From the Departments of Physiology and
Medicine, Gastrointestinal Division, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205 and
§ Department of Physiology, College of Medicine, Pusan
National University, Pusan 602-739, Republic of Korea
Received for publication, January 17, 2003, and in revised form, February 12, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Na+/H+
exchanger 3 (NHE3) kinase A regulatory protein (E3KARP) has been
implicated in cAMP- and Ca2+-dependent
inhibition of NHE3. In the current study, a new role of E3KARP is
demonstrated in the stimulation of NHE3 activity. Lysophosphatidic acid
(LPA) is a mediator of the restitution phase of inflammation but has
not been studied for effects on sodium absorption. LPA has no effect on
NHE3 activity in opossum kidney (OK) proximal tubule cells, which lack
expression of endogenous E3KARP. However, in OK cells exogenously
expressing E3KARP, LPA stimulated NHE3 activity. Consistent with the
stimulatory effect on NHE3 activity, LPA treatment increased the
surface NHE3 amount, which occurred by accelerating exocytic
trafficking (endocytic recycling) to the apical plasma membrane. These
LPA effects only occurred in OK cells transfected with E3KARP. The
LPA-induced increases of NHE3 activity, surface NHE3 amounts, and
exocytosis were completely inhibited by pretreatment with the PI
3-kinase inhibitor, LY294002. LPA stimulation of the phosphorylation of Akt was used as an assay for PI 3-kinase activity. LY294002 completely prevented the LPA-induced increase in Akt phosphorylation, which is
consistent with the inhibitory effect of LY294002 on the LPA stimulation of NHE3 activity. The LPA-induced phosphorylation of Akt
was the same in OK cells with and without E3KARP. These results show
that LPA stimulates NHE3 in the apical surface of OK cells by a
mechanism that is dependent on both E3KARP and PI 3-kinase. This is the
first demonstration that rapid stimulation of NHE3 activity is
dependent on an apical membrane PDZ domain protein.
Na+/H+ exchanger 3 (NHE3)1 plays an essential
role in NaCl and NaHCO3 absorption in ileum,
colon, gallbladder, and proximal tubule of kidney (1-3). NHE3
cycles between the plasma membrane and recycling endosomal compartment
under basal conditions (4-9). NHE3 is both rapidly stimulated and
inhibited by several growth factors, neurotransmitters, and hormones
that are released as part of digestion (1). These act by altering the
amount of the NHE3 in the plasma membrane, as well as by changes in the NHE3 turnover number (1). For instance, epidermal growth factor and clonidine stimulate ileal sodium absorption by increasing the
percentage of total NHE3 in the ileal brush border (BB) (10). In PS120
fibroblasts stably transfected with NHE3, the surface NHE3 amounts are
stimulated by treatment with fibroblast growth factor (4). In opossum
kidney proximal tubule cells exogenously transfected with the
endothelin Phosphatidylinositol (PI) 3-kinase has been implicated in stimulation
of several plasma membrane transport processes, including those of
GLUT4 and NHE3 (1, 4-9, 13-15). PI 3-kinase activation is associated
with translocation of these transporters from intracellular storage
sites to the plasma membrane. Activation of PI 3-kinase results in
increased intracellular levels of 3'-phosphorylated inositol
phospholipids and induction of signaling responses, including the
activation of the Ser/Thr protein kinase Akt (6, 16). PI 3-kinase and
Akt are intermediates in the insulin-induced GLUT4 translocation to
plasma membranes, and a constitutively active Akt mutant stimulates
GLUT4 translocation (13, 17). PI 3-kinase regulates the basal and
stimulated level of plasma membrane NHE3 amount and NHE3 transport
activity. In PS120 and OK cells, basal NHE3 activity and surface amount
are decreased by ~50% by the PI 3-kinase inhibitors, wortmannin and
LY294002 (4, 7). Also, the epidermal growth factor- or fibroblast
growth factor-induced increase of NHE3 activity in PS120 fibroblasts,
Caco-2 cells, and ileal BB is inhibited by these PI 3-kinase inhibitors
(4, 18). Consistent with these results, we reported recently (6) that
constitutively active mutants of PI 3-kinase and Akt stimulated NHE3
activity in PS120 fibroblasts by increasing the NHE3 amount in the
plasma membrane, and a peptide stimulator of PI 3-kinase had the same
effect in OK cells (6). These results suggest that PI 3-kinase and Akt
activation, which is stimulated by PI 3-kinase, play necessary and
sufficient roles in the stimulation of exocytosis of NHE3 in PS120 fibroblasts.
NHERF and E3KARP, which are scaffolding proteins, possess two PDZ
domains and an ERM-binding domain (19). Both have been implicated in cAMP-dependent inhibition of NHE3 activity
(19), whereas only E3KARP is involved in Ca2+ dependent
inhibition of NHE3 (20). These PDZ domain proteins interact with NHE3
through their C-terminally extended second PDZ domain, and via their C
termini also associate with ezrin, which links to the actin
cytoskeleton and also acts as an anchoring protein for protein kinase
AII (21). Therefore, both NHERF and E3KARP physically link NHE3 to
protein kinase AII for the acute inhibition of NHE3 activity through
protein kinase A-dependent phosphorylation. The protein
kinase A-dependent phosphorylation of NHE3 is necessary for
cAMP-stimulated endocytosis of NHE3 (22). It has not been studied
whether these PDZ domain proteins are involved in rapid stimulation of
NHE3 activity and in the endocytic recycling of NHE3 to the plasma membrane.
Lysophosphatidic acid (LPA) is an inflammatory mediator (23-26). It is
a bioactive phospholipid released by activated platelets, fibroblasts,
leukocytes, phagocytes, and endothelial cells. LPA is released in high
concentrations at the site of injury in the gastrointestinal tract and
proximal tubule. LPA enhances intestinal restitution and wound
healing/remodeling (27-29). In opossum kidney proximal tubule cells,
LPA also induces proliferation, although on a more prolonged time
scale, through the activation of PI 3-kinase and the extracellular
signal-regulated kinase (ERK) (30). In this study, we demonstrate the
first evidence that LPA stimulates epithelial sodium absorption, acting
by increasing NHE3 activity and the amount of NHE3 on the plasma
membrane. This occurs by stimulation of exocytic trafficking by
activation of PI 3-kinase. E3KARP but not NHERF is necessary for
LPA-induced stimulation of NHE3 activity and increase of NHE3 in the
plasma membrane. This is the initial demonstration of E3KARP being
involved in rapid stimulation of sodium absorption.
