Am J Physiol Renal Physiol 299: F559–F567, 2010.
First published June 24, 2010; doi:10.1152/ajprenal.00617.2009.
Vasopressin increases phosphorylation of Ser84 and Ser486 in Slc14a2
collecting duct urea transporters
Shelly Hwang,1 Ruwan Gunaratne,1 Markus M. Rinschen,1 Ming-Jiun Yu,1 Trairak Pisitkun,1
Jason D. Hoffert,1 Robert A. Fenton,2 Mark A. Knepper,1 and Chung-Lin Chou1
1
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, Maryland; and 2The Water and Salt Research Center, Department of Anatomy, Aarhus University, Aarhus, Denmark
Submitted 26 October 2009; accepted in final form 21 June 2010
Hwang S, Gunaratne R, Rinschen MM, Yu M-J, Pisitkun T,
Hoffert JD, Fenton RA, Knepper MA, Chou C-L. Vasopressin in-
creases phosphorylation of Ser84 and Ser486 in Slc14a2 collecting duct
urea transporters. Am J Physiol Renal Physiol 299: F559 –F567, 2010.
First published June 24, 2010; doi:10.1152/ajprenal.00617.2009.—
Vasopressin-regulated urea transport in the renal inner medullary
collecting duct (IMCD) is mediated by two urea channel proteins,
UT-A1 and UT-A3, derived from the same gene (Slc14a2) by alter-
native splicing. The NH2-terminal 459 amino acids are the same in
both proteins. To study UT-A1/3 phosphorylation, we made phospho-
specific antibodies to UT-A sequences targeting phospho-serines at
positions 84 and 486, sites identified previously by protein mass
spectrometry. Both antibodies proved specific, recognizing only the
phosphorylated forms of UT-A1 and -A3. Immunoblotting of rat
IMCD suspensions or whole inner medullas showed that the V2R-
selective vasopressin analog 1-deamino-8-D-arginine vasopressin
(dDAVP) increases phosphorylation at Ser84 (in UT-A1 and UT-A3)
and Ser486 (in UT-A1) by about eightfold. Time course studies in rat
IMCD suspensions showed maximum phosphorylation within 1 min
of dDAVP exposure, consistent with the time course of vasopressin-
stimulated phosphorylation of the vasopressin-sensitive water channel
aquaporin-2 at Ser256. Confocal immunofluorescence in Brattleboro
rat medullary tissue showed labeling limited to the IMCD, which
increased markedly in response to dDAVP. Immuno-electron micros-
copy studies showed that both phosphorylated forms were present
mainly in intracellular compartments in the presence of vasopressin.
These studies demonstrate regulated phosphorylation of both UT-A1
and UT-A3 in response to vasopressin in a manner consistent with
coordinate regulation of UT-A and aquaporin-2 in the renal IMCD.
The findings add to prior evidence for vasopressin-induced phosphor-
ylation of UT-A1, providing evidence that UT-A3 may be regulated
by phosphorylation as well.
phospho-specific antibody; confocal microscopy; immunofluores-
cence immunocytochemistry; immuno-electron microscopy
THE RENAL INNER MEDULLARY collecting duct (IMCD) is the sole
site of expression of two urea transport proteins, UT-A1 and
UT-A3 (Fig. 1), which are derived from a single gene
(Slc14a2) by alternative splicing. Based on recent X-ray crys-
tallography studies, these proteins probably function as urea
channels (20), rather than carriers as previously believed.
These urea channels mediate transepithelial urea transport,
which allows rapid equilibration of urea between the lumen of
the IMCD and the inner medullary interstitium. Thus, urea,
which is generated as the chief nitrogen waste product from
protein metabolism, can be excreted at high rates without
