INVITED REVIEW

Cell biology of vasopressin-regulated aquaporin-2 trafficking

Hanne B. Moeller & Robert A. Fenton
Received: 28 May 2012 / Revised: 10 June 2012 / Accepted: 11 June 2012 / Published online: 29 June 2012
#Springer-Verlag 2012
Abstract Whole-body water balance is predominantly con-
trolled by the kidneys, which have the ability to concentrate or
dilute the urine in the face of altered fluid and solute intake.
Regulated water excretion is controlled by various hormones
and signaling molecules, with the antidiuretic hormone argi-
nine vasopressin (AVP) playing an essential role, predomi-
nantly via its modulatory effects on the function of the water
channel aquaporin-2 (AQP2). The clinical conditions, central
and nephrogenic diabetes insipidus, emphasize the importance
of the AVP-AQP2 axis. In this article, we summarize the most
important and recent studies on AVP-regulated trafficking of
AQP2, with focus on the cellular components mediating (1)
AQP2 vesicle targeting to the principal cell apical plasma
membrane, (2) docking and fusion of AQP2-containing
vesicles, (3) regulated removal of AQP2 from the plasma
membrane, and (4) posttranslational modifications of AQP2
that control several of these processes. Insight into the
molecular mechanisms responsible for regulated AQP2 traf-
ficking is proving to be fundamental for development of novel
therapies for water balance disorders.
Keywords AQP2
.
Water channel
.
Vasopressin
.
Protein
trafficking
.
Posttranslational modifications
.
Phosphorylation
.
Ubiquitination
Maintaining body water balance
Maintaining body water balance, even when challenged
with various water intakes, water losses, or varying body
salt concentrations is a basic physiological necessity. Main-
taining water homeostasis lies in the kidney's ability to
concentrate urine and can be attributed to the vasopressin/
V2-receptor/aquaporin-2 axis that has evolutionary co-de-
veloped over millions of years [41]. In the kidney, approx-
imately 180 L/day of blood is filtered by the glomerulus.
However, less than 1 % of the filtered water is excreted as
final urine. The osmolality of this urine can vary between
50 mOsm/kg in the absence of the antidiuretic hormone
arginine vasopressin (AVP) and 1,200 mOsm/kg in the
presence of AVP [1]. These large differences in urine osmo-
lality are due to the reabsorption of water across the tubular
epithelium of the nephron via water channels, so-called
aquaporins (AQPs). This is a passive process that is driven
by osmotic gradients generated, predominantly, via counter-
current multiplication in the thick ascending limb of Henle's
loop. Under normal conditions, approximately 90 % of the
filtered volume of water is reabsorbed in the proximal tubule
and the thin descending limb of Henle's loop via constitu-
tively expressed AQP1 water channels. Acute regulation of
AQP1 is controversial [13]. Water reabsorption in the con-
necting tubule (CNT) and collecting duct (CD) is under the
control of AVP and other signaling molecules and occurs via
the apical AQP2 and the basolateral AQP3 and AQP4 water
channels. Thus, it is in these segments that fine tuning of
water excretion occurs, and as such, the essential regulation
of whole-body water homeostasis [76].
Abnormalities in water homeostasis emphasize the essen-
tial role of regulated AQP2 trafficking. Although not the
focus of this review (see [52, 76]), defective or dysregulated
AQP2 targeting and synthesis underlies a variety of clinical
H. B. Moeller
:
R. A. Fenton (*)
Department of Biomedicine and Center for Interactions of Proteins
in Epithelial Transport (InterPrET), Aarhus University,
Bldg. 1233 Wilhelm Meyers Alle,
Aarhus 8000, Denmark
e-mail: RoFe@ana.au.dk
H. B. Moeller
e-mail: hbmo@ana.au.dk
Pflugers Arch - Eur J Physiol (2012) 464:133144
DOI 10.1007/s00424-012-1129-4
conditions. Examples include inherited or acquired forms of
nephrogenic diabetes insipidus, resulting in loss of body
water [1, 52], or the serious complication of water retention
that can occur in heart disease and liver cirrhosis [52].
Cellular effects of vasopressin in the connecting tubule
and collecting duct
AQP2 is expressed in kidney CNT cells, CD principal cells,
and inner medullary collecting duct (IMCD) cells. Apical
plasma membrane abundance of AQP2 is the rate-limiting
step and controls the reabsorption of water; a result of
regulated exocytosis of subapical AQP2 bearing vesicles
and regulated AQP2 retrieval from the plasma membrane.
Together, these two processes carefully balance the levels of
apical membrane AQP2. Total cellular expression of AQP2
and apical plasma membrane localization are mainly con-
trolled by AVP [78, 125], although other stimuli can play a
role. Upon minor increases in the osmolality of the blood
(1 %), AVP is released into the circulation from the posterior
pituitary gland [93]. In the principal cells, AVP binds to the
basolateral Gs-protein-coupled V2-receptor [17]. This inter-
action initiates a complex intracellular signaling cascade
resulting in activation of adenylate cyclase (most likely
AC6 [94]), increased intracellular cAMP levels, activation
of PKA and other kinases, and phosphorylation of AQP2.
These events cause both the translocation of AQP2-bearing
vesicles to the apical plasma membrane and slow down
AQP2 endocytic retrieval, thus promoting CD water reab-
sorption [49, 76] (Fig. 1a). AVP stimulation also results in
increased intracellular Ca
2+
levels [15, 102]. Although the
mechanism for this is not completely clear, calmodulin-
dependent release of Ca
2+
from ryanodine-sensitive intra-
cellular stores plays a role [10]. There are some indications
that calcium is necessary for AQP2 insertion into the plasma
membrane. In isolated perfused tubules, inhibition of the
AVP-induced calcium response prevents AQP2 trafficking
but does not affect intracellular cAMP levels [10]. Although
the nonselective requirement of Ca
2+
for membrane vesicle
fusion cannot be discounted, activation of the exchange
protein directly activated by cAMP (Epac) also triggers
intracellular Ca
2+
mobilization and apical insertion of
AQP2 in the CD [128] (Fig. 1b).
