From the
Among water channel proteins (aquaporins), aquaporin-collecting
duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin
causes cAMP production in the renal collecting duct cells, and this is
believed to lead to exocytic insertion of water channel into the apical
membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for
cAMP-dependent protein kinase, residues at positions 253-256
( Arg- Arg-Gln- Ser). To determine the role of
this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by
site-directed mutagenesis. In Xenopus oocytes injected with
wild-type or mutated AQP-CD cRNAs, osmotic water permeability
(Pf) was 4.8-7.7 times higher than Pf of water-injected
oocytes. Incubation with cAMP plus forskolin or direct cAMP injection
into the oocytes increased Pf of wild-type, but not mutated,
AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression
were similar in wild and mutated types as identified by Western blot
analysis. In vitro phosphorylation studies of AQP-CD proteins
expressed in oocyte showed that cAMP-dependent protein kinase
phosphorylated wild-type, but not mutated, AQP-CD proteins.
Phosphoamino acid analysis revealed that this phosphorylation occurred
at the serine residue. Moreover, phosphorylation of AQP-CD protein in
intact rat kidney medulla tissues was stimulated by incubation with
cAMP. Our data suggest that cAMP stimulates water permeability of
AQP-CD by phosphorylation. This process may contribute to the
vasopressin-regulated water permeability of collecting duct in addition
to the apical insertion of AQP-CD by exocytosis.
A water channel of collecting duct, aquaporin-collecting duct
(AQP-CD),
Phosphorylation is an important regulatory process responsible for
the modulation of channel proteins (see Ref. 5 for review). The same
holds true for MIP family members as well. For example, cAMP-dependent
protein kinase phosphorylates MIP possibly at Ser-243
(6, 7) . Phosphorylation increases the voltage-dependent
closure of MIP channel
(6) . When Ser-262 in nodulin-26, a MIP
family in the peribacteroid membrane of symbiotic root nodules, is
phosphorylated by a Ca
As reported previously
(1, 3) , the expression
of wild-type AQP-CD markedly increased Pf of oocytes. We tested the
effect of cAMP on Pf of AQP-CD-expressing oocytes. Fig. 1 A depicts the time course of oocyte Pf after incubation with cAMP.
Wild-type AQP-CD-expressing oocytes were incubated with
8-(4-chlorophenylthio)-cAMP plus forskolin (
Immunoblot
of wild-type AQP-CD proteins expressed in the oocytes exhibited an
apparent molecular mass of a 29-kDa and a broad 35-45-kDa band as
previously reported (Fig. 2, lane2). A band at
35-45-kDa was thought to be a glycosylated form of 29-kDa AQP-CD
protein
(1, 2, 14) . Similar amounts of AQP-CD
proteins were expressed in oocytes injected with wild-type and mutated
AQP-CD RNAs (Fig. 2, lanes2-7).
Identical bands were detected when membrane fraction from inner
medulla, where AQP-CD was most abundantly present
(1, 2) , was applied (Fig. 2, lane8).
We thank Dr. Miwako K. Homma for valuable discussions
and Dr. K. Tomita for encouragement of this work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
was cloned from the rat kidney using
a reverse transcribed polymerase chain reaction-based strategy
(1) . The cDNA for AQP-CD encodes 271-amino-acid protein
( M
28,928) with 59 and 42% sequence identity with
rat eye lens major intrinsic protein (MIP) and human
AQP-channel-forming integral membrane protein (AQP-CHIP), respectively.
Because AQP-CD is immunohistochemically confined to the apical region
of the collecting duct cells, it was suggested to be a
vasopressin-sensitive water channel
(1) . Recent studies
demonstrated that AQP-CD is present exclusively in principal cells and
inner medullary collecting duct cells
(2, 3) . AQP-CD is
diffusely distributed in the cytoplasm in euhydrated condition, whereas
apical staining of AQP-CD is intensified in dehydrated condition
(2, 3, 4) . Moreover, the immunoblot of the
collecting duct cell membranes demonstrated an increased expression of
AOP-CD protein in dehydrated rat
(2, 4) .