impairing urine-concentrating ability (4, 12). Vasopressin
Address for reprint requests and other correspondence: M. A. Knepper, 10
Center Dr., MSC 1603, Bldg. 10, Rm. 6N260, National Institutes of Health,
Bethesda, MD 20892-1603 (e-mail: knep@helix.nih.gov).
http://www.ajprenal.org
strongly regulates the urea permeability of the IMCD (34).
However, the mechanisms involved in this regulation remain
unclear. [32P]-labeling experiments revealed that vasopressin
signaling increases phosphorylation of UT-A1 (5, 44). Candi-
date phosphorylation sites (Ser486 and Ser499) in UT-A1 have
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been mutated and shown to be involved in urea transport
regulation, although sites in UT-A3 have not been investigated
in this manner (6). More recently, mass spectrometry-based
phosphoproteomic studies identified several sites in addition to
Ser486 and Ser499 (2, 16), namely Ser10, Ser62, Ser63, and
Ser84 (2). Among these sites, three have been demonstrated by
mass spectrometry to be regulated by vasopressin: Ser84,
Ser486, and Ser499 (2, 6). Of these, Ser84 is present in both
UT-A1 and UT-A3, whereas the other two sites are only
present in UT-A1 (Fig. 1).
To investigate regulated phosphorylation at Ser84, we pro-
duced a phospho-specific antibody to this site and used it in
immunoblotting and immunohistochemical experiments in
rats. The results show that vasopressin stimulates phosphory-
lation of both UT-A1 and UT-A3 at Ser84 with a time course
similar to that of vasopressin-induced phosphorylation of aqua-
porin-2 at Ser256. In addition, to confirm the role of the Ser486
site in UT-A1 and investigate its regulation by vasopressin, we
produced a phospho-specific antibody to this site and con-
firmed that phosphorylation of this site is also increased by
vasopressin.
METHODS
Experimental animals. Pathogen-free male Sprague-Dawley rats
(Taconic Farms, Germantown, NY) or homozygous Brattleboro rats
(Harland Sprague-Dawley, Indianapolis, IN) weighing 150 –200 g
were used in these studies. All experiments were conducted in accord
with animal protocol H-0110R1 approved by the Animal Care and
Use Committee of the National Heart, Lung, and Blood Institute or the
boards of the Institute of Anatomy and Institute of Clinical Medicine,
Aarhus University, according to the licenses for use of experimental
animals issued by the Danish Ministry of Justice.
Phospho-specific UT-A antibodies. Rabbit polyclonal phospho-
specific UT-A1/3 antibodies were generated against 12-amino acid
synthetic phosphopeptides surrounding rat Ser84 or Ser486 (see
NP_062220.2 for sequence). For each antibody, two rabbits were
immunized and affinity-purified (PhosphoSolutions, Aurora, CA),
P7281 and P7282 for pSer84-UT-A1/3; P7283 and P7284 for
pSer486-UT-A1. The specificities of these antibodies were tested by
dot blotting. We also used a previously characterized rabbit antibody
L446 targeted for the NH2-terminal tail of both UT-A1 and UT-A3
(7). IgG concentration of each antibody was quantified by a NanoDrop
spectrophotometer (model ND-1000, NanoDrop Technologies, Wil-
mington, DE; P7281, 50 ␮g/ml; P7282, 60 ␮g/ml; P7283, 90 ␮g/ml;
P7284, 70 ␮g/ml; L446, 140 ␮g/ml). Other antibodies used in this
paper were rabbit anti-aquaporin-2 (AQP2; K5007) (14), rabbit anti-
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F560 UREA CHANNEL PHOSPHORYLATION AT Ser84 AND Ser486