Although AVP is the major regulator of AQP2 trafficking
and is the focus of this review, it must be emphasized that a
variety of other molecules/hormones can influence AQP2
membrane accumulation and cellular expression, e.g., prosta-
glandins [87, 115, 130], bradykinin [107], dopamine [3], argi-
nine/NO [4], ANP [47], oxytocin [9], and angiotensin II [56].
In addition to regulating AQP2 trafficking, AVP also
affects AQP2 expression levels via multiple mechanisms.
AVP increases AQP2 transcription [26, 116]. This occurs
via PKA-induced phosphorylation of the cAMP-responsive
element-binding protein that subsequently increases AQP2
transcription via a cAMP responsive element (CRE) [35, 64,
126] (Fig. 1c). Recent studies have suggested that the PKA-
induced CRE pathway is responsible for the initial increase
in AQP2 transcription following AVP stimulation, but the
long term effect occurs via a different pathway and may
involve Epac [50]. AVP may also regulate AQP2 protein
abundance by stabilization of the protein and reduced deg-
radation [70, 72].
AQP2-trafficking to the apical plasma membrane
Following translation, AQP2 is folded into its monomeric
conformation, and subsequently a tetrameric complex, in the
endoplasmic reticulum. These tetramers are transported to
the golgi complex [30] and, similarly to AQP1, are believed
to constitute the AQP2 functional unit with each monomer
being an independent pore for water [71]. Two out of four
AQP2 monomers are complex N-glycosylated in the Golgi
apparatus before the channels are transported through the
trans-Golgi network (TGN) to different subcellular compart-
ments. Although AQP2 is predominantly associated with
the apical plasma membrane, it must be mentioned that, to
some extent, AQP2 is also associated with the basolateral
plasma membrane [12, 75, 121]. A large proportion of
AQP2 that exits the TGN is stored in some form of endo-
somal transport vesicle and upon relevant stimulus is trans-
ported to the apical plasma membrane [78]. In addition to
regulated trafficking, AQP2 recycles constitutively between
cell surface and intracellular vesicles independently of hor-
monal stimulation [24, 60, 104].
Although there is a complex interplay of several regula-
tory pathways, for simplicity, we consider total plasma
membrane abundance of AQP2 a result of (1) regulated
vesicular trafficking to the membrane, (2) docking and
fusion of vesicles with the apical plasma membrane (exocy-
tosis), and (3) removal of the water channel from the mem-
brane (endocytosis) in the remainder of this review. It has
become clear that this regulation is not merely a result of
regulation of general transport processes but requires that
AQP2 itself interacts with and modulates other proteins in
addition to AQP2 itself being subjected to regulated post-
translational modifications (PTMs).
Vesicular transport to the membranea role for actin
The role of the actin cytoskeleton for AQP2 trafficking has
been extensively studied, and modulating the filamentous
actin network affects AQP2 trafficking in vitro [73, 82, 83,
109]. Actin may be involved at various levels of AQP2
134 Pflugers Arch - Eur J Physiol (2012) 464:133144
regulation, including reservation of the intracellular storage
pool of AQP2 and in vesicular transport [83]. For AQP2
exocytosis, two distinct roles for actin have been proposed
[19, 83]. First, actin filaments are suggested to provide a
track for guided movements of AQP2-containing vesicles to
the apical plasma membrane. Second, the apical actin net-
work is suggested to constitute a physical barrier that
holds subapical vesicles and prevents their exocytosis
[14]. AVP can depolymerize actin filaments in both the toad
bladder and apically in IMCD cells and AQP2-transfected
CD8 cells, resulting in exocytosis of AQP2-carrying
vesicles [14, 28, 48, 99, 108]. Recently, it was demonstrated
that AVP/forskolin-mediated F-actin depolymerization
occurred locally and was closely related to AQP2 plasma
membrane accumulation [129].
Although the mechanisms for AVP effects on the actin
barrier have been examined, the results are not completely
clear and likely are multifactoral. Inhibition/inactivation of
RhoA, a small GTP-binding protein that participates in
polymerization of actin, results in AQP2 membrane accu-
mulation [48, 57, 108, 110], whereas Rho activation stabil-
izes F-actin and inhibits AQP2 membrane accumulation
[107]. Linking these observations to the AVP effect, forsko-
lin treatment stabilizes the inactive form of RhoA in CD8
cells [110]. Traditionally, this membrane accumulation has
been taken as a role for actin in AQP2 translocation
Fig. 1 Regulated trafficking events of AQP2. a Adenylate cyclase (AC)
is activated upon AVP binding to the Gs-protein-coupled basolateral AVP
type 2 receptor (V2R), resulting in increased intracellular cAMP levels
and activation of PKA. This promotes apical plasma membrane accumu-
lation of tetrameric AQP2 by increasing exocytosis of subapical AQP2-
bearing vesicles and decreasing AQP2 endocytosis from the plasma
membrane. Upon removal of AVP, AQP2 is internalized and can be stored
in subapical vesicles. Upon restimulation, AQP2 can recycle to the
membrane. b Increased intracellular Ca
2+
aids AQP2 trafficking. AVP
stimulation results in increased intracellular Ca
2+
levels via Ca
2+
release
from calmodulin-dependent ryanodine-sensitive intracellular stores. Ad-
ditionally, activation of the exchange protein directly activated by cAMP
(Epac) can also trigger Ca
2+
mobilization and apical membrane expres-
sion of AQP2. The role of increased Ca
2+
in AQP2 trafficking remains
unclear. c AVP regulates AQP2 protein abundance. AVP increases AQP2
transcription via a CRE. Long-term AVP stimulation may regulate tran-
scription via Epac. AQP2 protein abundance is also regulated by an AVP-
induced decrease in AQP2 degradation. d Apical depolymerization of
actin in response to AVP allows vesicle fusion of AQP2-bearing vesicles
with the apical plasma membrane. AQP2 itself may be involved in
regulation of this process. AVP triggers cAMP signaling that induces
phosphorylation of AQP2 at Ser256. This phosphorylation dissociates G-
actin (globular) from AQP2 and promotes AQP2 interaction with tropo-
myocin 5b (TM5b). This sequesters TM5b fromF-actin (filamentous) and
induces destabilization of the F-actin network, allowing vesicle transport
to the membrane. e Docking and fusion of AQP2-bearing vesicles with
the apical plasma membrane is mediated via SNARE mechanisms.