-dependent
calmodulin-independent protein kinase, malate uptake is stimulated
(8, 9) . In the kidney, the presence of three AQPs,
AQP-CHIP, AQP-CD, and AQP 3 has been observed
(1, 10, 11) . Of these AQPs, a potential
phosphorylation site for cAMP-dependent protein kinase is present in
AQP-CD
(1, 12) , whereas other AQPs lack such a site.
Because the role of this site is not established at present, we
examined whether phosphorylation by cAMP-dependent protein kinase is
involved in the regulation of AQP-CD.
Site-directed Mutagenesis and in Vitro RNA
Synthesis
An in vitro mutagenesis system (Promega) was
used for site-directed mutagenesis. Ser-256 in the amino acid sequence
of AQP-CD cDNA was altered to Ala (S256A), Leu (S256L), Thr (S256T),
Asp (S256D), or Glu (S256E) with mutation primers,
GCGGCGGCAGGCGGTGGAGCTCC, GCGGCGGCAGTTGGTGGAGCTCC,
GCGGCGGCAGACGGTGGAGCTCC, GCGGCGGCAGGACGTGGAGCTCC, and
GCGGCGGCAGGAGGTGGAGCTCC, respectively. Mutations were confirmed by a
fluorescence DNA sequencer (Applied Biosystems 373A). Capped RNA
transcripts were synthesized in vitro with T3 RNA polymerase
using NotI-digested AQP-CD expression vector DNA
(3) .
Measurement of Osmotic Water Permeability of
Oocyte
Oocytes at stages V-VI were obtained from
Xenopuslaevis(1) . Each oocyte was injected
with either 40 nl of water or 4 ng of wild-type or mutated AQP-CD
cRNAs. After 48 h of incubation, the osmotic water permeability (Pf) of
oocyte was measured at 20 °C from the time course of osmotic cell
swelling as described
(1, 3) . Because the time course
of cell swelling was principally linear during the initial 15 s, Pf of
oocytes was calculated from this 15-s response. In some experiments,
oocytes were incubated in Barth's buffer containing 0.5
mM 8-(4-chlorophenylthio)-cAMP plus 50 µM
forskolin, or oocytes were directly injected with 20 nl of 1
mM cAMP before the assay.
Western Blot Analysis
Oocyte membranes were
isolated as described
(13) . The membrane fraction of rat inner
medulla was prepared as described
(14) . Western blot was
performed as described
(14) using an affinity-purified antibody
against 15 COOH-terminal amino acids of AQP-CD
(3) . In our
previous studies, Western blot with this anti-AQP-CD antibody
specifically identified a band at 29 kDa, which was an expected
molecular size of AQP-CD protein
(3, 4) . Moreover,
immunohistochemical study of the collecting duct with the antibody
revealed that the staining was confined to the apical membrane of the
principal cells and the inner medullary collecting duct cells
(3) , both of which are known to be vasopressin-responsive cells
(2, 20) . These results suggested that the antibody used
in this study was specific for AQP-CD protein.
In Vitro Phosphorylation
Phosphorylated membrane
proteins of oocyte and kidney medulla were analyzed by SDS-PAGE and
autoradiography essentially as described
(21) . Protein
G-agarose (Protein G PLUS-Agarose, Oncogene Science) and
affinity-purified anti-AQP-CD antibody (1:200 dilution) were used for
immunoprecipitation. Immune complexes were resuspended in a 50-µl
volume of phosphorylation buffer and incubated with 0.2 µg of
catalytic subunit of cAMP-dependent protein kinase (Sigma; specific
activity, 39 nmol of phosphate/min/mg of protein) in the presence of
0.5 mM ATP containing 10 µCi of
[-
P]ATP (6000 Ci/mmol, DuPont NEN). In one
protocol, the incubation was performed in the presence of 10
µM H89 (Seikagaku Corp.), an inhibitor of cAMP-dependent
protein kinase
(15, 16) . Autoradiograms were quantified
by a dual-wavelength densitometer (Shimadzu CS-9000).
Quantitative Evaluation of AQP-CD Protein Expressed on
the Oocyte Membrane
To quantify AQP-CD protein on the oocyte
membrane, an anti-AQP-CD antibody against the external domain of AQP-CD
was made using synthetic peptide corresponding to amino acid residues
at positions 113-127 as described
(3) .