Fig. 1. Phospho-Ser84 UT-A1/3 and phospho-Ser486 UT-A1 antibody recognition sites. The schematic drawing of UT-A1 and UT-A3 shows that the
NH2-terminal 1– 459 amino acids of UT-A1 are identical to UT-A3. The filled boxes represent hydrophobic regions that contain transmembrane domains of the
protein. The residue numbers indicated here and elsewhere in this paper correspond to the rat sequence (RefSeq no. NP_062220.2). The antibodies recognizing
phosphorylated Ser84 of both UT-A1 and UT-A3 (P7181 and P7282) were raised against a synthetic phosphopeptide targeting to Ser84. The antibody recognizing

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phosphorylated Ser486 of UT-A1 (P7284) was raised against a synthetic phosphopeptide targeting to Ser486. A previously characterized antibody recognizing total
UT-A1 and UT-A3 (L446) was raised against amino acids 56 –78 (7, 38).

phospho-Ser256-AQP2 (14), and chicken anti-AQP2 (LL265) (3). All
of these antibodies have been previously characterized.
IMCD suspensions. IMCD suspensions were prepared as previ-
ously described with slight modification (9). After rats were eutha-
nized, kidney inner medullas were dissected, minced, and digested
into suspensions by incubation at 37°C for 70 –90 min in digestion
solution (250 mM sucrose, 10 mM triethanolamine, pH 7.6) contain-
ing collagenase B (3 mg/ml; Roche, Indianapolis, IN) and hyaluron-
idase (1,400 U/ml; Worthington, Lakewood, NJ). The resulting inner
medullary suspension (whole IM) was subjected to three low-speed
centrifugations (at 70 g, 30 s) to separate the IMCD-enriched fraction
in the pellet from the non-IMCD fraction in the supernatant. When all
three fractions were used, they were harvested by centrifugation at
1,000 g for 5 min and washed once. The pellets were resuspended in
1⫻ Laemmli buffer (1.5% SDS, 10 mM Tris, pH 6.8) containing
protease and phosphatase inhibitors and passed through a DNA
shredder (Qiagen, Valencia, CA). After protein concentration deter-
mination by the BCA method (Thermo Scientific, Rockford, IL),
samples were stored in the presence of 6% glycerol and 40 mM DTT
after heating at 60°C for 15 min. When IMCD suspensions were used
in physiological experiments, the IMCD-enriched pellet was gently
washed and resuspended in bicarbonate-buffered solution containing
(in mM) 118 NaCl, 4 Na2HPO4, 5 KCl, 25 NaHCO3, 2 CaCl2, 1.2
MgSO4, and 5.5 glucose (290 mosmol/kgH2O). IMCD suspensions
were allowed to equilibrate with gentle stirring with an aid of a
micro-stirring bar at 37°C with 95% air-5% CO2 supply for 10 min
before use. Time course experiments were carried out by exposing
IMCD suspensions to 1-deamino-8-D-arginine vasopressin (dDAVP)
or vehicle for various lengths of time (0, 1, 5, 15, 30 min). Hormone
incubation was terminated by spinning the suspensions at 14,000 rpm
for 1 min to harvest the pellet containing IMCD segments. Samples
were resuspended in 1⫻ Laemmli buffer and treated as described
above.
Semiquantitative immunoblotting. Proteins were resolved by SDS-
PAGE on polyacrylamide gels (Criterion, Bio-Rad, Hercules, CA) and
transferred electrophoretically onto nitrocellulose membranes. Mem-
branes were blocked for 30 min with Odyssey blocking buffer (Li-
Cor, Lincoln, NE), rinsed, and probed with the respective affinity-
purified antibodies at proper dilution (in Odyssey blocking buffer
containing 0.1% Tween 20) overnight at 4°C. After 1-h incubation
with secondary antibody (Alexa Fluor 680 goat anti-rabbit immuno-
globulin G; Invitrogen, Carlsbad, CA) at 1:5,000 dilution, sites of
antibody-antigen reaction were detected using an Odyssey infrared
imager (Li-Cor).
Perfusion fixation of rat kidneys. Rats under anesthesia were
surgically prepared for retrograde perfusion of the kidneys via the
abdominal aorta. The kidneys were first perfused with PBS for 10 s to
wash out the blood, followed by ice-cold 4% paraformaldehyde for 5
min. The fixed kidneys were trimmed to expose all three major
regions (cortex, outer medulla, and inner medulla), embedded in
paraffin, and sectioned (4 ␮m) for immunofluorescence studies.
Immunofluorescence confocal microscopy. Immunostaining was
performed as previously described (43). In brief, paraffin-embedded
whole kidney sections were dewaxed using xylene and rehydrated
sequentially in 100, 95, 90, and 70% ethanol. Antigen retrieval was
performed with microwave treatment for 15 min in TEG buffer (10
mM Tris and 0.5 mM EGTA, pH 9.0) followed by neutralization in 50
mM NH4Cl (in PBS). Blocking was performed using 1% BSA, 0.2%
gelatin, and 0.05% saponin in PBS. Incubation with the primary
antibody (diluted in 0.1% BSA and 0.3% Triton X-100 in PBS) was
performed overnight (4°C). After being washed with 0.1% BSA, 0.2%
gelatin, and 0.05% saponin in PBS, tissue sections were incubated for
1 h with secondary antibody (conjugated with either Alexa 488 or
Alexa 568; Invitrogen) diluted in 0.1% BSA and 0.3% Triton X-100
in PBS. After subsequent washes with PBS, nuclei were counter-
stained with DAPI (4 ␮l of 0.2 mg/ml DAPI stock solution diluted in
10 ml PBS). The sections were then preserved in fluorescence mount-
ing medium (S3023, Dako North America). For peptide blocking
controls, antibody was preincubated with appropriate peptides at a
1:25 molar ratio for 2 h at 4°C before use. Sections were also
incubated without primary antibody as a negative control. Confocal
fluorescence images were acquired using a Zeiss LSM 510 META
microscope and software (Carl Zeiss MicroImaging, Thornwood, NY).
Immunogold-electron microscopy. We carried out immunogold
labeling of rat renal inner medulla tissues following the procedure
described by Moeller et al. (27), with rats treated with dDAVP for 60
min and control rats. Anti-pS84 (no. 7281, dilution 1:50), anti-pS486
(no. 7284, dilution 1:50), and anti-UT-A1/3 (L446, dilution, 1:300)
were used.
Statistical analysis. Data are presented as means ⫾ SE. All statis-
tical comparisons were made by t-tests. P ⬍ 0.05 was considered
significant.
RESULTS
Specificities of phospho-specific UT-A1/3 antibodies. Figure
1 shows the locations of phosphoserines targeted by the phos-
pho-specific antibodies. Figure 2 shows the results of dot
blotting testing the specificities of the antibodies. As shown,
phospho-Ser84 UT-A1/3 antibody P7282 recognized only the
phosphopeptide targeted to Ser84 of UT-A1/3 but not the

