3 Duke University Medical Center, Durham, North Carolina; and 4Department of Veterans Affairs Medical Center, Baltimore, Maryland Running title: PTH transiently increases percent mobile fraction of Npt2a. Address correspondence to: Edward J. Weinman, MD, Department Medicine, Division Nephrology, University Maryland, School of Medicine, Room N3W143, UHM, 22 .South Greene Street, Baltimore, MD 21202, FAX: 410-706-4195, [email protected]. Copyright © 2009 by the American Physiological Society. Email: Abstract. The renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including the 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 EGFP-Npt2a in proximal tubule-like 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 approximately 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 two-fold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to over 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. Key Words. Sodium-dependent Phosphate Transporter Type 2a, Fluorescence Recovery After Photobleaching (FRAP), Parathyroid Hormone, OK Cells, PDZ domains Introduction. 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 physiologic 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 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 physiologic adaptation to phosphate deprivation in intact animals and to exposure of proximal tubule cells to low phosphate media (5,6,20). Recent experiments have indicated also that the Npt2a/NHERF-1 complex is uniquely the target of 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 NHERF1/ezrin/actin complex is not known but we speculate that the mobility of Npt2a might be altered, at least transiently, prior to 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 on 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 it adaptor proteins such as NHERF-1. Materials and Methods. Native 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 NHERF1 containing a serine to aspartic acid mutation at position 77 using 10 μg/ml Blasticidin to maintain selection pressure (21). The level of expression of wild type and mutated NHERF-1 were similar to the NHERF-1 levels in native OK cells as determined by immunoblot of whole cell lysates (data not shown). Native OK cells were cultured on sterile Nunc Lab-Tek Two Chambered #1 German borosilicate coverglasses in DMEM/F12 media supplemented with 10% fetal bovine serum, 100 units/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/F media was supplemented with 5% fetal bovine serum to facilitate polarization and adherence to the cover slips. 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, Zurich). Each well was transfected with 2µg of one of the above pEGFP cDNA vectors using 4µl Lipofectamine 2000 in Opti-MEM media for 18 hours. The media was then changed to serum free D-MEM/F12 containing no phenol red or antibiotics, and the cells were allowed to grow for an additional 48 hours 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 100x 1.4 N.A. Plan-Apochromat oil immersion objective with a pixel size of 512 X 512 nm. For quantitative measurement of the mobile fraction and diffusion coefficient, we examined Fluorescence Recovery After Photobleaching (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 = [(F - F0)/(Fi –F0)] x 100 (%) To calculate the effective diffusion constant (Deff), the experimental data was 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 non-bleached regions. This loss was generally less than 10% and an example of wild-type EGFP-Npt2a-TRL and EGFPNpt2a-AAA are provided in Figure 2A and 2B. The mobile fraction, expressed as a percent, was calculated using Microsoft Excel and curve fitting analysis performed using Origin 6.0 (Microcal) software to calculate the effective diffusion coefficient. Where studied, cells were treated with Bis(sulfosuccinimidyl) suberate (BS3), a water-soluble cross-linking reagent (10 mM) at 4°C for 30 minutes, after which the cells were washed once with PBS and the cross-linking reaction quenched by incubating the cells for twenty minutes with a solution of Tris-HCl 50 mM in PBS. To examine the effect of PTH, cells were treated with PTH (10–7 M) and FRAP performed within 10 min, the earliest time points possible, up to 50 min. For each cell, 2 or more ROIs were identified and the results averaged to yield a single value per cell. All data are shown as Mean of means ± SEM. N=the number of cells analyzed in each group. Statistical comparison was performed using Analysis of Variance. 