AQP2-bearing vesicles contain specific v-SNAREs that bind to specific
t-SNAREs on the apical plasma membrane, a process requiring the bind-
ing of the ATPase soluble N-ethylmaleimide-sensitive factor. Munc18
inhibits the SNARE-mediated membrane fusion. f After AVP washout,
AQP2 localizes to clathrin-coated pits and undergoes clathrin-mediated
endocytosis. Adirect AQP2 interaction with Hsp70/Hsc70 suggests a role
for these proteins in uncoating of the endocytic vesicles. g Phosphoryla-
tion of AQP2 determines the intracellular localization. AVP-induced
phosphorylation at Ser256 and Ser269 is involved in retaining AQP2 in
endocytosis-resistant membrane domains. The mechanism behind this is
possibly a reduced interaction with the endocytic machinery. The phos-
phatase PP1 is involved in dephosphorylation of AQP2. Interaction with
the protein MAL promotes retention of AQP2 in the apical plasma
membrane. h AQP2 is ubiquitinated with one or more ubiquitin proteins
at Lys270. Ubiquitination occurs in the membrane after removal of AVP
stimulation and mediates AQP2 internalization and degradation via
lysosomes
Pflugers Arch - Eur J Physiol (2012) 464:133144 135
although this has not been directly examined. Furthermore,
recent studies have suggested that membrane accumulation
via inactivation of RhoA is due to an effect of the actin
cytoskeleton-inhibiting endocytosis [57] rather than exocyto-
sis. Activity of another protein, SPA-1, a GTPase-activating
enzyme for Rap1 that is involved in assembly of F-actin, is
required for forskolin-mediated AQP2 trafficking [79]. SPA-1
binds directly to AQP2 and affects the assembly of F-actin
through crosstalk with Rho family GTPases [79]. Supporting
this role, AQP2 trafficking was impaired in SPA-1-deficient
mice [79]. Although the above studies support a role of
actin depolymeriation for AQP2 trafficking, disruption of
the actin cytoskeleton with cytochalasin D inhibited AQP2
translocation and water permeability in toad urinary blad-
der [19, 105]. Thus, it is likely that a complete disruption
of the actin cytoskeleton inhibits AQP2 trafficking, where-
as partial depolymerization enhances trafficking. Addition-
ally, as actin polymerization and actin coating of fusing
transport vesicles can act as stabilizers during exocytosis
[100], a nonspecific effect of actin depolymerization can-
not be discounted.
Rather than AQP2 being passively transported to the
membrane via regulation of the actin cytoskeleton, some
studies have demonstrated that AQP2 itself can directly
modulate the local actin cytoskeleton and influence its
own vesicle transport. AQP2 directly binds to actin [81]
and a reciprocal AQP2 interaction between AQP2, G-
actin, and tropomyosin 5b, which depends on the AQP2
phosphorylation status, has been demonstrated [80]. This
interaction is believed to catalyze F-actin reorganization
and open a pathway for the local release of AQP2 vesicles
to the plasma membrane following AVP stimulation (see
Fig. 1d). AVP and forskolin can mediate a burst of exocy-
tosis that is observed only in cells expressing AQP2 [85],
and very recently, it was shown that AQP2 itself is neces-
sary for AVP-mediated actin filament depolymerization
[129]. In addition to AVP, actin reorganization and AQP2
membrane expression can be affected by other pathways
[91].
Docking and fusion with the membrane
The specificity of docking and fusing of AQP2-bearing
vesicles is mediated by SNARE (Soluble N-ethylmaleimide-
sensitive factor attachment protein receptors) mechanisms
[95, 101]. Multiple components of the SNARE system are
present in the CD principal cell (Fig. 1e). The v-SNARE
proteins vesicle-associated membrane protein (VAMP)-2 and
VAMP-3 are found in AQP2-containing vesicles [2, 20, 38,
59, 77], and t-SNARES are observed in the apical membrane
of CD principal cells (syntaxin-4) [63] and in the apical
plasma membrane of MCD4 renal cells (syntaxin 3 and
SNAP25). Another SNARE, SNAP23, colocalizes with
AQP2 in the CD [37]. cAMP-mediated AQP2 targeting
to the plasma membrane is inhibited by tetanus toxin,
suggesting a role of v-SNARES in AQP2 docking [22].
In MCD4 cells, functional studies demonstrated that
knockdown of VAMP 2, VAMP 3, syntaxin 3, and
SNAP23 inhibited AQP2 fusion at the apical membrane
[89]. In addition, Munc18 (a protein-inhibiting SNARE-
mediated membrane fusion) inhibits the AVP effect on
AQP2 trafficking and knockdown increases AQP2 mem-
brane accumulation [89]. The SNARE-associated protein
Snapin serves as a linker between AQP2 and the t-
SNARE complex and can aid trafficking from storage
vesicles to the apical plasma membrane by association
with syntaxin-3 and SNAP23 [66].