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 UREA CHANNEL PHOSPHORYLATION AT Ser84 AND Ser486
Fig. 2. Specificities of phospho-Ser84 UT-A1/3 antibody (P7281 and P7282)
and phospho-Ser486 UT-A1 antibody (P7283 and P7284). In each dot blot,
0.01 ␮g of phosphopeptides was loaded to the upper end of the nitrocellulose
membrane and corresponding nonphosphopeptides were loaded to the lower
end. All 4 antibodies were used at a 1:1,000 dilution (see IgG concentrations
in METHODS). Antibody P7282 and P7284 recognized phosphopeptides but not
nonphosphopeptides.
corresponding nonphosphopeptide. Antibody P7281 was sim-
ilar in specificity and produced only a very faint nonphos-
phopeptide signal that is barely visible in Fig. 2. Antibody
P7282 was consequently used in immunoblotting. Antibody
P7281, however, yielded better immunostaining in tissue sec-
tions than P7282, so it was used in immunofluorescence studies
only with appropriate labeling controls. Similarly, one phos-
pho-Ser486-UT-A1 antibody (P7284) showed specificity for its
phosphopeptide. Antibody P7284 was used in both immuno-
blotting and immunofluorescence studies.
Phospho-UT-A1 and UT-A3 localization in rat kidney. Figure 3A
shows immunoblots testing for the presence of phosphorylated
UT-A1 and UT-A3 proteins in a whole inner medullary sus-
pension and two subfractions, non-IMCD and IMCD-enriched
(first 3 lanes of each blot). The results show that all three
phosphorylation sites, pSer84-UT-A1, pSer84-UT-A3, and
pSer486-UT-A1, are enriched in the IMCD fraction, consistent
with the known exclusive distribution of UT-A1 and UT-A3 in
IMCD (30, 38). Previous studies of UT-A1 demonstrated that
it exists in the IMCD chiefly at two distinct molecular weights,
97 and 117 kDa (on 10% polyacrylamide gel), that represent
two distinct levels of glycosylation (deglycosylated UT-A1 ran
at 88 kDa.) (7). In Fig. 3A, these two previously identified
bands are indicated by arrows labeled “UT-A1.” Also, previous
studies of UT-A3 demonstrated that it is present chiefly at two
distinct molecular weights, 67 and 44 kDa, that represent two
distinct levels of glycosylation (deglycosylated UT-A3 ran at
40 kDa) (38). In Fig. 3A, these previously identified bands are
indicated by arrows labeled “UT-A3.” Both phospho-specific
antibodies P7282 and P7284 recognized pSer84-UT-A1 or
pSer486-UT-A1 as a higher band at 117 kDa and a lower band
at 97 kDa, consistent with previous observations showing that
these bands represent two different glycosylation states (7).
The phospho-specific antibody P7282 also recognized pSer84-
UT-A3 at two molecular weights, ⬃67 and ⬃44 kDa, consis-
tent with what was reported previously for UT-A3 (38) and
confirmed here in an immunoblot using an antibody to total
UT-A1/3 (antibody LL446; Fig. 3A, right). Furthermore, as
shown in the last two lanes of each blot in Fig. 3A, V2
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receptor-selective vasopressin analog dDAVP (1 nM, 30 min)
strongly increased the abundance of pSer84-UT-A1, pSer84-
UT-A3, and pSer486-UT-A1, but not total UT-A1/3, in IMCD,
confirming that phosphorylation of Ser84 of UT-A1 and
UT-A3 and Ser486 of UT-A1 is regulated by vasopressin.
Figure 3B confirms that the tubule fractionation procedure
successfully enriched the collecting duct-specific marker
AQP2 and deenriched the loop of Henle marker AQP1 in the
IMCD fractions. Preadsorption controls confirm the specifici-
ties of the phospho-specific antibodies (Fig. 3C), showing that
the same bands described above were ablated by preincubation
with the specific phospho-peptide. Note that in the pS486 blot,
there is a pair of bands at ⬃75 and ⬃55 kDa (labeled with
asterisks) that appear to be compatible with similar bands seen
previously with the same antibody after limited chymotrypsin
cleavage of UT-A1 (7). Thus, it appears possible that a small
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fraction of total UT-A1 may be subject to physiological pro-
teolytic cleavage of the middle loop downstream from Ser486.
Immunofluorescence labeling showed that both total and
phosphorylated UT-A1/3 (both sites) are specifically localized
to IMCD cells, with no labeling of other cell types (Fig. 4).
Within the IMCD cell, phospho-UT-A1/3 were found in both
the cytoplasm and apical region of the cell, a result consistent
with previous immunolocalization studies of UT-A1 (30) or
UT-A3 (38).
Time course of UT-A1 and UT-A3 phosphorylation. Previ-
ously, using [32P] radioisotope labeling in IMCD suspensions,
Zhang et al. (44) demonstrated that vasopressin could rapidly
(within 2–5 min) increase phosphorylation of UT-A1. To test
whether vasopressin stimulates phosphorylation of Ser84 or
Ser486 of UT-A1 or UT-A3 in a similar fashion, IMCD
suspensions were incubated with dDAVP for various lengths of
time. As shown in Fig. 5A, there was a substantial increase in
phosphorylation of UT-A1 (117- and 97-kDa bands) at Ser84
and Ser486 in response to dDAVP, beginning at the shortest
incubation time (1 min) and persisting throughout the entire
experimental period (30 min). (Note that the ratio of the upper
UT-A1 band density to the lower UT-A1 band density appears
to be greater for Ser486-phosphorylated UT-A1 than for Ser84-
phosphorylated UT-A1.) Similarly, a rapid increase in phos-
phorylation can be seen at Ser84 in UT-A3. In contrast, there
was no change in the abundance of total UT-A1 or UT-A3 in
response to dDAVP. Phosphorylation of AQP2 at Ser256 was
also increased within 1 min of dDAVP addition as previously
demonstrated (8, 15). Figure 5B summarizes the time course
data graphically, showing the magnitude of UT-A1 and UT-A3
phosphorylation induced by dDAVP as log2(dDAVP:control)
as a function of time (n ⫽ 3).
In vivo UT-A1 and UT-A3 phosphorylation in response to
vasopressin. We next tested whether these phosphorylation
events can occur in the animal. The experiment was conducted
on six Sprague-Dawley rats given 200 mM sucrose for drink-
ing for 16 h before the experiment to lower the baseline level
of phospho-UT-A. Thereafter, three rats received an intramus-
cular injection of 2 nmol dDAVP 1 h before euthanasia and the
other three rats received saline as control. Rats receiving
dDAVP treatment had a large increase in urine osmolality
(before: 184 ⫾ 13, after: 1,389 ⫾ 65 mosmol/kgH2O, n ⫽ 3,
P ⬍ 0.0001), whereas the control rats had only a small increase
in urine osmolality over the 1-h waiting period (before: 155 ⫾
3, after: 297 ⫾ 3 mosmol/kgH2O, n ⫽ 3, P ⬍ 0.05). Immu-
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F562 UREA CHANNEL PHOSPHORYLATION AT Ser84 AND Ser486