Immunohistochemical study in the rat kidney and Western blot analysis
of AQP-CD protein expressed in oocytes with this antibody showed
essentially similar results as obtained by an anti-AQP-CD antibody
against the COOH-terminal domain of AQP-CD
(3) . Oocytes
(10-20 in each group) were incubated in Barth's buffer with
or without 0.5 mM 8-(4-chlorophenylthio)-cAMP plus 50
µM forskolin at 20 °C. After incubation for 1 h in the
presence of 2% bovine serum albumin at 4 °C, oocytes were further
incubated for 1 h with the anti-AQP-CD antibody (1:50 dilution). After
three washes, oocytes were incubated with I-protein A for
1 h at 4 °C, and the radioactivity was measured by a scintillation
counter.
Phosphoamino Acid Analysis
After wild-type AQP-CD
proteins were phosphorylated in vitro for 10 min as above, the
P-labeled protein bands were eluted from SDS gel using
standard methodology as described
(17) .
In Vivo Phosphorylation
Tissues of rat inner
medulla were minced with a razor blade and suspended in Krebs-HEPES
medium (130 mM NaCl, 5 mM KCl, 1 mM
CaCl, 1 mM MgSO
, 10 mM
glucose, 25 mM Tris-HEPES buffer, pH 7.4) oxygenated with 95%
O
, 5% CO
. After 30 min, the tissue suspensions
were distributed into wells of a 24-well plate and incubated for 2 h in
the presence of 0.5 mCi/ml [
P]orthophosphate
(DuPont NEN) at 37 °C under a 5% CO
atmosphere. The
samples were further incubated in the absence or the presence of 0.5
mM 8-(4-chlorophenylthio)-cAMP. The samples were
immunoprecipitated with anti-AQP-CD antibody and analyzed by SDS-PAGE
and autoradiography as above.
) or were directly
injected with 20 pmol of cAMP at time 0 (
). The responses of Pf
to these maneuvers were essentially similar; Pf increased at a linear
rate up to 15 min and peaked at 30 min. Fig. 1 B summarizes measurement of oocyte Pf. Pf of water-injected oocytes
(control) was 21 ± 2 (S.E.)
10
cm/s.
Expression of wild-type AQP-CD increased Pf 7.7-fold. Pf increased
further when wild-type AQP-CD-expressing oocytes were incubated for 30
min with cAMP plus forskolin (+56%) or were directly injected with
cAMP 30 min before Pf measurement (+43%). Consistent with our
results, it was reported recently that incubation with cAMP increased
Pf in oocytes injected with poly(A
) mRNA from rat
renal medulla
(18) . We mutated Ser at position 256, a potential
phosphorylation site for cAMP-dependent protein kinase, to Ala, Leu,
Thr, Asp, or Glu. Pf of these oocytes ranged from 102 to 153
10
cm/s, indicating that the mutated AQP-CDs also
possess water channel activities. However, the incubation with cAMP
plus forskolin failed to increase Pf in every type of mutated AQP-CD
oocyte. Moreover, the direct injection of cAMP had no effect on Pf in
S256A, S256T, and S256E oocytes, suggesting that the residue at Ser-256
is required for cAMP-induced Pf stimulation.
Figure 1:
Pf of oocytes. A, changes of
oocyte Pf after cAMP treatments. Oocytes were injected with 4 ng of
wild-type AQP-CD cRNA 48 h prior to experiments. Oocytes were incubated
with cAMP plus forskolin () or were injected directly with cAMP
(
) at time 0 as described under ``Experimental
Procedures.'' After the treatments, osmotic cell swelling of the
oocytes was monitored and Pf was calculated. Each point represents
means ± S.E. of 8-10 oocytes. B, values of oocyte
Pf. Oocytes were injected water ( Control), with wild-type
AQP-CD cRNA ( Wild-type), or with mutated AQP-CD cRNAs
( S256X, Ser-256 was replaced by residue X) 48 h prior
to experiments. Pf is shown as means ± S.E. cAMP + FSK, incubated for 30 min with 0.5 mM
8-(4-chlorophenylthio)-cAMP plus 50 µM forskolin; cAMP
Injection, injected with 20 nl of 1 mM cAMP 30 min before
experiments. The number of oocytes is shown in
parentheses.