Cell Culture and Generation of Stable Cell Lines--
OK/E3V
(NHE3 epitope tagged on the C terminus with the vesicular stomatitis
virus G-protein (VSV-G epitope)) cells (generously provided by J. Noel,
University of Montreal), as described (7, 31), were maintained in high
glucose Dulbecco's modified Eagle's medium supplemented with 25 mM NaHCO3, 10 mM HEPES, 1 mM sodium pyruvate, 10% fetal bovine serum, 100 units/ml
penicillin, and 50 µg/ml streptomycin in a 5% CO2/95%
O2 humidified incubator at 37 °C. OK/E3V cell lines were
selected for Na+/H+ exchanger activity (every
other passage) by exposing cells to an acid load consisting of 50 mM NH4Cl/94 mM NaCl solution for 1 h, followed by an isotonic 2 mM Na+
solution as described (31). A clonal cell line of OK/E3V expressing E3KARP (OK/E3V/E3KARP) was generated by stable transfection of OK/E3V
cells with a pcDNA3.1 neomycin resistance plasmid (Invitrogen) bearing E3KARP. Transfection was performed with LipofectAMINE (Invitrogen) as described (7), and cells were maintained in growth
medium containing 400 µg/ml G418 to maintain the selection pressure.
Cells resistant to the selection pressure were selected through six
passages prior to study.
Measurement of Na+/H+
Exchange--
Cellular Na+/H+ exchange
activity in OK cell lines was determined fluorometrically using the
intracellular pH-sensitive dye acetoxymethyl ester of
2'7'-bis(carboxyethyl)5-6-carboxy-fluorescein (BCECF-AM, 5 µM; Molecular Probes, Eugene, OR), as described
previously (7, 31). OK cells were plated on glass coverslips and grown until they reached 90% confluency. They were then placed in serum-free medium for 2 days before transport was studied (11, 12). For acidification and dye loading, cells were incubated in Na+
solution (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 25 mM
glucose, 20 mM HEPES, pH 7.4) containing BCECF-AM and 40 mM NH4Cl for 30 min at 37 °C. Cells were
initially perfused with TMA+ solution (130 mM
tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM
NaH2PO4, 25 mM glucose, 20 mM HEPES, pH 7.4), which was then switched to
Na+ solution for the sodium-dependent
pHi recovery. At the end of each experiment, the
fluorescence ratio was calibrated to pHi using the high
potassium/nigericin method using 130 mM
KCl/pHi buffer plus 10 µM
nigericin titrated to pH 6.0, 6.3, and 7.2. Na+/H+ exchange activity data were calculated
as the product of Na+-dependent changes in
pHi times the buffering capacity at each pHi
and were analyzed by nonlinear regression using Origin (Microcal Software) to estimate Vmax and
K'(H+)i (32). In some cases,
Na+/H+ exchange activity was described as the
rate of initial sodium-dependent alkalinization over a
minimum of 9 s, as determined by least square analysis.
Biotinylation and Immunoblotting--
The amounts of NHE3 on the
plasma membrane were measured by biotinylation of cell surface proteins
as described (7, 32). OK cells as described above were serum-starved
for 2 days and then exposed to LPA at 37 °C for the indicated times.
All subsequent steps were performed at 4 °C. Cells were rinsed twice
with ice-cold phosphate-buffered saline (150 mM NaCl, 20 mM Na2HPO4, pH 7.4) and once in
borate buffer (154 mM NaCl, 1.0 mM boric acid,
7.2 mM KCl, and 1.8 mM CaCl2, pH
9.0). Cells were then exposed to 0.5 mg/ml sulfo-NHS-SS-biotin
in borate buffer for 40 min with horizontal shaking. After labeling,
cells were washed with quenching buffer (20 mM Tris-HCl,
120 mM NaCl, pH 7.4) to scavenge the unreacted biotin.
Cells were washed three times with ice-cold phosphate-buffered saline
and lysed in 1 ml of N+ buffer (60 mM
HEPES-NaOH, pH 7.4, 150 mM NaCl, 3 mM KCl, 5 mM EDTA, 3 mM EGTA, and 1% Triton X-100) by
sonication for 20 s. Insoluble cell debris was removed by
centrifugation for 20 min at 12,000 × g. Supernatant
representing the total NHE3 fraction was incubated with
streptavidin-agarose for 2 h and then the resultant beads were
washed four times in N+ buffer to remove nonspecifically
bound proteins. The proteins bound to streptavidin-agarose beads, which
represent surface fractions, were solubilized with Laemmli buffer. The
total and surface fractions were resolved by SDS-PAGE and transferred
to nitrocellulose membrane. After blocking with 5% nonfat milk,
the blots were probed with a monoclonal anti-VSV-G antibody (P5D4) as
the primary antibody and horseradish peroxidase-conjugated anti-mouse
as the secondary antibody, and bands were visualized by enhanced
chemiluminescence (33). The densities of NHE3 protein bands were
quantitated by scanning densitometer and ImageQuant software.
Endocytic Internalization--
Endocytosis was measured by a
protocol slightly modified from the reduced GSH-resistant
endocytosis assay described previously by us (20). Cells were labeled
with 1.5 mg/ml sulfo-NHS-SS-biotin for 40 min and quenched at 4 °C.
Cells were then warmed to 37 °C, treated with LPA, dopamine
(positive control) (34), or vehicle for 30 min at 37 °C, and rinsed
with ice-cold phosphate-buffered saline twice at 4 °C. Surface
biotin was cleaved by washing with 50 mM Tris-HCl, 150 mM GSH, pH 8.8. The freshly endocytosed proteins bearing
biotin were protected from cleavage with GSH. Cells were solubilized in
N+ buffer, and biotinylated proteins were retrieved and
assayed for endocytosed NHE3 as described above.
Exocytic Insertion (Endocytic Recycling) of NHE3 to the Apical
Plasma Membrane--
To measure exocytic insertion of NHE3, NHS
reactive sites on the cell surface were first blocked by pretreatment
with sulfo-NHS-acetate as described, with some slight modifications
(12, 34). Cells were rinsed with PBS-Ca-Mg (ice-cold phosphate-buffered
saline (PBS) with 0.1 mM Ca2+ and 1 mM Mg2+) two times at 4 °C. The apical
surface was then exposed to 1.5 mg/ml sulfo-NHS-acetate in PBS-Ca-Mg
for 2 h. After quenching for 20 min, cells were rinsed with PBS at
37 °C and treated with LPA or vehicle for 30 min. Cells were then
treated with 0.5 mg/ml sulfo-NHS-SS-biotin and lysed with
N+ buffer. The biotinylated fraction, which represents
newly inserted surface proteins, was precipitated by streptavidin
agarose, and the precipitate was subjected to SDS-PAGE and Western
blotting with anti-VSV-G antibody as described above.