Fig. 3. Immunoblotting using anti-phospho-Ser84

UT-A1 and UT-A3 and anti-phospho-Ser486
UT-A1 antibodies in rat inner medullary samples.
A: in each blot, whole inner medullary suspension
(whole IM) and 2 subfractions, non-inner medul-
lary collecting duct (non-IMCD) and IMCD,
were loaded (first 3 lanes) to test the presence of
phosphorylated or nonphosphorylated UT-A1 and
UT-A3 in IMCD. In addition, IMCD samples
with and without treatment of 1-deamino-8-D-
arginine vasopressin (dDAVP; 1 nM, 30 min)
were loaded (last 2 lanes) to test the effect of
vasopressin on phosphorylation of UT-A1 and
UT-A3 on targeted sites. Samples were loaded on
a 4 –15% polyacrylamide gel. Loading was 30 ␮g
per lane except for dDAVP-treated IMCD which
was 20 ␮g. Antibodies P7282, P7284, and L446
labeled UT-A1 as 117- and 97-kDa bands (ar-
rows) as previously found (7). Antibodies P7282
and L446 labeled UT-A3 as broad ladder-like 67-

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and 44-kDa bands (arrows), as prevously found
(38). All antibodies, P7282, P7284, and L446,
were used at a 1:1,000 dilution. B: an enrichment
test for IMCD suspension preparation using col-
lecting duct aquaporin (AQP2) as a marker for
IMCD and thin descending limb aquaporin
(AQP1) as a marker for non-IMCD. C: pread-
sorption control. Twenty micrograms IMCD sam-
ples treated with dDAVP were loaded per lane on
a 12.5% polyacrylamide gel. Membranes were
incubated with antibody with or without pread-
sorption with appropriate peptides for 4 h at room
temperature before use. Peptide concentrations:
0.1 ␮g/ml. *Bands compatible with physiological
proteolytic cleavage of UT-A1 (7).

noblotting was performed on whole homogenates of the ter-
minal 80% of kidney inner medulla (the papilla). The results
show that inner medullas of dDAVP-treated rats have signifi-
cantly higher abundances of all three phospho-UT-A proteins,
pSer84-UT-A1, pSer84-UT-A3, and pSer486-UT-A1, than in-
ner medullas from control rats (Fig. 6A). Figure 6B graphically
depicts the magnitude of UT-A phosphorylation induced by
dDAVP expressed as log2(dDAVP:control).
An in vivo dDAVP response experiment was also conducted
in Brattleboro rats, a natural strain of rat that lacks endog-
enously circulating vasopressin, to examine the immunolocal-
ization of phosphorylated UT-A1/3 in kidney inner medulla
before and after vasopressin stimulation. Figure 7 shows a
seemingly “all or none” pattern of pSer84-UT-A1/3 in cyto-
plasm and the apical region of Brattleboro rat IMCD after
vasopressin stimulation (Fig. 7, A and B). Vasopressin-induced
redistribution of AQP2 from intracellular domain to apical
plasma membrane was also shown as a positive control (Fig.
7C). Preabsorption with the Ser84 phosphopeptide fully ab-
lated the signal (Fig. 7A). Similar results were obtained for
pSer486-UT-A1 as shown in Fig. 7, D–F. Again, preabsorption
with the Ser486 phosphopeptide fully ablated the signal (Fig. 7D).
Immunogold-EM localization of phosphorylated urea chan-
nel proteins. Immunogold-EM localization of pSer84- and
pSer486-phosphorylated urea transporters in rat inner medulla
showed no appreciable labeling in the absence of dDAVP
pretreatment (not shown). After 60-min dDAVP (1 ng) treat-
ment of rats, there was abundant labeling of IMCD cells, the
majority of which was intracellular (Fig. 8). A large proportion
of the gold particles was observed in the Golgi complexes and
Golgi vesicles in the apical region of IMCD cells. Only a few
gold particles were observed in direct association with the
apical plasma membrane. Some pSer-84 gold particles were
observed within the basolateral membrane infoldings (not
shown). Using a UT-A antibody that recognizes both UT-A1
and UT-A3 total protein, abundant plasma membrane labeling,

Fig. 4. Immunofluorescence localization of phos-

phorylated UT-A1/3 in Sprague-Dawley rat kidney
inner medulla. Phospho-Ser84 UT-A1/3 (left) and
phospho-Ser486 UT-A1 proteins (middle), as well
as total UT-A1/3 (right), are present only in col-
lecting duct cells. P7281 and P7284 antibodies
were used at a 1:200 dilution; L446 was used at a
1:100 dilution. (Preadsorption controls are shown
below in Figs. 7 and 8.)