In renal collecting
duct, the vasopressin binding to basolateral vasopressin V receptor triggers a series of cellular events that lead to an
increase in apical water permeability. These events include cAMP
production and subsequent activation of cAMP-dependent protein kinase.
In the final step, it is hypothesized that AQP-CD is shuttled by
exocytosis and fuses with the apical membrane (shuttle hypothesis)
(19, 20) . We examined whether cAMP stimulates the
exocytosis of AQP-CD in oocytes. The amount of AQP-CD protein on the
oocyte membrane was quantified using an anti-AQP-CD antibody against
the external domain of AQP-CD and
I-protein A. First of
all,
I radioactivity of water-injected and
AQP-CD-expressing oocytes was measured after incubation without cAMP.
In three independent experiments, the mean radioactivity of
water-injected oocytes was 23.3% (range, 17.9-27.3%) of that of
AQP-CD-expressing oocytes. The radioactivity of water-injected oocytes
was thought to be derived from a nonspecific binding of
I-protein A to the oocyte membrane. This nonspecific
component was subtracted from the radioactivity of AQP-CD-expressing
oocytes to quantify the amount of AQP-CD on the oocyte membranes. When
the radioactivity of AQP-CD-expressing oocytes after incubation without
cAMP was taken as 100%, the radioactivity (the mean of three separate
experiments) changed 97.8, 107.0, 103.2, and 104.1% after 7.5-, 15-,
30-, and 60-min incubation with cAMP, respectively. These results
suggest that exocytosis of AQP-CD contributes little, if any, to
cAMP-induced Pf stimulation in AQP-CD-expressing oocytes.
Figure 2:
Western blot analysis of oocyte and inner
medulla membrane proteins probed with an affinity-purified antibody
against AQP-CD. Membranes were prepared from 10 oocytes injected with
water ( lane1), cRNAs of wild-type ( lane2), or mutated AQP-CDs ( lanes 3-7) or were
prepared from rat inner medulla ( lane8). Mutated
AQP-CDs were S256A ( lane3), S256L ( lane4), S256T ( lane5), S256D ( lane6), and S256E ( lane7).
To examine whether the cAMP effect on Pf of
wild-type AQP-CD-expressing oocytes is mediated by phosphorylation,
wild-type AQP-CD proteins were recovered from oocyte membranes and
incubated for 5-30 min with a catalytic subunit of cAMP-dependent
protein kinase plus [-
P]ATP
(Fig. 3 A). We observed two bands with an apparent
molecular mass of 29 kDa and 35-48 kDa, which corresponded to the
bands identified by Western blots (Fig. 2, lane2).
P incorporation into AQP-CD increased as
the incubation time extended from 5 to 20 min. Analysis of
Fig. 3A by densitometry showed that the density of broad
35-45-kDa bands was 3.9-5.5 times higher than of that of
29-kDa bands. In Western blot of wild-type AQP-CD protein
(Fig. 2, lane2), the density of the
35-45-kDa band was similar (93%) to that of the 29-kDa band.
These results suggested that glycosylated AQP-CD proteins were
preferentially phosphorylated for some reason. This reason is not clear
at present. The stoichiometry of phosphorylation of glycosylated and
unglycosylated AQP-CD proteins may be different.
Figure 3:In vitro phosphorylation of
oocyte and inner medulla membrane proteins. A, time course of
phosphorylation. Membranes from wild-type AQP-CD-expressing oocytes
were immunoprecipitated with an affinity-purified anti-AQP-CD antibody.
The samples were incubated for 5 min ( lane1), 10 min
( lane2), 20 min ( lane3), or 30
min ( lane4) with catalytic subunit of cAMP-dependent
protein kinase and [-
P]ATP. Membranes from
20 oocytes were applied in each lane. B, membranes
from wild-type AQP-CD-expressing oocytes ( lanes2,
9, and 10), water-injected oocytes ( lane1), mutated AQP-CD-expressing oocytes ( lanes
3-7), and inner medulla ( lane8) were
analyzed. The membranes were incubated in the presence of H89, an
inhibitor of cAMP-dependent protein kinase ( lane9),
or in the absence of catalytic subunit of cAMP-dependent protein kinase
( lane10).