Many other proteins are involved in AQP2 trafficking and
exocytosis, but their precise role and how they interact with
AQP2 (or AQP2-containing vesicles) remains to be fully
established. Annexin-2 is a member of a protein family which
associates with membrane phospholipids in a calcium-
dependent manner. Annexin-2 localizes to the plasma mem-
brane in response to forskolin stimulation in cultured cells
[112] and has been shown to interact with/or associate with
AQP2 [82, 132]. Inhibition of annexin II impairs water
permeability in response to cAMP in cultured cells, and
it has been proposed that this is due to reduced AQP2
vesicle fusion [112]. In addition to increases in cAMP
mediated by Gs proteins and adenylate cyclases, members
of the Gi family have been shown to be required for
AQP2 trafficking, although their precise role remains to
be determined [97, 118]. Finally, multiple GTPases play
key roles in regulation of vesicle transport between cellu-
lar compartments [86]. Rab GTPases belonging to this
family of proteins and Rab3, which is involved in exo-
cytic pathways [84], have been identified in AQP2-bearing
vesicles [59]. Recent studies suggest that the Akt substrate
protein AS160 is involved in AQP2 translocation via its
effects on Rab proteins [40, 46].
Removal of AQP2 from the membrane
After AVP exposure, AQP2 localizes to endocytosis-resistant
membrane domains and, upon AVP washout, AQP2 is inter-
nalized [5]. Other stimuli that, under specific conditions,
can cause internalization of AQP2 include PGE2 and
dopamine [74]. It is suggested that AVP removal results
in a release of an endocytic block that maintains AQP2
at the cell surface [5], but the molecular mechanisms
behind this remain to be fully established. A body of
evidence supports the internalization of AQP2 via a
clathrin-mediated pathway (Fig. 1f). AQP2 accumulates
in clathrin-coated pits, where it interacts with the adaptor
136 Pflugers Arch - Eur J Physiol (2012) 464:133144
protein AP2 (a component of clathrin-coated vesicles) [61]
before it is internalized [5, 7, 8, 60, 96, 103]. Inhibition of
dynamin, a protein involved in pinching off clathrin-
coated pits, induces membrane accumulation of AQP2,
suggesting that the clathrin-mediated endocytosis of
AQP2 occurs via a dynamin-dependent pathway [104].
Finally, heat shock protein 70 (hsp70) and heat shock
cognate 70 (hsc70), which are involved in uncoating of
clathrin-coated vesicles and trafficking, both directly inter-
act with AQP2. Inhibition of hsc70 results in AQP2
membrane accumulation [61].
Actin may also play a role in AQP2 endocytosis [83].
Studies on the effects of simvastatin in Brattleboro rats
(which genetically lack AVP) and parallel cell line studies
demonstrated that inhibition of RhoA occurs in parallel with
inhibited endocytosis, also suggesting involvement of the
actin network in AQP2 internalization [57]. Furthermore,
moesin, part of the ezrin/radixin/moesin (ERM) protein
complex that interacts with PDZ domains and induce
delayed protein internalization, can regulate AQP2 surface
expression [109].
After internalization, AQP2 is localized to EEA1-
positive early endosomes before it is transferred to
Rab11-positive storage vesicles [106]. Upon restimulation
with AVP, AQP2 can be recycled to the apical membrane
(the membrane shuttle hypothesis) [122]. Alternatively,
internalized AQP2 can follow the route of degradation
via multivesicular bodies and lysosomes [26, 43, 67].
LIP5 interacts with and is responsible for sorting of
AQP2 to multivesicular bodies [119], (Fig. 1h). AQP2
in multivesicular bodies can also be reexcreted into the
urine as exosomes [88]. Although ubiquitinated, it is
controversial if proteasomal degradation of AQP2 occurs.
Studies using treatment of cells with the proteasomal
inhibitor lactacystin suggested a role for proteasomal
degradation of AQP2 by increasing AQP2 abundance
[26]. However, whether this effect is a direct effect on
AQP2 degradation or an indirect effect is not known.
Another study suggested that AQP2 is polyubiquitinated
(although not directly shown), and this mediates AQP2
degradation via the proteasomal pathway [72]. It must be
emphasized that a direct association of AQP2 and the
proteasomes has not been demonstrated.
Similarly to exocytosis, the posttranslational status of
AQP2 itself is an active player in regulation of endo-
cytosis. For example, phosphorylation and ubiquitina-
tion of AQP2 are important in determining the process
of internalization. Although few studies have directly
addressed the dynamics of AQP2 endocytosis, it is now
clear that phosphorylation and ubiquitination of the
COOH-tail of AQP2 are dual players in determining
the rate of internalization and AQP2 membrane abun-
dance [6, 43, 61, 70, 92].
Posttranslational modifications of AQP2
Phosphorylation
AQP2 is polyphosphorylated at the COOH terminus
(Fig. 2). Ser256 was the first AQP2 phosphorylation site
identified and has been extensively characterized using
phosphospecific antibodies, AQP2-mutant-expressing cell
models, and functional studies on oocytes [11, 21, 42].
Almost a decade later, phosphoproteomic analysis identi-
fied further AQP2 phosphorylation at Ser261, Ser264,
and Ser269 [34]. All phosphorylation sites are highly
conserved among species [69]. A number of other phos-
phorylation sites for various kinases in AQP2 have been
predicted, but whether these are truly phosphorylated
residues in vivo remains to be established [6]. Which
phosphatases are directly responsible for AQP2 dephos-
phorylation is unclear, but a role for PP1 has been
suggested [70, 132].
Regulation of phosphorylation
The levels of phosphorylation at all known AQP2 phosphor-
ylation sites (Ser256, Ser261, Ser264, and Ser269) are reg-
ulated by AVP. In the presence of AVP, phosphorylation of
Ser256, Ser264, and Ser269 increases; whereas phosphory-
lation of Ser261 is higher in the absence of AVP [32].
