Download PTH transiently increases the percent mobile fraction of Npt2a in OK

Am J Physiol Renal Physiol 297: F1560–F1565, 2009.
First published 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. Weinman,1,4 Deborah Steplock,1 Boyoung Cha,2 Olga Kovbasnjuk,2 Nicholas A. Frost,1
Rochelle Cunningham,1,4 Shirish Shenolikar,3 Thomas A. Blanpied,1 and Mark Donowitz2
1
University of Maryland School of Medicine, 2Johns Hopkins University School of Medicine, Baltimore; 3Duke University
Medical Center, Durham, North Carolina; and 4Department of Veterans Affairs Medical Center, Baltimore, Maryland
Submitted 4 November 2008; accepted in final form 28 September 2009
sodium-dependent phosphate transporter type 2a; fluorescence recovery after photobleaching; parathyroid hormone; OK cells; PDZ domains
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-
RECENT EXPERIMENTS HAVE INDICATED
Address for reprint requests and other correspondence: E. J. Weinman, Dept.
of Medicine, Div. of Nephrology, Univ. of Maryland School of Medicine, Rm.
N3W143, UHM, 22 South Greene St., Baltimore, MD 21202 (e-mail:
[email protected]).
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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 NHERF1/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
␮l 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 ␮g/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 ␮g/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
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Weinman EJ, Steplock D, Cha B, Kovbasnjuk O, Frost NA, Cunningham R, Shenolikar S, Blanpied TA, Donowitz M. PTH transiently
increases the percent mobile fraction of Npt2a in OK cells as determined by
FRAP. Am J Physiol Renal Physiol 297: F1560–F1565, 2009. First published September 30, 2009; doi:10.1152/ajprenal.90657.2008.—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-Npt2aAAA) 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.
PTH TRANSIENTLY INCREASES PERCENT MOBILE FRACTION OF Npt2a
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 ␮g of one of the above pEGFP cDNA
vectors using 4 ␮l 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
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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 ␮m wide and 2– 4 ␮m
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
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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-Npt2aAAA as well as the magnitude of loss of fluorescence intensity in non-photobleached regions of the
cell.
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PTH TRANSIENTLY INCREASES PERCENT MOBILE FRACTION OF Npt2a
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 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 䡠 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
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
M f ⫽ [(F ⬁ ⫺ F 0)/(F i ⫺ F 0)] ⫻ 100 (%)
To calculate the effective diffusion constant (Deff), the experimental data were fit to the empirical formula (2, 8)
F(t) ⫽ F 0 ⫹ F ⬁ {1 ⫺[w 2(w 2 ⫹ 4␲D efft) ⫺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 ␮m.
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
Table 1. Percent mobile fraction and effective diffusion constant of EGFP-Npt2a-TRL, EGFP-Npt2a-AAA, and EGFP-GPI
expressed in native OK cells
Mf, %
Deff, ⫻10⫺11 cm2/s
EGFP-GPI
EGFP-Npt2a-TRL
EGFP-Npt2a-AAA
EGFP-Npt2a-TRL⫹BS3
60⫾7* (n⫽4)
NC
17⫾2 (n⫽6)
2.7⫾0.5
38⫾5* (n⫽4)
2.7⫾0.2
1⫾1* (n⫽6)
NC
Values are means ⫾ SE; n ⫽ total number of cells studied. Wild-type glycosylphosphatidylinositol (GPI), wild-type sodium-dependent phosphate transporter
2a [Npt2a; enhanced green fluorescent protein (EGFP)-Npt2a-TRL], or Npt2a containing a C-terminal mutation of TRL to AAA (EGFP-Npt2a-AAA) was
expressed in native opossum kidney (OK) cells, and the mobile fraction (Mf) and effective diffusion constants (Deff) were determined using fluorescence recovery
after photobleaching (FRAP). The effect of the water-soluble cross-linker BS3 on EGFP-Npt2a-TRL was also determined. NC, not calculated. *P ⬍ 0.05
compared with EGFP-Npt2a-AAA.