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 UREA CHANNEL PHOSPHORYLATION AT Ser84 AND Ser486
Fig. 5. Time course of UT-A1 and UT-A3 phosphorylation in response to
dDAVP in rat IMCD suspensions. A: representative immunoblots for pSer84-
UT-A1, pSer84-UT-A3, pSer486-UT-A1, and total UT-A1/3 in control and
dDAVP-treated IMCD suspensions prepared from Sprague-Dawley rats. At
each time point, IMCD treated with 1 nM dDAVP had a substantially higher
UT-A1 and UT-A3 phosphorylation level compared with its control, whereas
the total UT-A1 or UT-A3 did not significantly change. Phosphorylation of
AQP2 at Ser256 was also measured as a positive control to demonstrate the
efficacy of the dDAVP. Thirty micrograms of IMCD protein were loaded per
lane on a 12.5% polyacrylamide gel. B: summary line graph depicts the
changes in protein abundance of each protein, expressed as log2(dDAVP:
control; n ⫽ 3). Band densities were quantified by drawing a single integration
box around both bands for UT-A1 and both bands for UT-A3.
AJP-Renal Physiol • VOL 299 • SEPTEMBER 2010 •
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including both the apical plasma membrane and basolateral
plasma membranes, was detected.
DISCUSSION
In this paper, we used two new phospho-specific antibodies
recognizing phosphorylated serines in the collecting duct urea
transporters UT-A1 and UT-A3 to investigate vasopressin-
mediated regulation of urea transporter phosphorylation. One
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Fig. 6. In vivo phosphorylation of UT-A1 and UT-A3 in response to dDAVP.
Experiment was done on 6 water-loaded Sprague-Dawley rats. Three rats
received intramuscular injection of 2 nmol dDAVP 1 h before euthanasia and
3 rats received saline only as control. Terminal inner medullas were dissected
and homogenized for immunoblotting. A: immunoblots displaying phosphor-
ylation changes in response to dDAVP in whole inner medulla homogenates.
Thirty micrograms of sample protein were loaded per lane on a 12.5%
polyacrylamide gel. Inner medullary whole homegenates from dDAVP-treated
rats had significantly higher levels of pSer84-UT-A1, pSer84-UT-A3, and
pSer486-UT-A1 than control rats. B: summary of quantification of immuno-
blots for phospho-UT-A1 and UT-A3 presented as log2(dDAVP:control). Band
densities were quantified by drawing a single integration box around both
bands for UT-A1 and both bands for UT-A3.
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F564 UREA CHANNEL PHOSPHORYLATION AT Ser84 AND Ser486

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Fig. 7. Immunofluorescence localization of pSer84-UT-A1/3 (A–C) and pS486-UT-A1 (D–F) in Brattleboro rat kidney inner medulla– effect of dDAVP. A: test
of antibody (P7281) specificity using a peptide blocking assay (blue: nuclei, green: pSer84-UT-A1/3). Tissue sections were preincubated with antibody with or
without preadsorption with Ser84 phosphopeptide for 2 h at 4°C before use. P7281 antibody was used at a 1:50 dilution. Peptide concentration: 0.5 ␮g/ml.
Phosphorylation at Ser84 was detected in kidney sections obtained from dDAVP-treated rats but not vehicle-treated rats. Preadsorption with immunizing
phosphopeptide ablated signal. B: immunolocalization of pSer84-UT-A1/3 and total UT-A1/3 in consecutive kidney sections. pSer84-UT-A1/3 and UT-A1/3
were detected in collecting ducts only. Both antibodies were used at a 1:50 dilution. C: double labeling for pSer84-UT-A1/3 (green) and AQP2 (red). After
dDAVP treatment, AQP2 is mainly apical, whereas pSer84-UT-A1/3 is localized both apically and intracellularly. AQP2 antibody was used at a 1:100 dilution.
A–C: identical confocal microscope settings were used to visualize vehicle and dDAVP-treated samples. Immunofluorescence using pS486-UT-A1 antibody was
done in a manner similar to pS84-UT-A1/3 antibody as described above. D: test of antibody (P7284) specificity using a peptide-blocking assay (blue: nuclei,
green: pSer486-UT-A1). Phosphorylation at Ser486 was detected in kidney sections obtained from dDAVP-treated rats but not vehicle-treated rats. Preadsorption
with immunizing phosphopeptide ablated signal. E: immunolabeling of pSer486-UT-A1 and total UT-A1/3 in consecutive kidney sections. pSer486-UT-A1/3
and UT-A1/3 were detected in collecting ducts only. F: double labeling for pSer486-UT-A1 (green) and AQP2 (red). After dDAVP treatment, AQP2 is mainly
apical, whereas pSer486-UT-A1 is localized both apically and intracellularly.