Protein kinase A (PKA) is responsible for Ser256 phosphor-
ylation, but other kinases could also be involved [69]. AQP2
phosphorylation is a hierarchal mechanism. Ser256 phos-
phorylation precedes phosphorylation of Ser264 and Ser269
[32], and an intact Ser256 site is also necessary for phos-
phorylation of Ser264 and Ser269 [32, 68]. Phosphorylation
of Ser256, Ser264, and Ser269 occurs within minutes of
agonist stimulation and is sustained as long as the agonist is
present [32, 87]. Relative quantification of AQP2 phospho-
forms in rat IMCD and mpkCCD cells demonstrated that
baseline levels of phosphorylation at Ser256 were constitu-
tively high and did not significantly increase after dDAVP
treatment, whereas large increases in pS269 abundance in
response to dDAVP were observed [124]. Additionally,
phosphorylation of Ser269 is only detected when the agonist
is present [67]. Combined with cell data, this suggests that
although an intact Ser256 site is required for AQP2 traffick-
ing, an increase in Ser256 phosphorylation is not essential.
Phosphorylation and AQP2 membrane targeting can be
regulated by extracellular tonicity. Cultured renal CD8
cells, exposed to hypotonic medium, have decreased
AQP2 Ser256 phosphorylation and AQP2 membrane ex-
pression [113]. Contrastingly, hypertonicity enhances
AQP2 membrane accumulation. Although Ser256 phos-
phorylation is required for this effect, it occurs indepen-
dently of cAMP, suggesting that kinases other than PKA
Pflugers Arch - Eur J Physiol (2012) 464:133144 137
could be responsible for the effects of tonicity regulated
Ser256 phosphorylation [27]. MAPK family members are
candidates for the hypertonic regulation of AQP2 phos-
phorylation and trafficking.
Localization of phosphorylated AQP2
AQP2 phosphoforms are localized to different cell organ-
elles, aiding our understanding of the potential regulatory
role of phosphorylation, e.g., for sorting AQP2 to spe-
cific cell compartments. Phosphorylation at Ser269
(pS269-AQP2) is only detected in the apical plasma
membrane of principal cells and is not observed in any
intracellular organelles [67]. Phosphorylated Ser256
(pS256-AQP2) is detected in both intracellular vesicles
and the apical plasma membrane [11]. Phosphorylated
Ser261 (pS261-AQP2) is predominantly localized within
intracellular compartments, possibly the Golgi and lyso-
somes [33]. Phosphorylated Ser264 (pS264-AQP2) can
be observed intracellularly but also in both the apical
and basolateral plasma membranes of principal cells after
acute dDAVP treatment [18].
In cell lines expressing phosphorylation deficient
AQP2 at Ser256 (AQP2-S256A), AQP2 is predominant-
ly within the cell, even when means are taken to in-
crease cAMP with forskolin [45, 90, 120]. It must be
noted that this mutation does not prevent a constitutive
AQP2 recycling pathway through the plasma membrane
[60]. Mimicking phosphorylation at Ser256 (AQP2-
S256D) or Ser269 (S269D-AQP2) localizes AQP2 pre-
dominantly to the plasma membrane in basal, unstimu-
lated conditions [70, 74, 120]. However, S269A-AQP2
retains the ability to accumulate in the plasma mem-
brane in response to forskolin treatment [70]. Taken
together, these studies strongly support a role for
Ser256 and Ser269 in AQP2 membrane targeting.
Some discrepancies in the localization of AQP2-S261A/
D mutants have been described. In one study of MDCK
cells, these forms are localized in intracellular compartments
even in the presence of forskolin [114]. In other MDCK
Fig. 2 Models of AQP2 membrane organization. a Schematic model
of AQP2 monomer in the plasma membrane showing the full length
amino acid sequence of human AQP2. The NPA motifs are indicated in
orange. Several posttranslational modifications are illustrated: glyco-
sylation at an extracellular loop; phosphorylation at Ser256, Ser261,
Ser264, and Ser269; and ubiquitination at Lys270. The last three amino
acids of AQP2 constitute a PDZ ligand. b Schematic illustration of
AQP2 topography in the plasma membrane. AQP2 is believed to be
organized in tetramers in the membrane where each monomer consti-
tutes a single water channel. c AQP2 consists of six transmembrane
domains connected by intracellular and extracellular loops. The NH2
terminus and the COOH terminus are located in the cytoplasm. In the
hourglass model, two NPA motifs in the intracellular and extracellular
loops are thought to dock in the membrane and form the water pore
138 Pflugers Arch - Eur J Physiol (2012) 464:133144
models [58, 70] and in LLC-PK1 cells [62], S261 mutants
are localized within the cell but translocated to the plasma
membrane in response to stimulation.
The role of phosphorylation in AQP2 trafficking
What are the specific roles of dynamic and regulated AQP2
phosphorylation? The establishment of an answer to this
question is severely complicated by (1) the number of pos-
sible combinations of different phosphorylated forms of
AQP2, i.e., 16 different possible combinations; (2) the rel-
ative abundance of each phosphorylation site; (3) the num-
ber of various phosphorylation combinations that can occur
within an AQP2 tetramer; (4) the number of possible protein
kinases that can target each site under various conditions;
and (5) the number of different phosphatases that could be
involved in regulation. Furthermore, several studies that
have assessed the role of AQP2 phosphorylation have made
use of various phosphorylation-mimicking mutants, e.g.,
S256D-AQP2. Whether these mutants truly recapitulate
the in vivo effects of phosphorylation is open to interpreta-
tion, as the static negative charge of the mutants are likely,
in reality, to not be observed in vivo where the phosphory-
lation of an individual site is likely to be more dynamic.