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 down stream 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 (Figure 1, 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 the Cterminal PDZ binding domain on the mobility of Npt2a, we studied Npt2a in which the Cterminal 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 (Figure 1, and Table 1). Thus, the association between Npt2a and its PDZ binding proteins plays a significant role in limiting the lateral mobility of Npt2a. In accord 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-GPI used here to reflect a putatively non-fixed protein with significant differences in the transmembrane domains compared to 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). Npt2a binds to NHERF-1. The C-terminus of NHERF-1 binds ezrin which in turn, binds actin. Accordingly, this multi-protein 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 Figure 3A and Table 2A, 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 ten minutes, a time in which changes in the cell surface abundance of Npt2a in the plasma membrane are not clearly evident. Over the ensuing 20 to 50 min after PTH treatment, however, there is a decrease in the cell surface abundance of Npt2a, the appearance of EGFP-Npt2a-TRL in sub-apical membrane vesicles, and a return of the percent mobile fraction to baseline and below. Prior studies by Bonjour and colleagues 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 (1). 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 two-fold increase in the percent mobile fraction was observed, suggesting the important role played by PDZ adaptor proteins on 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 (Figure 3B, Table 2B). 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 3A, 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 per cent mobile fraction of EGFP-Npt2a-TRL. As compared to OKH 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 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 3B). 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 3C). 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. 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). Grant Support. These studies were supported by grants from the National Institutes of Health DK55881 (EJW and SS) and MH080046 (TB), Research Service, Department of Veterans Affairs (EJW), and the Kidney Foundation of Maryland (RC). R. Cunningham is a recipient of a Harold Amos Faculty Development Award from the Robert Wood Johnson Foundation and NA Frost was support by an Integrative Membrane Biology training grant (5T32-GM008181). Additional support was provided by grants from the National Institutes of Health DK26523 (MD), DK61765 (MD), DK072084 (MD) and DK64388 (The Hopkins Basic Disease Development Core Center) and the Hopkins Center for Epithelial Disorders. Disclosures. None References. 1. Caverzasio J, Rizzoli R, Bonjour JP. Sodium-dependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line. J Biol Chem 261:3233-3237, 1986. 2. Cha B, Kenworthy A, Murtazina R, Donowitz M. The lateral mobility of NHE3 on the apical membrane of renal epithelial OK cells is limited by the PDZ domain proteins NHERF1/2, but is dependent on an intact actin cytoskeleton as determined by FRAP. J Cell Sci 117:3353-3365, 2004. 3. Cole JA, Forte LR, Krause WJ, Thorne PK. Clonal sublines that are morphologically and functionally distinct from parental OK cells. Am J Renal Physiol 256:F672-679, 1989. 4. Cunningham R, Steplock D, Wang F, Huang H, E X, Shenolikar S, Weinman EJ. 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Nashiki K, Taketani Y, Takeichi T, Sawada N, Yamamoto H, Ichikawa M, Arai H, Miyamoto K, Takeda E. Role of membrane microdomains in PTHmediated down-regulation of NaPi-IIa in opossum kidney cells. Kidney Int 68:1137-1147, 2005. 18. Shenolikar S, Voltz J, Minkoff CM, Wade J, Weinman EJ. Targeted disruption of the mouse gene encoding a PDZ domain-containing protein adaptor, NHERF-1, promotes Npt2 internalization and renal phosphate wasting. Proc Nat Acad Sci USA 99:11470-11475, 2002. 19. Voltz JW, Brush M, Sikes S, Steplock D, Weinman EJ, Shenolikar S. Phosphorylation of PDZ1 domain attenuates NHERF-1 binding to cellular targets. J Biol Chem 282:33879-33887, 2007. 