Establishment of OK/E3V Cells Stably Expressing
E3KARP--
To address the question whether E3KARP is involved in
stimulation of NHE3, we established an OK cell line stably expressing both NHE3V and E3KARP (OK/E3V/E3KARP cells) as described under "Experimental Procedures." The expression levels of NHERF and E3KARP in the clonal cell lines were determined. As shown in Fig. 1, E3KARP is expressed in OK/E3V/E3KARP
cells but not in parental OK or OK/E3V cells. Endogenous NHERF is
expressed in these OK cells in similar amounts. The level of E3KARP
expression is less in OK/E3V/E3KARP cells than in the PS120/E3V/E3KARP
cells we have described previously (20). Endogenous NHERF is highly
expressed in OK/E3V/E3KARP cells, in contrast to the minimal expression of endogenous NHERF in PS120 cells (35).
LPA Stimulates NHE3 Activity in an E3KARP-dependent
Fashion--
Growth factors, including epidermal growth factor and
fibroblast growth factor, increase NHE3 activity in Caco-2 intestinal epithelial cells and in PS120 fibroblasts, respectively, and endothelin stimulates NHE3 in OK cells (4, 7, 11, 12). To determine whether LPA,
as an example of the mediators of the restitution phase of
inflammation, affects NHE3 activity in proximal tubule epithelial
cells, we treated OK/E3V and OK/E3V/E3KARP cells with LPA for 30 min at
37 °C and then measured NHE3 activity. As shown in Fig.
2B, LPA treatment (100 µM, 30 min at 37 °C) caused ~50% increase in NHE3
Vmax (1533 ± 63 µM/s,
control versus 2285 ± 76 µM/s,
LPA-treated cells, p < 0.01) in OK/E3V/E3KARP cells.
In contrast, NHE3 activity was not significantly affected by LPA treatment in OK/E3V cells (Fig. 2A). The dose and time
dependence of the LPA-induced stimulation of NHE3 activity were
determined. As shown in Fig.
3A, LPA significantly
increased NHE3 activity as early as 10 min, and the
LPA-dependent activation increased up to 60 min. LPA
activated NHE3 in a concentration-dependent manner from
10 LPA Increases the Surface Amount of NHE3 in E3KARP-containing
Cells--
To determine whether the LPA-induced increase of NHE3
activity was because of an increase of amount of plasma membrane NHE3 protein, we measured the surface NHE3 amounts by cell surface biotinylation. Plasma membrane proteins were biotinylated by reaction with sulfo-NHS-SS biotin at 4 °C, and biotinylated proteins were isolated by precipitation with immobilized streptavidin-agarose. As
shown in Fig. 4A, treatment
with 10 The LPA Increase in Surface NHE3 Amount Is Because of Increased
Exocytic Insertion--
The net increase in surface NHE3 amount could
be because of a decrease in endocytic internalization and/or an
increase in exocytic insertion of NHE3. To clarify which mechanism was
involved in the increase of surface NHE3 amount, the rates of
endocytosis and exocytosis of NHE3 in OK/E3V/E3KARP cells were
measured. To quantitate the exocytic insertion, all plasma membrane
reactive sites were blocked by pretreatment with sulfo-NHS-acetate
(12). Cells were then treated with LPA or vehicle for 30 min, followed by biotinylation of cell surface proteins. Biotinylated NHE3 represents NHE3 that was initially located in intracellular compartments and then
inserted on the plasma membrane after treatment with LPA. After
treatment with LPA for 30 min, biotinylated amounts of NHE3 were
increased in OK/E3V/E3KARP cells (Fig.
5A). LPA increased the NHE3
exocytic trafficking by ~60% (Fig. 5A, inset on
right). These results indicate that exocytic trafficking of NHE3
accounts for the LPA-induced increase of surface NHE3 amount with 30 min of LPA exposure.
To quantitate the endocytic internalization of NHE3, apical membrane
proteins were labeled with sulfo-NHS-SS-biotin before treatment with
LPA or vehicle. After biotinylation for 40 min at 4 °C, cells were
exposed to reduced GSH, which removes biotin from surface proteins.
With this protocol, biotinylated NHE3 represents the pool of NHE3 that
was initially present on the apical membrane and then was endocytosed,
thus protecting it from GSH. Consistent with a previous report (34),
there was increased NHE3 internalized from plasma membrane after
treatment with dopamine for 30 min (positive control). In contrast, LPA
treatment for 30 min had no effect on the amount of endocytosed NHE3
(Fig. 5, B and inset on right). These results
suggest that endocytosis of NHE3 is not involved in the LPA-induced (30 min) increase of surface NHE3 amount.
LPA-induced NHE3 Stimulation Is PI
3-Kinase-dependent--
PI 3-kinase has been implicated
in some basal and stimulated regulation of NHE3 activity in fibroblasts
and Caco-2 cells and basal NHE3 activity in OK cells based on
biochemical studies using specific inhibitors of PI 3-kinase,
wortmannin and LY294002, and peptide stimulation of PI 3-kinase (4-7).
We also reported recently (6) that constitutively active mutants of PI
3-kinase and Akt stimulated basal NHE3 activity by increasing surface
NHE3 amounts in PS120 fibroblasts. To determine whether PI 3-kinase and
Akt were involved in the LPA-induced activation of NHE3, we examined the effect of the PI 3-kinase inhibitor, LY294002, on the LPA-induced NHE3 activation in OK/E3V/E3KARP cells. As shown in Fig.
6, pretreatment with 50 µM
LY294002 inhibited basal NHE3 activity by 50%, which is consistent
with previous reports in OK cells (7). In addition, LY294002
pretreatment completely blocked the LPA-induced stimulation of NHE3
activity. These results indicate that PI 3-kinase is necessary for the
LPA-induced stimulation of NHE3, as well as in basal NHE3 activity.
PI 3-Kinase Is Necessary for the LPA-induced Increase
of Plasma Membrane Amount and Exocytic Trafficking of NHE3--
LPA
stimulates the NHE3 activity through increasing the amounts of NHE3
localized on the apical plasma membrane in OK/E3V/E3KARP cells (Fig.
4B). To determine whether the increase of surface NHE3
amounts requires the activation of PI 3-kinase, we next examined the
effect of LY294002 on the surface NHE3 amounts in OK/E3V/E3KARP cells.
