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

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. Defective PTH regulation of NHE3 activity and phosphate
adaptation in cultured NHERF-/- renal proximal tubule cells. J Biol Chem 279:
37815-37821, 2004.
5. Cunningham R, E X, Steplock D, Shenolikar S, Weinman EJ. Defective
PTH regulation of sodium-dependent phosphate transport in NHERF-1-/renal proximal tubule cells and wild-type cells adapted to low phosphate
media. Am J Physiol Renal Physiol 289:F933-F938, 2005.
6. Cunningham R, Steplock D, E X, Biswas RS, Wang F, Shenolikar S,
Weinman EJ. Adenoviral expression of NHERF-1 in NHERF-1 null mouse
renal proximal tubule cells restores Npt2a regulation by low phosphate
media and parathyroid hormone. Am J Physiol Renal Physiol 291:F896F901, 2006.
7. Déliot N, Hernando N, Horst-Liu Z, Gisler SM, Capuano P, Wagner CA,
Bacic D, O'Brien S, Biber J, Murer H. Parathyroid hormone treatment
induces dissociation of type IIa Na+-P(i) cotransporter-Na+/H+ exchanger
regulatory factor-1 complexes. Am J Physiol Cell Physiol 289:C159-C167,
2005.
8. Ellenberg J, Siggia ED, Moreira JE, Smith CL, Presley JF, Worman HJ,
Lippincott-Schwartz J. Nuclear membrane dynamics and reassembly in
living cells: targeting of an inner nuclear membrane protein in interphase and
mitosis. J Cell Biol 138:1193-1206, 1997.
9. Haggie PM, Stanton BA, Verkman AS. Increased diffusional mobility of
CFTR at the plasma membrane after deletion of its C-terminal PDZ binding
motif. J Biol Chem 279:5494-5500, 2004.
10. Hernando N, Déliot N, Gisler SM, Lederer E, Weinman EJ, Biber J,
Murer H. PDZ-domain interactions and apical expression of type IIa Na/P(i)
cotransporters. Proc Natl Acad Sci U S A 99:11957-11962, 2002.
11. Hernando N, Gisler SM, Pribanic S, Déliot N, Capuano P, Wagner CA,
Moe OW, Biber J, Murer H. NaPi-IIa and interacting partners. J Physiol.
2005 567:21-26, 2005.
12. Khadeer MA, Tang Z, Eiden MV, Chellaiah MA, Weinman EJ, Gupta A.
Two families of Na+-dependent phosphate transporters in the osteoclast.
Cellular distributions and protein interactions. Am J Physiol Cell Physiol 284:
C1633-C1644, 2003.
13. Khundmiri SJ, Weinman EJ, Steplock D, Cole J, Ahmad A, Baumann
PD, Barati M, Rane MJ, Lederer ED. Parathyroid hormone regulation of
NA+,K+-ATPase requires the PDZ 1 domain of sodium hydrogen exchanger
regulatory factor-1 in opossum kidney cells. J Am Soc Nephrol 16:259825607, 2005.
14. Lederer ED, Khundmiri SJ, Weinman, EJ. Role of NHERF-1 in regulation
of the activity of Na-K ATP’ase and sodium-phosphate cotransport in
epithelial cells. J Am Soc Neph 14: 1711-1719, 2003.
15. Levi M, Jameson DM, van der Meer BW. Role of BBM lipid composition
and fluidity in impaired renal Pi transport in aged rat. Am J Physiol Renal
Physiol 256:F85-94, 1989.
16. Mahon MJ. Ezrin promotes functional expression and parathyroid hormonemediated regulation of the sodium-phosphate cotransporter 2a in LLC-PK1
cells. Am J Physiol Renal Physiol 294:F667-F675, 2008.
17. 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