20. Weinman EJ, Bodetti A, Akom M, Cunningham R, Wang F, Wang Y, Liu, J, Steplock D, Shenolikar S, Wade JB. NHERF-1 is required for renal adaptation to a low phosphate diet. Am J Physiol Renal Physiol 285: F1225F1232, 2003. 21. Weinman EJ, Biswas, RS, Peng Q, Shen L, Turner CL, E X, Steplock D, Shenolikar S, Cunningham R. Parathyroid hormone inhibits renal phosphate transport by phosphorylation of serine 77 of sodium-hydrogen exchanger regulatory factor-1. J Clin Invest 117:3412-3420, 2007. Figure Legends. Figure 1. Representative FRAP curves for EGFP-fusion proteins representing GPI, wildtype 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 BS3. Figure 2. Representative figures showing pre-bleach and post-bleach fluorescence in native OK cells expressing EGFP-Npt2a-TRL (Panel A) or EGFP-Npt2a-TRL (Panel 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. Figure 3. The effect of PTH as a function of time (min) on the Percent Mobile Fraction of EGFP-Npt2a-TRL (Panel A) and EGFP-Npt2a-AAA (Panel B). Results are expressed as the Mean of means ± SEM. * = p < 0.05 versus Control. Tables. Table 1. Percent Mobile Fraction (Mf) and Effective Diffusion Constant (Deff) of EGFPNpt2a-TRL, EGFP-Npt2a-AAA, and EGFP-GPI expressed in native OK cells. EGFP-GPI Mf (%) Deff (x10-11 cm2/sec) EGFP-Npt2a- EGFP-Npt2a- EGFP-Npt2a- TRL AAA TRL plus BS3 60 ± 7* (n=4) 17 ± 2 (n=6) 38 ± 5* (n=4) 1 ±1 * (n=6) NC 2.7 ± 0.5 2.7 ± 0.2 NC Wild-type GPI, wild-type Npt2a (EGFP-Npt2a-TRL), or Npt2a containing a Cterminal mutation of TRL to AAA (EGFP-Npt2a-AAA) were expressed in native OK cells and the Mobile Fraction (Mf) and Effective Diffusion Constants determined using FRAP. The effect of the water soluble cross linker BS3 on EGFP-Npt2a-TRL was also determined. Results are expressed as the Mean of means ± SEM. N=total number of cells studied. * = p < 0.05 compared to EGFP-Npt2a-AAA. NC=not calculated. Table 2. The effect of PTH on the Percent Mobile Fraction (Mf) and Effective Diffusion Constant (Deff) of EGFP-Npt2a-TRL and EGFP-Npt2a-AAA expressed in native OK cells. A. EGFP-Npt2a-TRL Mf (%) Deff (x10-11 cm2/sec) Control 1-10 min 11-20 min 21-30 min >30 min 18 ± 1 30 ± 3* 13 ± 2* 16 ± 3 18 ± 2 (n=13) (n=8) (n=11) (n=7) (n=7) 3.4 ± 0.3 3.8 ± 0.4 3.3 ± 0.1 3.2 ± 0.2 3.2 ± 0.1 B. EGFP-Npt2a-AAA Mf (%) Deff (x10-11 cm2/sec) Control 1-10 min 11-20 min 21-30 min >30 38 ± 4 33 ± 4 35 ± 7 36 ± 6 38 ±4 (n=11) (n=11) (n=6) (n=6) (n=5) 2.9 ± 0.3 2.1 ± 0.3 2.2 ± 0.3 3.3 ± 0.3 2.5 ± 0.2 The effect of PTH as a function of time of treatment on the Mobile Fraction (Mf) and Effective Diffusion Constant of wild-type Npt2a (EGFP-Npt2a-TRL) (Table 2A) or EGFP-Npt2a-AAA (Table 2B) expressed in native OK cells. Results are expressed as the Mean of means ± SEM. N=total number of cells studied. * = p< 0.05 compared to non-treated Control cells. Table 3. The effect of PTH on the Percent Mobile Fraction (Mf) and Effective Diffusion Constant (Deff) 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. A. OK-H cells transfected with the empty pcDNA6/His vector. Mf (%) Deff (x10-11 cm2/sec) Control 1-10 min 11-20 min 21-30 min >30 min 48 ± 4 45 ± 4 48 ± 3 49 ± 5 NT (n=7) (n=6) (n=7) (n=6) 2.7 ± 0.5 2.9 ± 0.4 2.9 ± 0.4 2.6 ± 0.2 NT B. OK-H cells expressing wild-type NHERF-1. Mf (%) Deff (x10-11 cm2/sec) Control 1-10 min 11-20 min 21-30 min >30 39 ± 2 50 ± 3* 44 ± 5 40 ± 4 NT (n=11) (n=9) (n=7) (n=9) 3.0 ± 0.7 2.8 ± 0.6 3.4 ± 0.3 2.7 ± 0.3 NT c. OK-H cells expressing NHERF-1 containing a S77D mutation. Mf (%) Deff (x10-11 cm2/sec) Control 1-10 min 11-20 min 21-30 min >30 51 ± 1 50 ± 3 50 ± 3 51 ± 4 NT (n=7) (n=5) (n=5) (n=5) 3.3 ± 0.7 3.1 ± 0.6 2.8 ± 0.5 2.8 ± 0.3 NT The effect of PTH as a function of time of treatment on the Mobile Fraction (Mf) and Effective Diffusion Constant of wild-type Npt2a (EGFP-Npt2a-TRL) expressed in OK-H cells transfected with the empty pcDNA6/His vector (Panel A), wild-type NHERF1 (Panel B), or NHERF-1 containing a S77D mutation (Panel C). Results are expressed as the Mean of means ± SEM. N=total number of cells studied. NT = Not Tested. * = p< 0.05 compared to non-treated Control cells. Figure 1. 120% N o r m a liz e d F lu o r e s c e n c e 100% 80% EGFP-GPI 60% EGFP-Npt2a-AAA 40% 20% EGFP-Npt2a-TRL EGFP-Npt2a-TRL + BS3 0% 0 100 200 300 400 Time (sec) 500 600 700 Figure 2A. EGFP-Npt2a-TRL Figure 2B EGFP-Npt2a-AAA Figure 3A. * M o b ile F r a c tio n (% ) 40 * 30 20 ** 10 0 Control 1-10 11-20 PTH (min) 21-30 >30 Figure 3B. M o b ile F r a c tio n (% ) 50 40 30 20 10 0 Control 1-10 11-20 PTH (min) 21-30 >30