As shown in Fig. 7A, the
amounts of NHE3 localized on the plasma membrane were increased by LPA
treatment, whereas LY294002 pretreatment completely blocked the
LPA-induced increase of surface NHE3 amounts. LY294002 also caused a
small but significant decrease in basal surface NHE3 amount. These
changes in surface NHE3 parallel the inhibitory effect of LY294002 on
the LPA-stimulated, as well as basal, NHE3 activity.
The LPA-induced increase of surface NHE3 amount is because
of enhanced exocytic trafficking of NHE3 to the apical plasma membrane (Fig. 5, A and B). Therefore, we next measured
the effect of LY294002 pretreatment on the LPA-induced increase of
exocytic trafficking of NHE3. As shown in Fig. 7B, the
LPA-induced increase of exocytic trafficking was completely prevented
by pretreatment with LY294002. This shows that PI 3-kinase plays a
necessary role in the LPA-dependent increase of NHE3
exocytosis to the apical plasma membrane. In addition, LY294002
pretreatment slightly reduced the basal rate of exocytic
trafficking of NHE3 in the absence of LPA treatment.
The E3KARP Dependence of LPA-stimulated NHE3 Activity Is Not
Because of E3KARP-dependent Activation of PI
3-Kinase/Akt--
The mechanism of the E3KARP dependence of
LPA stimulation of NHE3 is not explained. The hypothesis was tested
that E3KARP increased the LPA stimulation of PI 3-kinase as the
mechanism by which E3KARP led to LPA stimulation of NHE3. The assay
used for PI 3-kinase activity was activation of Akt as assessed as amount of phosphorylated Akt. We examined the phosphorylation of Akt
after treatment with LPA in both OK/E3V and OK/E3V/E3KARP cells by
using phospho-specific antibodies for Akt, which specifically recognize
activated Akt, and anti-Akt antibodies for total Akt (Sigma). As shown
in Fig. 8 in both OK/E3V and
OK/E3V/E3KARP cells, Akt was phosphorylated by 1 min of LPA exposure,
with maximal phosphorylation at 10 min. The Akt phosphorylation was
sustained up to 30 min. Also, the magnitude of stimulation of Akt
phosphorylation relative to basal Akt and the level of basal Akt
phosphorylation were similar in OK/E3V and OK/E3V/E3KARP cells.
The LPA-induced phosphorylation of Akt was totally prevented by
pretreatment with LY294002, consistent with the inhibitory effect of
LY294002 on NHE3 activity and the exocytic trafficking of NHE3. Thus,
although PI 3-kinase activity is required for the LPA-induced
stimulation of NHE3 activity and exocytic trafficking of NHE3, these
results show that PI 3-kinase/Akt activation do not explain why E3KARP
is necessary for LPA stimulation of NHE3.
LPA Activates NHE3 Activity by an ERK-independent Manner--
ERK
has been implicated in growth factor-induced regulation of NHE1 and in
acid-induced NHE3 activation (36, 37). In OK cells,
LPA-dependent activation of ERK, as well as activation of
PI 3-kinase, has been reported (30). To test whether ERK is involved in
the LPA-induced NHE3 activation, we examined the effect of PD98059, a
specific inhibitor of ERK, on the LPA-dependent activation
of NHE3 in OK/E3V/E3KARP cells. As shown in Fig.
9, basal NHE3 activity and the
LPA-stimulated NHE3 activities were not affected by pretreatment with
PD98059. We observed that the LPA-induced ERK phosphorylation is not
altered by LY294002 treatment (data not shown). These results show that
the LPA-induced ERK phosphorylation is mediated by a mechanism that is
not PI 3-kinase dependent. Also LPA activation of ERK is not involved
in LPA stimulation of NHE3. Thus LPA activates multiple signaling
pathways in OK cells, only some of which are involved in NHE3 and PI
3-kinase stimulation.
In these studies, LPA was shown to stimulate NHE3 activity
and to increase the percent of total NHE3 on the plasma membrane by
increasing exocytic trafficking. These three related events were all
dependent on the presence of E3KARP. Unlike some other reported
regulation of NHE3 that could be mediated by either E3KARP or NHERF
(cAMP) (19), the LPA stimulation of NHE3 activity only occurred in the
presence of E3KARP and not NHERF. This specificity in an effect of
E3KARP has only been reported previously (20) for Ca2+
dependent inhibition of NHE3, serum/glucocorticoid-inducible kinase
1-dependent stimulation of NHE3 (38), and activation of
phospholipase C- These studies show that E3KARP is necessary to allow LPA-stimulated
exocytic trafficking (also called endocytic recycling) of NHE3. A role
for PDZ proteins in trafficking of transport proteins has been
identified only recently. One type of involvement is via plasma
membrane retention (40). In neurons, NMDA receptor and potassium
channel Kv1.4 binding to PSD-95 increases plasma membrane retention
(less endocytosis) and leads to a longer half-life (41). In Madin-Darby
canine kidney cells, the PDZ domain binding C-terminal amino acids of
CFTR and podocalyxin are necessary for their BB localization,
whereas those of the potassium channel Kir 2.3 and
D-aminobutyric acid transporter are required for their basolateral membrane sorting. Removing the PDZ domain-binding motif from CFTR leads to equal BB and basolateral membrane localization because of decreased BB retention (40).
PDZ domain binding is also associated with increased endocytosis or
exocytic trafficking of some transport proteins (41). Endocytosis of
the activin II receptor and PI 3-kinase is necessary for exocytic trafficking of NHE3 under basal
and LPA-stimulated conditions in OK/E3KARP cells. Although constitutively active PI 3-kinase and Akt have been shown to increase the plasma membrane amount and activity of NHE3 in PS120 fibroblasts (6), the current studies demonstrated that PI 3-kinase activity was
increased by LPA (using Akt activity to track PI 3-kinase activity) and
that stimulation of PI 3-kinase activity was necessary for LPA to
stimulate NHE3 activity and its exocytic insertion. The assay for PI
3-kinase could not be done by immunoprecipitating PI 3-kinase,
as is usually performed, because the commercially available anti-p85
and p110 antibodies did not immunoprecipitate opossum kidney PI
3-kinase. Concerning the pool of PI 3-kinase involved in stimulation of
NHE3, although we showed that rapid growth factor stimulation of ileal
BB NHE3 activity and amount were associated with an increase in ileal
BB PI 3-kinase amount and activity (18), we did not establish that it
was an increase in the apical membrane PI 3-kinase that was necessary
for LPA stimulation of NHE3 activity. Other possibilities are that
activation of PI 3-kinase in the recycling or early endosomal
compartments are the relevant pools. Although PI 3-kinase plays an
essential role in the LPA-induced stimulation of NHE3, the LPA-induced
Akt activation is not affected by the expression of exogenous E3KARP, inconsistent with the E3KARP dependence of the LPA-induced NHE3 stimulation. The lack of Akt inhibitors prevents us from concluding that the activated Akt is involved in LPA stimulation of NHE3 activity,
and it is possible that LPA-induced stimulation of NHE3 occurs by a PI
3-kinase-dependent but an Akt-independent pathway.