Membrane accumulation of AQP2
As described, AQP2 apical plasma membrane abundance
depends on the balance of transport to (exocytosis) and from
the membrane (endocytosis). Although the Ser256 phospho-
form of AQP2 exists both within the cell and in the plasma
membrane [11], it is clear that this specific phosphorylation
site is critical for AVP-induced plasma membrane accumu-
lation of AQP2 [21, 32, 45, 65]. However, it is unclear
whether Ser256 phosphorylation actually induces exocyto-
sis. Although AVP and forskolin can mediate a burst of
exocytosis in cells expressing AQP2 [85], the effects of
S256 mutation on this exocytic burst are not significantly
different [85]. Cell assays of internalization have postulated
a role for phosphorylation sites in AQP2 membrane reten-
tion, with both phosphorylation at Ser256 and Ser269 play-
ing roles in retaining AQP2 in the plasma membrane by
reducing endocytosis [70, 92] (Fig. 1g). One proposed
mechanism behind this phenomenon is that phosphoryla-
tion at these residues reduces interaction of AQP2 with
key members of the endocytic machinery or retains
AQP2 in endocytosis-resistant membrane domains [61,
70]. Again, the data from these studies are predominantly
generated in AQP2-mutant cell lines; thus, caution must
be exercised when interpreting the results. Future studies
that can assess the role of individual phosphorylation
sites on AQP2 endocytosis without using mutant cell
lines would be informative.
Water permeabilitychannel gating
Some mammalian or plant aquaporins are gated by phos-
phorylation, i.e., the phosphorylation induces a conforma-
tional change in the channel structure, resulting in opening/
closing of the water pore and allowing alterations in the flux
of water [23, 29, 39, 117, 131]. There are conflicting data on
the role of Ser256 phosphorylation as a gating mechanism
for mammalian AQP2. In reconstituted proteoliposomes,
PKA phosphorylation of Ser256 enhanced water permeabil-
ity compared to wildtype AQP2 [16]. Similar findings were
observed in a study on oocytes where cAMP was suggested
to regulate AQP2 water permeability [51]. Contrastingly,
studies on AQP2-containing apical endosomes from rat
IMCD cells did not suggest a role for PKA-mediated phos-
phorylation for enhancing water permeability [53]. Other
studies on oocytes have also suggested no effect of C-
terminal polyphosphorylation for regulation of single chan-
nel water permeability [42, 68]. Thus, whether phosphory-
lation results in gating of AQP2 remains debatable. A high-
resolution crystal structure of AQP2 in a phosphorylated/
nonphosphorylated state would be informative to address
this controversy.
Ubiquitination
Ubiquitination is a posttranslational modification (PTM)
where the protein ubiquitin (8 kDa, 76 AA) is covalently
bound to lysine residues in the target protein. This process
requires three enzymes: ub-activating enzyme (E1), ub-
conjugating enzyme (E3), and ub-ligating enzyme (E3)
[25]. Deubiquitination is rapid and mediated by deubiquiti-
nating enzymes. Kamsteeg et al. [43] were the first to
describe ubiquitination of AQP2, which was followed by
other studies [3, 44, 54, 119]. Ubiquitination of AQP2
occurs at a single residue, Lys270, which itself is further
ubiquitinated via K63 linked chains. In cells, AQP2 can
exist as monoubiquitinated or with up to four ubiquitin
moieties attached. Which enzymes are involved in ubiquiti-
nation of AQP2 remains unknown. Studies on E3 ligases
that are regulated in response to long-term AVP stimulation/
removal have suggested that Nedd4 and CUL5 could poten-
tially, directly or indirectly, influence AQP2 ubiquitination
[55]. AVP-induced changes in the abundance of Nedd4 were
also observed by quantitative proteomics [98]. However,
AQP2 lacks the PY-motif that Nedd4 usually requires for
target protein interaction; thus, whether Nedd4 regulates
AQP2 ubiquitination directly remains unclear.
In MDCK cells, ubiquitination of AQP2 is induced by
forskolin and increased (with a peak after 5 min) after
forskolin washout. Ubiquitination also increases in the pres-
ence of TPA, an activator of PKC. K63-linked polyubiquiti-
nation can regulate endocytosis [25], and the ubiquitin-
Pflugers Arch - Eur J Physiol (2012) 464:133144 139
deficient AQP2 mutant AQP2-Lys270Arg had a decreased
rate of endocytosis compared to wildtype AQP2 [43]. Fol-
lowing ubiquitination, AQP2 was targeted for degradation
via the lysosomal pathway. Thus, AQP2 ubiquitination is in
line with the well-established theory that monoubiquitination
of various cell surface receptors functions as an endosomal
sorting signal targeting them for lysosomal degradation [31]
(Fig. 1h).
Posttranslational crosstalking
Single PTMs are able to regulate protein function through
creating new protein-binding sites, mediating proteinpro-
tein interactions, or by causing allosteric changes in the
target protein. AQP2 itself provides an example that mem-
brane proteins can be highly modified in a dynamic fashion.
PTM crosstalking, e.g., phosphorylation and ubiquitination,
could, in principal, increase the information of the protein
substantially [36]. Certainly, the modifications taking place
in the small span at the AQP2 COOH terminus opens up for
the possibility of PTM crosstalk (Fig. 2). An important but
extremely challenging task is to determine which PTMs
coexist on AQP2 at any particular timepoint following a
particular stimulus and to assign the PTM to a specific
molecular and biological function. Although some progress
has been made on examining the functional role of coexist-
ing phosphorylation sites in cell systems, e.g., phosphoryla-
tion of Ser256 dominates over Ser261 in determining AQP2
membrane localization [62], the combinations of phosphor-
ylation, ubiquitination, or other PTMs coexisting at any
particular timepoint are enormous. For example, in cells
expressing Ser256D-K270R-ubi mutants, AQP2 had an in-
tracellular localization under forskolin and control condi-
tions, suggesting that Ser256 phosphorylation is overruled
by ubiquitination [114]. However, in this particular mutant,
the addition of the ubiquitin moiety is different from the in
vivo state where ubiquitin is attached as a side chain. Fur-
thermore, addition of ubiquitin in a linear arrangement
interrupts a potential PDZ ligand at the C-terminal tail of
AQP2 (Fig. 2), which may also play a role in AQP2 traf-
ficking. Thus, the relationship between phosphorylation
combined with ubiquitination and AQP2-trafficking is high-
ly complex [114], and interpretation of these technically
challenging studies is not straightforward.