In in vitro phosphorylation studies after 10-min incubation, the pattern of
labeling of rat inner medulla AQP-CD was identical to that of wild-type
AQP-CD expressed in oocytes (Fig. 3 B, lanes2 and 8). By contrast, no labeling was found in
any types of mutated AQP-CD proteins expressed in oocytes
(Fig. 3 B, lanes 3-7). The phosphorylation
of wild-type AQP-CD protein was inhibited by an inhibitor of
cAMP-dependent protein kinase, H89
(15, 16) (Fig. 3 B, lane 9). No
phosphorylation was achieved in the absence of the catalytic subunit of
cAMP-dependent protein kinase (Fig. 3 B, lane
10). For phosphoamino acid analysis, 29-kDa phosphoproteins were
eluted from SDS gel after electrophoresis of wild-type AQP-CD protein.
Phosphoserine was the only amino acid detected, indicating that AQP-CD
protein is phosphorylated at the serine residue (Fig. 4).
Figure 4:
Phosphoamino acid analysis.
P-Labeled wild-type AQP-CD proteins were eluted from SDS
gel. After acid hydrolysis, the sample was mixed with cold phosphoamino
acids and electrophoresed on a cellulose thin-layer chromatography
plate at pH 3.5 (water/acetic acid/pyridine, 945:50:5) at 1,000 V. The
positions of phosphoamino acids were visualized by
ninhydrin.
We
further tried to determine whether phosphorylation of AQP-CD occurs in
intact tissues. Bands of phosphorylated AQP-CD protein were weakly
detected by autoradiography in the control (no incubation with cAMP)
(Fig. 5, lane1). We speculate that endogenous
cAMP is involved in this phosphorylation. cAMP treatment enhanced the
band intensity in a time-dependent manner (Fig. 5, lanes2 and 3). Thus cAMP phosphorylates AQP-CD
protein of rat kidney medulla in vivo, suggesting that AQP-CD
protein acts as a physiological substrate for phosphorylation by
cAMP-dependent protein kinase.
Figure 5:
In
vivo phosphorylation. Tissues of rat inner medulla were suspended in
phosphate-free medium, and the suspensions were incubated with
[P]orthophosphate for 2 h.
8-(4-Chlorophenylthio)-cAMP was added to the incubation medium at a
final concentration of 0.5 mM and incubated for 10 min
( lane2) or 20 min ( lane3).
Alternatively, the suspension was incubated for 10 min in the absence
of cAMP ( lane1). The membrane pellets were
immunoprecipitated with an anti-AQP-CD antibody and analyzed by
SDS-PAGE and autoradiography.
We observed that exogenous cAMP
enhanced oocyte Pf in wild-type AQP-CD-expressing oocytes but not in
mutated AQP-CD-expressing oocytes, which were devoid of a potential
phosphorylation site for cAMP-dependent protein kinase (Ser-256).
cAMP-dependent protein kinase phosphorylated wild-type AQP-CD protein
in vitro at the serine residue. cAMP stimulated
phosphorylation of AQP-CD protein of rat kidney medulla in
vivo. The amount of AQP-CD protein expressed on the oocyte
membrane was not increased by cAMP treatment. These observations
suggest that the phosphorylation of AQP-CD protein at Ser-256 is
responsible for Pf elevation by cAMP. We speculate that vasopressin
increases Pf of in vivo collecting duct cells by
phosphorylation of AQP-CD protein in addition to by the stimulation of
exocytic AQP-CD insertion into apical membrane as proposed previously
(19, 20) . From a physiological viewpoint, both of these
mechanisms may have additive effects on the increase of collecting duct
Pf in vasopressin-stimulated hydroosmotic conditions and on increasing
water reabsorption. In the present study, the rate of Pf increase by
cAMP was relatively slow compared with the responses of other channel
proteins to cAMP
(22, 23, 24) , raising the
possibility that the cAMP-dependent phosphorylation may modulate the
function of AQP-CD protein by another mechanism besides the direct
phosphorylation at Ser-256. Further studies are necessary to determine
the precise mechanism whereby phosphorylation by cAMP-dependent protein
kinase increases Pf and the relative contributions of AQP-CD protein
phosphorylation and the exocytic AQP-CD insertion to
vasopressin-responsive Pf increase in in vivo collecting
tubules.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.