LPA-induced rapid stimulation of sodium absorption in any epithelial
cell has not been reported previously. LPA is an inflammatory mediator
that is increased in amount over a prolonged period in chronic
inflammation and has been considered to be involved in the recovery or
restitution phase of inflammation (23). Because decreased sodium
absorption and diarrhea often accompany acute inflammatory states, it
is not surprising that stimulation of sodium absorption would be part
of the recovery response. The concentration dependence of the LPA
stimulation of NHE3 parallels its concentration dependence on
proliferation, which is another of its restitutive responsives (30).
Although LPA appears to function in the recovery from inflammation,
LPA's stimulation of sodium absorption suggests that it might also be
useful in the treatment of diarrheal diseases. In fact, a preliminary
study indicated that LPA decreased the inflammation and necrosis in the
ethanol/trinitrobenzine sulfonic acid rectal instillation model of rat
distal colitis (47). Therefore, it will be interesting to determine
whether LPA stimulation of NHE3 activity is related to the recovery
from inflammation and diarrhea.
The mechanism by which E3KARP increases LPA-induced exocytic
trafficking of NHE3 is not known. One possible explanation is that
E3KARP increases or alters steps in LPA-induced signaling that are
needed to stimulate exocytosis of NHE3 to the apical plasma membrane.
LPA acts via multiple plasma membrane receptors and in other cells has
been shown to affect at least Gq/phospholipase C activation,
Gi/Ras activation and Gi/adenylate cyclase inhibition, and
G12/13/Rho activation (23-25). At least in some cells, LPA rapidly activates PI 3-kinase (including in OK cells) and tyrosine kinases, including Src. It also elevates
[Ca2+]i in OK cells with a maximum
effect at 100 µM LPA, IC50 2.5 × 10 Another explanation for the E3KARP dependence of the LPA-induced NHE3
activation is that E3KARP may regulate NHE3 by affecting a step in
exocytic trafficking. von Zastrow and co-workers (52, 53) have reviewed
several examples of receptors and transport proteins in which PDZ
proteins appear to act as molecular switches determining whether these
proteins move from the recycling compartment to the plasma membrane
versus moving to lysosomes for degradation. Therefore, it is
possible that E3KARP may act as a molecular switch by affecting
components of the exocytic machinery with involvement of products of PI
3-K activation. Despite nearly all the LPA stimulation of NHE3 being
prevented by LY294002, the fact that PI 3-K activation as judged by Akt
activity was not affected by the presence of E3KARP suggests that the
major effect of E3KARP on LPA stimulation of NHE3 was on the exocytic
machinery at steps that are independent of Akt activation.
Understanding LPA stimulation of NHE3 will require defining which
additional steps in signal transduction and/or the components in the
exocytic trafficking events initiated by LPA are necessary for
LPA-induced NHE3 exocytosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor, treatment with endothelin-1 increased the
surface NHE3 amount and NHE3 activity (11, 12) by an increase in
exocytosis. Thus modulation of surface NHE3 amounts is a major
mechanism in the acute stimulation of NHE3 activity by growth factors
and hormones.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
[in a new window]
Fig. 1.
Establishment of E3KARP expressing opossum
kidney/E3V proximal tubule cells. 20 µg of lysates from OK
(non-transfected), OK/E3V, OK/E3V/E3KARP, and PS120/E3V/E3KARP cells
were separated by SDS-PAGE, and the presence of NHERF and E3KARP were
probed with anti-NHERF (upper panel) and anti-E3KARP
(lower panel) antibodies, respectively. Representative data
from three similar experiments are shown.
6 M to 10
4 M
(Fig. 3B), with no effect at 10
7
M. This dose dependence of LPA on NHE3 activity is similar
to the concentration dependence of LPA on proliferation of OK cells (30). These results show that LPA stimulates NHE3 activity by an
E3KARP-dependent mechanism.
View larger version (22K):
[in a new window]
Fig. 2.
LPA stimulates NHE3 activity by an
E3KARP-dependent mechanism. OK/E3V (A) and
OK/E3V/E3KARP (B) cells were loaded with BCECF-AM in
Na+ solution, alone or containing 100 µM LPA
for 30 min. NHE3 activity was then assessed as the rate of
Na+-dependent cell pH recovery from an acid
load. Similar data were obtained from four similar experiments.
View larger version (11K):
[in a new window]
Fig. 3.
Time- and dose-dependent effects
of LPA on NHE3 activity. A, OK/E3V/E3KARP cells were
treated with 100 µM LPA for the indicated times and then
the rate of Na+-dependent pH recovery at
pHi 6.4 was measured. B, OK/E3V/E3KARP cells
were treated with the indicated concentrations of LPA for 30 min. For
determination of pHi recovery rate
( pHi/
s), slopes (initial rates) were calculated along
the pHi recovery by linear least square analysis over a
minimum of 9 s. n = 4. * indicates
p < 0.05 versus control.
4 M LPA for 60 min did not affect the
surface abundance of NHE3 in OK/E3V cells (non-E3KARP expressing). In
contrast, in OK/E3V/E3KARP cells, LPA treatment caused an increase in
apical membrane NHE3 protein abundance in a time-dependent
manner (Fig. 4B), consistent with the
time-dependent stimulation of NHE3 activity by LPA
treatment shown in Fig. 3B. 10 min of LPA exposure
significantly increased the amount of surface NHE3. The LPA-induced
increase in the surface NHE3 amount occurred without any change in
total cellular NHE3 abundance. These results suggest that the
LPA-induced activation of NHE3 is because of the increase of the amount
of surface NHE3.
View larger version (19K):
[in a new window]
Fig. 4.
LPA increases surface NHE3 amount in the
presence but not absence of E3KARP. OK/E3V (A) and
OK/E3V/E3KARP (B) cells were serum-deprived for 48 h
and then treated with 100 µM LPA for the indicated times.
Monolayers were then placed at 4 °C and biotinylated, and surface
proteins were retrieved from the cell lysate by streptavidin
precipitation. NHE3 protein abundance was quantified by immunoblot with
anti-VSV-G antibody. Representative data from three similar experiments
are shown.
View larger version (28K):
[in a new window]
Fig. 5.