Summary
In this review, we have focused on the intracellular traffick-
ing mechanisms of AQP2 and how both exocytosis and
endocytosis events of the channel are regulated via AVP.
Our understanding of AQP2 trafficking and function are still
incomplete; in addition to water transport, additional novel
roles for AQP2 are now emerging. For example, AQP2
contains an integrin-binding motif (Arg-Gly-Asp; RGD) in
the external C-loop that can interact with beta 1 integrin
[111, 127] and modulate both AQP2 expression [111] and
cell migration during tubulogenesis [127]. This novel func-
tion may explain the abundance of AQP2 in the basolateral
plasma membrane and is in line with a previous study
suggesting that beta1 integrin is required for kidney collect-
ing duct morphogenesis [123]. Thus, despite substantial
progress in the past 15 years, ongoing studies on AQP2
are likely to continue to provide novel ideas and major
advances in our understanding on membrane protein traf-
ficking and function.
Acknowledgments The work in the laboratories of the authors is
supported by the Danish Medical Research Council, the Novo Nordisk
Foundation, the Lundbeck Foundation, the Carlsberg Foundation, and
the Aarhus University Research Foundation. Ken P. Kragsfeldt, Aarhus
University Hospital, Aarhus, Denmark is thanked for his help with the
figures.
References
1. Babey M, Kopp P, Robertson GL (2011) Familial forms of
diabetes insipidus: clinical and molecular characteristics. Nat
Rev Endocrinol 7(12):701714
2. Barile M, Pisitkun T, Yu MJ, Chou CL, Verbalis MJ, Shen RF,
Knepper MA (2005) Large scale protein identification in intra-
cellular aquaporin-2 vesicles from renal inner medullary collect-
ing duct. Mol Cell Proteomics 4(8):10951106
3. Boone M, Kortenoeven ML, Robben JH, Tamma G, Deen PM
(2011) Counteracting vasopressin-mediated water reabsorption
by ATP, dopamine, and phorbol esters: mechanisms of action.
Am J Physiol Renal Physiol 300(3):F761771
4. Bouley R, Breton S, Sun T, McLaughlin M, Nsumu NN, Lin HY,
Ausiello DA, Brown D (2000) Nitric oxide and atrial natriuretic
factor stimulate cGMP-dependent membrane insertion of aqua-
porin 2 in renal epithelial cells. J Clin Invest 106(9):11151126
5. Bouley R, Hawthorn G, Russo LM, Lin HY, Ausiello DA, Brown
D (2006) Aquaporin 2 (AQP2) and vasopressin type 2 receptor
(V2R) endocytosis in kidney epithelial cells: AQP2 is located in
'endocytosis-resistant' membrane domains after vasopressin treat-
ment. Biol Cell 98(4):215232
6. Brown D, Hasler U, Nunes P, Bouley R, Lu HA (2008) Phos-
phorylation events and the modulation of aquaporin 2 cell surface
expression. Curr Opin Nephrol Hypertens 17(5):491498
7. Brown D, Orci L (1983) Vasopressin stimulates formation of
coated pits in rat kidney collecting ducts. Nature 302
(5905):253255
8. Brown D, Weyer P, Orci L (1988) Vasopressin stimulates endo-
cytosis in kidney collecting duct principal cells. Eur J Cell Biol
46(2):336341
9. Chou CL, DiGiovanni SR, Luther A, Lolait SJ, Knepper MA
(1995) Oxytocin as an antidiuretic hormone. II. Role of V2
vasopressin receptor. Am J Physiol 269 (1 Pt 2):F78-85
10. Chou CL, Yip KP, Michea L, Kador K, Ferraris JD, Wade JB,
Knepper MA (2000) Regulation of aquaporin-2 trafficking by
vasopressin in the renal collecting duct. Roles of ryanodine-
sensitive Ca2+ stores and calmodulin. J Biol Chem 275
(47):3683936846
140 Pflugers Arch - Eur J Physiol (2012) 464:133144
11. Christensen BM, Zelenina M, Aperia A, Nielsen S (2000) Local-
ization and regulation of PKA-phosphorylated AQP2 in response
to V(2)-receptor agonist/antagonist treatment. Am J Physiol Re-
nal Physiol 278(1):F2942
12. Coleman RA, Wu DC, Liu J, Wade JB (2000) Expression of
aquaporins in the renal connecting tubule. Am J Physiol Renal
Physiol 279(5):F874883
13. Conner MT, Conner AC, Bland CE, Taylor LH, Brown JE, Parri
HR, Bill RM (2012) Rapid aquaporin translocation regulates
cellular water flow: the mechanism of hypotonicity-induced
sub-cellular localization of the aquaporin 1 water channel. J Biol
Chem. doi:10.1074/jbc.M111.329219
14. Ding GH, Franki N, Condeelis J, Hays RM (1991) Vasopressin
depolymerizes F-actin in toad bladder epithelial cells. Am J
Physiol 260(1 Pt 1):C916
15. Ecelbarger CA, Chou CL, Lolait SJ, Knepper MA, DiGiovanni
SR (1996) Evidence for dual signaling pathways for V2 vaso-
pressin receptor in rat inner medullary collecting duct. Am J
Physiol 270(4 Pt 2):F623633
16. Eto K, Noda Y, Horikawa S, Uchida S, Sasaki S (2010) Phos-
phorylation of aquaporin-2 regulates its water permeability. J Biol
Chem 285(52):4077740784
17. Fenton RA, Brond L, Nielsen S, Praetorius J (2007) Cellular and
subcellular distribution of the type II vasopressin receptor in