antibody recognizes the phosphorylated Ser84 site present in
both UT-A1 and UT-A3, which was demonstrated by LC-
MS/MS analysis of rat collecting ducts by Bansal et al. (2). The
second antibody recognizes the phosphorylated Ser486 site,
present in UT-A1 but not UT-A3, which was demonstrated
originally by Hoffert et al. (16) and has been extensively
studied by Blount et al. (6). The latter study used mutational
analysis to infer a role for the Ser486 phosphorylation in the
regulation of UT-A1.
UT-A1 and UT-A3 are proteins of 929 and 460 amino acids
(Fig. 1) that are derived from the same gene (Slc14a2) as a
result of incorporation of alternative exons that contain either
a stop codon in the case of the shorter UT-A3 (exon 12) or a
full open reading frame that allows splicing to exon 14 in the
case of the longer UT-A1 (23 exons) (28, 33, 35). Since exons
1–11 are the same in UT-A1 and UT-A3, the corresponding
amino acid sequence is the same for the first 459 amino acids
of both proteins. Our L446 antibody was made to recognize the
NH2-terminal tail of both proteins allowing assessment of both
UT-A1 and UT-A3 abundances on one immunoblot. The two
are discriminated on the basis of size (Fig. 3). Figure 1
illustrates the relationship between the two proteins and labels
the two phosphorylation sites under study in this paper, viz
Ser84 and Ser486. It is at present unclear whether UT-A1 and
UT-A3 function independently as separate transporters or are
part of a more complex structure combining UT-A1 and
UT-A3 in a single heteromeric transporter. However, both can
function independently as homomeric transporters with heter-
ologous expression in oocytes (6, 13, 24) or cultured cells (6,
36). UT-A1 and UT-A3 have been shown to be coregulated, at
least in long-term regulation of transporter abundance. Chronic
conditions associated with downregulation of both UT-A1 and
UT-A3 include potassium depletion (19), ureteral obstruction
(21), extracellular fluid volume expansion (41), and glucocor-
ticoid excess (22). Also, Dahl salt-sensitive rats show increased
levels of both UT-A1 and UT-A3 compared with control rats
(11). However, studies of different hydration states in animals
showed that UT-A1 and UT-A3 expression can be regulated
differently (1, 23).
Since Ser84 is present in both UT-A1 and UT-A3, it was of
particular interest to determine whether the increased abun-
dance of phosphorylated Ser84 demonstrated by mass spec-
trometry (2) was due to vasopressin-induced phosphorylation
of either UT-A1, UT-A3, or both. The results shown in Figs. 3
and 5 demonstrated that indeed phosphorylation of this site is
markedly increased in both urea transporters in response to the

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 UREA CHANNEL PHOSPHORYLATION AT Ser84 AND Ser486 F565

Fig. 8. Immunogold electron microscopy dem-

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onstrates that pSer84 UT-A and pSer486 UT-A
are predominantly associated with intracellular
compartments. A: overview of an IMCD cell in
midregion of inner medulla labeled with
pSer84 UT-A. B: higher magnification of the
area highlighted in A. Few gold particles are
directly associated with the apical plasma
membrane (apm). Abundant labeling is ob-
served in the subapical Golgi apparatus (g) and
intracellular vesicles. C: further example of the
association of pSer84 UT-A with the subapical
Golgi apparatus. D: overview of an IMCD cell
in midregion of inner medulla labeled with
pSer486 UT-A. E: higher magnification of the
area highlighted in D. Few gold particles are
directly associated with the apm. Abundant
labeling is observed in subapical vesicles.
F: pSer486 UT-A is also abundant in the sub-
apical Golgi apparatus. G: overview of a prin-
cipal cell in IMCD labeled with L446 (UT-
A1/3 specific antibody). Inset: area highlighted
in box 1 and demonstrates labeling of the
subapical Golgi apparatus. H: area corresponds
to box 2 in G and demonstrates abundant la-
beling of the apical plasma membrane. I: area
corresponds to box 3 in G and demonstrates
that some gold particles are also observed in
the basolateral plasma membrane. In all im-
ages, gold particles are 10 nm in diameter.

V2 receptor vasopressin analog dDAVP. Furthermore, this of both Ser84 and Ser486 of UT-A1, and Ser84 of UT-A3, in
result demonstrates regulated phosphorylation of UT-A3, IMCD suspensions indicates a very rapid response, within 1
which has not been known previously to be a target for min of vasopressin addition. This finding is coherent with time
regulated phosphorylation. The time course of phosphorylation course studies of urea transport in isolated, perfused IMCD