LPA increases exocytic insertion but does not
alter endocytosis of NHE3. A, NHE3 endocytic recycling.
Cells were labeled with sulfo-NHS-acetate and then maintained at
4 °C or treated with 100 µM LPA or vehicle for 30 min
at 37 °C. Cells were then decreased to 4 °C and surface
biotinylation was then performed with sulfo-NHS-SS-biotin, biotinylated
proteins were isolated by precipitation with streptavidin-bound
agarose, and NHE3 protein abundance was measured by immunoblot analysis
with anti-VSV-G antibody. 4 °C control is on cells kept at 4 °C
throughout the experiment. Inset (right), NHE3
protein exocytosed to the plasma membrane was quantitated from three
experiments and described as mean ± S.E. * indicates
p < 0.05 compared with control. B, NHE3
endocytosis. Cell surface biotinylation was performed with
sulfo-NHS-SS-biotin at 4 °C, after which cells were maintained at
4 °C or were treated with vehicle, 100 µM LPA, or 100 µM dopamine for 30 min at 37 °C. Cells were then
treated with 150 mM reduced GSH, biotinylated proteins were
precipitated with streptavidin-bound agarose, and NHE3 protein
abundance protected from GSH was measured by immunoblot analysis.
4 °C control cells were exposed to sulfo-NHS-SS-biotin and
maintained at 4 °C until GSH exposure. LPA did not alter NHE3
endocytosis. Inset (right), relative densities of
NHE3 protected from GSH were quantitated from three experiments and
described as mean ± S.E. * indicates p < 0.05 compared with control. A positive endocytosis control was exposure of
OK/E3V/E3KARP cells to dopamine (100 µM) for 30 min at
37 °C, as previously described (34).
View larger version (23K):
[in a new window]
Fig. 6.
The PI 3-kinase inhibitor LY294002
prevents the LPA-induced increase of NHE3 activity. OK/E3V/E3KARP
cells were loaded with BCECF-AM in Na+ solution, alone or
containing 50 µM LY294002 for 15 min, and then treated
for 30 min with vehicle or 100 µM LPA. NHE3
activity was then assessed as the rate of
Na+-dependent cell pH recovery from an acid
load. Results are mean ± S.E. of Vmax and
K'(H+)i. Results at the right are
mean ± S.E. from eight similar experiments (comparison of
LY294002 versus LY294002 + LPA, ns).
View larger version (26K):
[in a new window]
Fig. 7.
LPA increases in the surface amounts and
exocytic insertion of NHE3 are PI 3-kinase-dependent.
A, surface NHE3 amount. OK/E3V/E3KARP cells were
serum-starved for 48 h and then pretreated with 50 µM LY294002 for 15 min and treated with 100 µM LPA for 30 min as indicated. Cell surface NHE3 amounts
were determined as described for Fig. 4. Representative data from one
experiment are shown on the left, and mean ± S.E. from
three similar experiments is shown on the right. * indicates
p < 0.05 compared with control. B, exocytic
insertion (endocytic recycling). Cells were labeled with
sulfo-NHS-acetate, after which cells were maintained at 4 °C or
pretreated with 50 µM LY294002 for 15 min and then
treated with 100 µM LPA for 30 min at 37 °C, as
indicated. Cell surface biotinylation was then performed with
sulfo-NHS-SS-biotin, and NHE3 protein abundance was measured by
immunoblot analysis with anti-VSV-G antibody. Representative data from
one experiment are shown on the left, and mean ± S.E.
from three similar experiments is shown on the right. *
indicates p < 0.05 compared with control. 4 °C
control data in B are as in Fig. 5.
View larger version (36K):
[in a new window]
Fig. 8.
LPA Increases Akt phosphorylation by a PI
3-kinase-dependent mechanism equally in the absence or
presence of E3KARP. A, OK/E3V and OK/E3V/E3KARP cells
were treated with 100 µM LPA for the times shown and/or
50 µM LY294002 as described for Fig. 7. 20 µg of
lysates from OK/E3V and OK/E3V/E3KARP were loaded on SDS-PAGE, and Akt
phosphorylation was determined by immunoblot analysis with anti-p-Akt
antibody (upper panel) and anti-Akt antibody (lower
panel), respectively. Representative data from two independent
experiments are shown with mean ± S.E. from four experiments with
0-, 10-, and 30-min LPA exposure shown in B, normalized to
Akt activity in the absence of LPA. C is results similar to
B (n = 4) but with 0, 1, 2.5, and 5 min of
LPA exposure. * indicates p < 0.05 compared with zero
time controls.
View larger version (26K):
[in a new window]
Fig. 9.
ERK is not involved in LPA-induced activation
of NHE3. OK/E3V/E3KARP cells were loaded with BCECF-AM in
Na+ solution, alone or containing 20 µM
PD98059 for 15 min, and then treated with vehicle or 100 µM LPA for 30 min. NHE3 activity was then assessed as the
rate of Na+-dependent cell pH recovery from an
acid load. Results from a representative experiment are shown.
Inset shows mean ± S.E. of eight similar
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 (39). The
Ca2+-dependent inhibition of NHE3 was because
of specific interaction of E3KARP but not NHERF with the cytoskeletal
protein
-actinin-4. In addition, E3KARP directly interacts with SGK1
and phospholipase C-
3 to account for the E3KARP
specificity. In contrast, NHERF and E3KARP both can reconstitute
cAMP inhibition of NHE3, because they both bind ezrin, which links
protein kinase AII to the PDZ domain protein·NHE3 complex.
Thus it is predicted that the specificity of E3KARP will be shown to be
because of specific binding of a protein involved in LPA signal
transduction that is necessary for NHE3 stimulation, although the
reason for the E3KARP dependence of the LPA-induced NHE3 activation has
not been identified.
1 adrenergic receptor require binding to
PDZ domain-containing proteins (42, 43). There are also examples in
which exocytic trafficking of proteins requires binding to PDZ domains.
The half-life of CFTR in BB of Madin-Darby canine kidney cells is
increased by an increased rate of exocytic trafficking that requires
the C-terminal PDZ protein-binding domain (40). Similarly, NHERF
binding increases exocytic trafficking of the
2 adrenergic and
k-opiod receptors following internalization (44, 45). In the absence of
NHERF binding, these receptors instead have increased lysosomal
trafficking (46). Thus PDZ domain protein binding can alter the fate of
endocytosed proteins. In the current study, we demonstrated that LPA
affects the exocytic insertion but not endocytosis of NHE3 in the
presence of E3KARP. This is the first report showing that exocytic
insertion of NHE3 can be regulated by a mechanism that is dependent on
a PDZ domain-containing protein.