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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 EGFPNpt2a-TRL expressed in cells treated with the water-soluble cross-linker
Bis(sulfosuccinimidyl) suberate (BS3).
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 EGFPNpt2a-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-
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PTH TRANSIENTLY INCREASES PERCENT MOBILE FRACTION OF Npt2a
Npt2a-AAA was significantly higher than wild-type EGFPNpt2a-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
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
EGFP-Npt2a-TRL
Control
Mf, %
Deff, ⫻10⫺11 cm2/s
1–10 min
18⫾1 (n⫽13)
3.4⫾0.3
30⫾3* (n⫽8)
3.8⫾0.4
11–20 min
21–30 min
⬎30 min
13⫾2* (n⫽11)
3.3⫾0.1
16⫾3 (n⫽7)
3.2⫾0.2
18⫾2 (n⫽7)
3.2⫾0.1
EGFP-Npt2a-AAA
Mf, %
Deff, ⫻10⫺11 cm2/s
Control
1–10 min
11–20 min
21–30 min
⬎30 min
38⫾4 (n⫽11)
2.9⫾0.3
33⫾4 (n⫽11)
2.1⫾0.3
35⫾7 (n⫽6)
2.2⫾0.3
36⫾6 (n⫽6)
3.3⫾0.3
38⫾4 (n⫽5)
2.5⫾0.2
Values are means ⫾ SE; n⫽total number of cells studied. The effect of parathyroid hormone (PTH) as a function of time of treatment on the mobile fraction
and effective diffusion constant of wild-type Npt2a (EGFP-Npt2a-TRL) or EGFP-Npt2a-AAA expressed in native OK cells is shown. *P ⬍ 0.05 compared with
nontreated control cells.
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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-Npt2aAAA (B). Results are expressed as means ⫾ SE. *P ⬍ 0.05 vs. control.
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
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.
The early increase in the mobile fraction of wild-type EGFPNpt2a-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-Npt2aTRL 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
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PTH TRANSIENTLY INCREASES PERCENT MOBILE FRACTION OF Npt2a
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
OK-H Cells Transfected with Empty pcDNA6/His Vector
Mf, %
Deff, ⫻10⫺11 cm2/s
Control
1–10 min
11–20 min
21–30 min
⬎30 min
48⫾4 (n⫽7)
2.7⫾0.5
45⫾4 (n⫽6)
2.9⫾0.4
48⫾3 (n⫽7)
2.9⫾0.4
49⫾5 (n⫽6)
2.6⫾0.2
NT
NT
Control
1–10 min
11–20 min
21–30 min
⬎30 min
39⫾2 (n⫽11)
3.0⫾0.7
50⫾3* (n⫽9)
2.8⫾0.6
44⫾5 (n⫽7)
3.4⫾0.3
40⫾4 (n⫽9)
2.7⫾0.3
NT
NT
OK-H Cells Expressing Wild-Type NHERF-1.
Mf, %
Deff, ⫻10⫺11 cm2/s
OK-H Cells Expressing NHERF-1 Containing a S77D Mutation
1–10 min
11–20 min
21–30 min
⬎30 min
51⫾1 (n⫽7)
3.3⫾0.7
50⫾3 (n⫽5)
3.1⫾0.6
50⫾3 (n⫽5)
2.8⫾0.5
51⫾4 (n⫽5)
2.8⫾0.3
NT
NT
Values are means ⫾ SE; n⫽total number of cells studied. Effect of PTH as a function of time of treatment on the mobile fraction and effective diffusion
constant of wild-type Npt2a (EGFP-Npt2a-TRL) expressed in OK-H cells transfected with the empty pcDNA6/His vector, wild-type sodium-hydrogen exchanger
regulatory factor-1 (NHERF-1), or NHERF-1 containing a S77D mutation is shown. NT, not tested. *P ⬍ 0.05 compared with nontreated control cells.
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 EGFPNpt2a-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
AJP-Renal Physiol • VOL
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.
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.
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Mf, %
Deff, ⫻10⫺11 cm2/s
Control
PTH TRANSIENTLY INCREASES PERCENT MOBILE FRACTION OF Npt2a
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