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F566 UREA CHANNEL PHOSPHORYLATION AT Ser84 AND Ser486
segments showing the initial rise in urea permeability ⬃40 s
after vasopressin exposure (40). It is also consistent with
UT-A1 phosphorylation studies using [32P] incorporation to
show increased phosphorylation in the same time frame (44).
The time course was similar for both sites and for Ser84 in both
UT-A1 and UT-A3, suggesting that the same signaling path-
ways were rate limiting in each case. In addition, the time
course of AQP2 phosphorylation at Ser256 has been shown to
be similar (8, 15). Previous studies suggested that this rate-
limiting process for the vasopressin response is at or before the
level of cAMP generation (29, 40).
We also confirmed the strong regulation of phosphorylation
at Ser84 and Ser486 using immunohistochemistry. In general,
with confocal immunofluorescence, only IMCD cells were
labeled and labeling included both the cell periphery and
intracellular regions. Phosphorylated UT-A1/3 is not restricted
to the apical plasma membrane, contrary to what was seen
previously in the case of phosphorylated AQP2 at Ser269 (14,
27). Thus, the results do not provide evidence pointing to a
specific role of phosphorylation of UT-A1 or UT-A3 in plasma
membrane retention or regulation of endocytosis as for Ser269
phosphorylation of AQP2 (26). Supporting this view were our
findings from immunogold electron microscopy using the two
antibodies that showed virtually exclusive presence of phos-
phorylated UT-A in intracellular compartments, with much of
the intracellular labeling associated with the Golgi apparatus
and Golgi vesicles in the apical region of the cells (Fig. 8). Our
data highlight the need for high-resolution imaging techniques
for confirmation of confocal microscopy studies, especially
when drawing conclusions regarding subcellular distribution. It
remains possible that the phosphorylation at either site plays a
role in exocytosis of UT-A1 or UT-A3 as proposed by Blount
et al. (6) and that the phosphorylation is rapidly removed on
delivery of the urea channels to the plasma membrane. Another
possibility is that some portion of plasma membrane UT
channels is phosphorylated, but that the phosphorylation is
masked by proteins that bind to the phosphorylation sites or
due to conformational changes in channel structure.
The amino acid sequence surrounding the Ser84 phosphoryla-
tion site is SKRRESELPRRA (Ser84 is underlined). As can be
seen, the Ser84 site is in a basic region of the protein (surrounded
by arginine and lysine moieties up- and downstream). Thus, it is
likely that Ser84 is phosphorylated by one or more “basophilic”
kinases (25). The amino acid sequence surrounding the Ser486
phosphorylation site is FPRRKSVFHIE (Ser486 is underlined).
Thus, Ser486 is also in a basic region with arginines and
lysines upstream from the phosphorylation site. This again
points to basophilic kinases as candidates for phosphorylation
of Ser486. Vasopressin is known to signal in part through
activation of the basophilic kinase protein kinase A (PKA).
The phosphorylation motif signature for PKA is x-(R/K)-(R/
K)-x-(S/T)-x (25). (This terminology gives the amino acid
sequence surrounding the target serine or threonine where R/K
means either arginine or lysine.) In collecting ducts, vasopres-
sin has also been found to signal via pathways involving
several additional kinases including myosin light chain kinase
(MLCK) (8), Akt or protein kinase B (31), and ERK1/2 (31).
Among these, only Akt has the phosphorylation motif signa-
ture to be a candidate for the two phosphorylation sites under
study in this paper, i.e., Ser84 and Ser486 of urea transporters.
The Akt phosphorylation target signature is R-x-R-x-x-(S/
AJP-Renal Physiol • VOL
T)-␾, where ␾ is a hydrophobic amino acid (25). MLCK has
only one known target, viz. myosin regulatory light chain A
and B isoforms where Thr18 and Ser19 are phosphorylated
(sequence PQRATSNVFA) (17, 18). The signature for Erk1/
2-mediated phosphorylation has been reported to be x-P-x-(S/
T)-P-x (proline moieties both up- and downstream from target
Ser or Thr) (25). No ERK1/2 candidate sites have been iden-
tified in UT-A1 or UT-A3 by mass spectrometry as of this
writing, although other critical sites may remain undiscovered
(42). In addition, because inhibitors of calmodulin block some
aspects of vasopressin signaling (10), it is worthwhile to
consider CaM-kinase II which is strongly expressed in the
IMCD (39) as a candidate for phosphorylation at Ser84 and
Ser486 of UT-A1/3. The CaM-kinase II target signature has
been reported to be x-R-x-x-(S/T)-x-x. Both Ser84 and Ser486
have a basic amino acid in the -3 position, making them
Downloaded from http://ajprenal.physiology.org/ at EBSCO on April 7, 2013
compatible with CaM-kinase II targets. Furthermore, Rinschen
et al. (32) recently reported that vasopressin activates CaM
kinase II in collecting duct cells. Finally, Stewart et al. (37)
proposed that urea transport by UT-A3 may be regulated via
phosphorylation by an acidophilic kinase casein kinase II. Its
target motif signature is x-x-(S/T)-x-x-(E/D)-x (25). The only
demonstrated phosphorylation sites in UT-A1/3 that are com-
patible with this motif are Ser-62 and Ser-63 (DLRSSDEDS),
although this site has not been found to show an increase in
phosphorylation in response to vasopressin (2).
PKA is capable of phosphorylating both Ser84 and Ser486
in vitro (2) but such a demonstration does not imply that PKA
is the only or even the chief kinase responsible for phosphor-
ylation at these sites. However, Zhang et al. (44) demonstrated
that the moderately specific PKA inhibitor H-89 blocked va-
sopressin-induced phosphorylation of UT-A1, pointing to a
role for PKA in regulation of sites present in UT-A1, presum-
ably Ser486 and/or Ser499.
ACKNOWLEDGMENTS
We acknowledge the professional skills and advice of Dr. C. A. Combs
[Light Microscopy Core Facility, National Heart, Lung, and Blood Institute
(NHLBI)] regarding light microscopy-related data in this paper. E. Merete
Locke is thanked for excellent technical assistance in immunogold electron
microscopy.
GRANTS
This study was supported by the Intramural Budget of the NHLBI (Project
Z01-HL-001285). S. Hwang was supported by an American Physiological
Society Undergraduate Summer Research Fellowship. M. M. Rinschen was
supported by the Biomedical Exchange Program, Hannover and the Braun
Foundation, Melsungen. R. A. Fenton is funded by the Danish Medical
Research Council, The Lundbeck Foundation, and the Novo Nordisk Founda-
tion.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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