6 M and transactivates epidermal growth
factor receptor, ERK (PI 3-kinase independent), Rho, and a
Cl
channel (26, 30, 48, 49). Which of these are linked to LPA stimulation of NHE3 is not yet known beyond the involvement of PI
3-kinase. However, we have demonstrated that not all signal transduction initiated by LPA in OK cells is involved in stimulation of
NHE3. In this study, we demonstrated that whereas LPA increased ERK
activity and phosphorylation, blocking LPA activation of ERK with
PD98059 did not alter LPA stimulation of NHE3, showing that ERK
activation is not involved in NHE3 stimulation. Although it is still
unknown which LPA-induced signaling steps are affected by E3KARP, the
previously reported specific interactions of E3KARP with phospholipase
C-
3,
-actinin-4, and serum/glucocorticoid-inducible protein
kinase (20, 38, 50) suggests that E3KARP may be involved in LPA-induced
NHE3 activation through direct interaction with a signaling molecule
that is regulated by LPA.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Ming Tse and Dr. Pann-Ghill Suh (Pohang University of Science and Technology) for helpful discussions. We acknowledge the secretarial assistance of H. McCann.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants RO1 DK26523 and PO1 DK44484, by Pusan National University, by the Hopkins Center for Epithelial Disorders, and by Grant O2-PJ1-PG3-20706-0004) from the Korea Health 21 R&D Projects Ministry of Health and Welfare, Republic of Korea (to J. H. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Contributed equally.
To whom correspondence should be addressed. Tel.:
410-955-9675; Fax: 410-955-9677; E-mail: mdonowit@jhmi.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M300580200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NHE3, Na+/H+ exchanger 3; NHERF, Na+/H+ exchanger regulatory factor; E3KARP, NHE3 kinase A regulatory protein; PI, phosphatidylinositol; LPA, lysophosphatidic acid; BCECF-AM, acetoxymethyl ester of 2'7'-bis(carboxyethyl)5-6-carboxyfluorescein; PBS, phosphate-buffered saline; BB, brush border; OK, opossum kidney; ERK, extracellular signal-regulated kinase; VSV-G, vesicular stomatitis virus G-protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Donowitz, M., and Tse, M. (2000) in Gastrointestinal Transport Molecular Physiology: Current Topics in Membranes (Barrett, K. E. , and Donowitz, M., eds), Vol. 50 , pp. 437-498, Academic Press, San Diego |
2. | Brett, C. L., Wei, Y., Donowitz, M., and Rao, R. (2002) Am. J. Physiol. 282, C1031-C1041 |
3. |
Numata, M.,
and Orlowski, J.
(2001)
J. Biol. Chem.
276,
17387-17394 |
4. |
Janecki, A. J.,
Janecki, M.,
Akhter, S.,
and Donowitz, M.
(2000)
J. Biol. Chem.
275,
8133-8142 |
5. |
Donowitz, M.,
Janecki, A.,
Akhter, S.,
Cavet, M. E.,
Sanchez, F.,
Lamprecht, G.,
Khurana, S.,
and Yun, C. H. C.
(2000)
Ann. N. Y. Acad. Sci.
915,
30-42 |
6. |
Lee-Kwon, W.,
Johns, D. C.,
Cha, B.,
Cavet, M.,
Park, J.,
Tsichlis, P.,
and Donowitz, M.
(2001)
J. Biol. Chem.
276,
31296-31304 |
7. | Akhter, S., Kovbasnjuk, O., Li, X., Cavet, M., Noel, J., Arpin, M., Hubbard, A., and Donowitz, M. (2002) Am. J. Physiol. 283, C927-C940 |
8. |
Kurashima, K.,
Szabo, E. Z.,
Lukacs, G.,
Orlowski, J.,
and Grinstein, S.
(1998)
J. Biol. Chem.
273,
20828-20836 |
9. |
D'Souza, S.,
Garcia-Cabado, A., Yu, F.,
Teter, K.,
Lukacs, G.,
Skorecki, K.,
Orlowski, J.,
and Grinstein, S.
(1998)
J. Biol. Chem.
273,
2035-2043 |
10. |
Li, X.,
Galli, T.,
Leu, S.,
Wade, J. B.,
Weinman, E. J.,
Leung, G.,
Cheong, A.,
Louvard, D.,
and Donowitz, M.
(2001)
J. Physiol.
537,
537-552 |
11. |
Laghmani, K.,
Preissig, R. A.,
Moe, O. W.,
Yanagisawa, A. M.,
and Alpern, R. J.
(2001)
J. Clin. Invest.
107,
1563-1569 |
12. | Peng, Y., Amemiya, M., Yong, X., Fan, L., Moe, O. W., Yin, H., Preissig, P. A., Yanagisawa, M., and Alpern, R. J. (2001) Am. J. Physiol. 280, F34-F42 |
13. | Czech, M. P., and Corvera, S. (1999) J. Biol. Chem. 274, 1865-1868[CrossRef] |
14. | Czech, M. P. (2002) Mol. Cell 9, 695-696[Medline] [Order article via Infotrieve] |
15. |
Calera, M. R.,
Martinez, C.,
Liu, H.,
Jack, A. K.,
Birnbaum, M. J.,
and Pilch, P. F.
(1998)
J. Biol. Chem.
273,
7201-7204 |
16. |
Cong, L. N.,
Chen, H.,
Li, Y.,
Zhou, L.,
McGibbon, M. A.,
Taylor, S. L.,
and Quon, M. J.
(1997)
Mol. Endocrinol.
11,
1881-1890 |
17. |
Foster, L. J.,
Li, D.,
Randhawa, V. K.,
and Klip, A.
(2001)
J. Biol. Chem.
276,
44212-44221 |
18. |
Khurana, S.,
Nath, S. K.,
Levine, S. A.,
Bowser, J. M.,
Tse, C. M.,
Cohen, M. E.,
and Donowitz, M.
(1996)
J. Biol. Chem.
271,
9919-9927 |
19. |
Yun, C. H. C.,
Oh, S.,
Zizak, M.,
Steplock, D.,
Tsao, S.,
Tse, C. M.,
Weinman, E. J.,
and Donowitz, M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3010-3015 |
20. |
Kim, J. H.,
Lee-Kwon, W.,
Park, J. B.,
Ruy, S. H.,
Yun, C.,
and Donowitz, M.
(2002)
J. Biol. Chem.
277,
23714-23724 |
21. |
Yun, C. H.,
Lamprecht, C.,
Forster, D. V.,
and Sidor, A.
(1998)
J. Biol. Chem.
273,
25856-25863 |
22. |
Zizak, M.,
Lamprecht, G.,
Steplock, D.,
Tariq, N.,
Shenolikar, S.,
Donowitz, M.,
Yun, C. H. C.,
and Weinman, E. J.
(1999)
J. Biol. Chem.
274,
24753-24758 |
23. | Xie, Y., Gibbs, T. C., and Meier, K. E. (2002) Biochim. Biophys. Acta 1582, 270-281[CrossRef][Medline] [Order article via Infotrieve] |
24. | Lynch, K. R., and Macdonald, T. L. (2002) Biochim. Biophys. Acta 1582, 289-294[Medline] [Order article via Infotrieve] |
25. | Sardar, V. M., Bautista, D. L., Fischer, D. J., Yokoyama, K., Nusser, N., Virag, T., Baker, D. L., Tigyi, G., and Parrill, A. L. (2002) Biochim. Biophys Acta 1582, 309-317[Medline] [Order article via Infotrieve] |
26. | Moolenaar, W. H., Kranenburg, O., Postma, F. R., and Zondag, G. C. M. (1997) Curr. Opin. Cell Biol. 9, 168-173[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Sturm, A.,
Becker, A.,
Schulte, K.-M.,
Goebell, H.,
and Dignass, A. U.
(1998)
Ann. N. Y. Acad. Sci.
859,
223-226 |
28. | Sturm, A., Sudermann, T., Schulte, K.-M., Goebell, H., and Dignass, A. U. (1999) Gastroenterology 117, 368-377[CrossRef][Medline] [Order article via Infotrieve] |
29. | Hines, O. J., Ryder, N., Chu, J., and McFadden, D. (2000) J. Surg. Res. 92, 23-28[CrossRef][Medline] [Order article via Infotrieve] |
30. | Dixon, R. J., and Brunskill, N. J. (1999) Kidney Int. 56, 2064-2075[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Levine, S. A.,
Montrose, M. H.,
Tse, C. M.,
and Donowitz, M.
(1993)
J. Biol. Chem.
268,
25527-25535 |
32. | Cavet, M. E., Akhter, S., Sanchez de Medina, F., Donowitz, M., and Tse, C. M. (1999) Am. J. Physiol. 277, C1111-C1121[Medline] [Order article via Infotrieve] |
33. | Hoogerwerf, W. A., Tsao, S. C., Devuyst, O., Levine, S. A., Yun, C. H., Yip, J. W., Cohen, M. E., Wilson, P. D., Lazenby, A. J., Tse, C. M., and Donowitz, M. (1996) Am. J. Physiol. 270, G29-G41[Medline] [Order article via Infotrieve] |
34. |
Hu, M. C.,
Fan, L.,
Crowder, L. A.,
Karim-Jimenez, Z.,
Murer, H.,
and Moe, O. W.
(2001)
J. Biol. Chem.
276,
26906-26269 |
35. |
Ahn, W.,
Kim, K. H.,
Lee, J. A.,
Kim, J. Y.,
Choi, J. Y.,
Moe, O. W.,
Milgram, S. L.,
Muallem, S.,
and Lee, M. G.
(2001)
J. Biol. Chem.
276,
17236-17243 |
36. | Gillis, D., Shrode, L. D., Krump, E., Howard, C. M., Rubie, E. A., Tibbles, L. A., Woodgett, J., and Grinstein, S. (2001) J. Membr. Biol. 181, 205-214[Medline] [Order article via Infotrieve] |
37. |
Bianchini, L.,
L'Allemain, G.,
and Pouyssegur, J.
(1997)
J. Biol. Chem.
272,
271-279 |
38. |
Yun, C. C.,
Chen, Y.,
and Lang, F.
(2002)
J. Biol. Chem.
277,
7676-7683 |
39. | Suh, P. G., Hwang, J. I., Ryu, S. H., Donowitz, M., and Kim, J. H. (2001) Biochem. Biophys. Res. Commun. 288, 1-7[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Swiatecka-Urban, A.,
Duhaime, M.,
Coutermarsh, B.,
Karlson, K. H.,
Collawn, J.,
Milewski, M.,
Cutting, G. R.,
Guggino, W. B.,
Langford, G.,
and Stanton, B. A.
(2002)
J. Biol. Chem.
277,
40099-40105 |
41. | Roche, K. W., Standley, S., McCallum, J., Ly, C. D., Ehlers, M. D., and Wenthold, R. J. (2001) Nat. Neurosci. 4, 794-802[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Xu, J.,
Paquet, M.,
Lau, A. G.,
Wood, J. D.,
Ross, C. A.,
and Hall, R. A.
(2001)
J. Biol. Chem.
276,
41310-41317 |
43. |
Matsuzaki, T.,
Hanai, S.,
Kishi, H.,
Liu, Z.,
Bao, Y.,
Kikuchi, A.,
Tsuchida, K.,
and Sugino, H.
(2002)
J. Biol. Chem.
277,
19008-19018 |
44. |
Li, J.-G.,
Chen, C.,
and Liu-Chen, L.-Y.
(2002)
J. Biol. Chem.
277,
27545-27552 |
45. | Cao, T. T., Deacon, H. W., Reczek, D., Bretscher, A., and von Zastrow, M. (1999) Nature 401, 286-290[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Gage, R. M.,
Kim, K.-A.,
Cao, T. T.,
and von Zastrow, M.
(2001)
J. Biol. Chem.
276,
44712-44720 |
47. | Sturm, A., Zeeh, J., Sudermann, T., Rath, H., Gerken, G., and Dignass, A. U. (2002) Digestion 66, 23-29[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Herrlich, A.,
Daub, H.,
Knebel, A.,
Herrlick, P.,
Ullrich, A.,
Schultz, G.,
and Gudermann, T.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8985-8990 |
49. | Dixon, R. J., Young, K., and Brunskill, N. J. (1999) Am. J. Physiol. 276, F191-F198[Medline] [Order article via Infotrieve] |
50. |
Hwang, J. I.,
Heo, K.,
Shin, K. J.,
Kim, E.,
Yun, C.,
Ryu, S. H.,
Shin, H. S.,
and Suh, P. G.
(2000)
J. Biol. Chem.
275,
16632-16637 |
51. | Deleted in proof |
52. | Carroll, R. C., Beattie, E. C, von Zastrow, M., and Malenka, R. C. (2001) Nat. Rev. Neurosci. 2, 315-324[CrossRef][Medline] [Order article via Infotrieve] |
53. | Sorkin, A., and von Zastrow, M. (2002) Nat. Rev. Mol. Biol. 3, 600-612[CrossRef] |