kidney. Am J Physiol Renal Physiol 293(3):F748760
18. Fenton RA, Moeller HB, Hoffert JD, Yu MJ, Nielsen S, Knepper
MA (2008) Acute regulation of aquaporin-2 phosphorylation at
Ser-264 by vasopressin. Proc Natl Acad Sci USA 105(8):3134
3139
19. Franki N, Ding G, Gao Y, Hays RM (1992) Effect of cytochalasin
D on the actin cytoskeleton of the toad bladder epithelial cell. Am
J Physiol 263(5 Pt 1):C9951000
20. Franki N, Macaluso F, Schubert W, Gunther L, Hays RM (1995)
Water channel-carrying vesicles in the rat IMCD contain cellu-
brevin. Am J Physiol 269(3 Pt 1):C797801
21. Fushimi K, Sasaki S, Marumo F (1997) Phosphorylation of serine
256 is required for cAMP-dependent regulatory exocytosis of the
aquaporin-2 water channel. J Biol Chem 272(23):1480014804
22. Gouraud S, Laera A, Calamita G, Carmosino M, Procino G,
Rossetto O, Mannucci R, Rosenthal W, Svelto M, Valenti G
(2002) Functional involvement of VAMP/synaptobrevin-2 in
cAMP-stimulated aquaporin 2 translocation in renal collecting
duct cells. J Cell Sci 115(Pt 18):36673674
23. Gunnarson E, Zelenina M, Axehult G, Song Y, Bondar A,
Krieger P, Brismar H, Zelenin S, Aperia A (2008) Identification
of a molecular target for glutamate regulation of astrocyte water
permeability. Glia 56(6):587596
24. Gustafson CE, Katsura T, McKee M, Bouley R, Casanova JE,
Brown D (2000) Recycling of AQP2 occurs through a
temperature- and bafilomycin-sensitive trans-Golgi-associated
compartment. Am J Physiol Renal Physiol 278(2):F317326
25. Haglund K, Dikic I (2005) Ubiquitylation and cell signaling.
EMBO J 24(19):33533359
26. Hasler U, Mordasini D, Bens M, Bianchi M, Cluzeaud F, Rous-
selot M, Vandewalle A, Feraille E, Martin PY (2002) Long term
regulation of aquaporin-2 expression in vasopressin-responsive
renal collecting duct principal cells. J Biol Chem 277(12):10379
10386
27. Hasler U, Nunes P, Bouley R, Lu HA, Matsuzaki T, Brown D
(2008) Acute hypertonicity alters aquaporin-2 trafficking and
induces a MAPK-dependent accumulation at the plasma mem-
brane of renal epithelial cells. J Biol Chem 283(39):26643
26661
28. Hays RM, Condeelis J, Gao Y, Simon H, Ding G, Franki N
(1993) The effect of vasopressin on the cytoskeleton of the
epithelial cell. Pediatr Nephrol 7(5):672679
29. Hedfalk K, Tornroth-Horsefield S, Nyblom M, Johanson U,
Kjellbom P, Neutze R (2006) Aquaporin gating. Curr Opin Struct
Biol 16(4):447456
30. Hendriks G, Koudijs M, van Balkom BW, Oorschot V,
Klumperman J, Deen PM, van der Sluijs P (2004) Glyco-
sylation is important for cell surface expression of the water
channel aquaporin-2 but is not essential for tetramerization
in the endoplasmic reticulum. J Biol Chem 279(4):2975
2983
31. Hicke L, Dunn R (2003) Regulation of membrane protein trans-
port by ubiquitin and ubiquitin-binding proteins. Annu Rev Cell
Dev Biol 19:141172
32. Hoffert JD, Fenton RA, Moeller HB, Simons B, Tchapyjnikov D,
McDill BW, Yu MJ, Pisitkun T, Chen F, Knepper MA (2008)
Vasopressin-stimulated increase in phosphorylation at Ser269
potentiates plasma membrane retention of aquaporin-2. J Biol
Chem 283(36):2461724627
33. Hoffert JD, Nielsen J, Yu MJ, Pisitkun T, Schleicher SM, Nielsen
S, Knepper MA (2007) Dynamics of aquaporin-2 serine-261
phosphorylation in response to short-term vasopressin treatment
in collecting duct. Am J Physiol Renal Physiol 292(2):F691700
34. Hoffert JD, Pisitkun T, Wang G, Shen RF, Knepper MA (2006)
Quantitative phosphoproteomics of vasopressin-sensitive renal
cells: regulation of aquaporin-2 phosphorylation at two sites. Proc
Natl Acad Sci USA 103(18):71597164
35. Hozawa S, Holtzman EJ, Ausiello DA (1996) cAMP motifs
regulating transcription in the aquaporin 2 gene. Am J Physiol
270(6 Pt 1):C16951702
36. Hunter T (2007) The age of crosstalk: phosphorylation, ubiquiti-
nation, and beyond. Mol Cell 28(5):730738
37. Inoue T, Nielsen S, Mandon B, Terris J, Kishore BK, Knepper
MA (1998) SNAP-23 in rat kidney: colocalization with
aquaporin-2 in collecting duct vesicles. Am J Physiol 275(5 Pt
2):F752760
38. Jo I, Harris HW, Amendt-Raduege AM, Majewski RR, Hammond
TG (1995) Rat kidney papilla contains abundant synaptobrevin
protein that participates in the fusion of antidiuretic hormone-
regulated water channel-containing endosomes in vitro. Proc Natl
Acad Sci USA 92(6):18761880
39. Johansson I, Karlsson M, Shukla VK, Chrispeels MJ, Larsson C,
Kjellbom P (1998) Water transport activity of the plasma mem-
brane aquaporin PM28A is regulated by phosphorylation. Plant
Cell 10(3):451459
40. Jung HJ, Kwon TH (2010) Membrane trafficking of collecting
duct water channel protein AQP2 regulated by Akt/AS160. Elec-
trolyte & Blood Pressure: E & BP 8(2):5965
41. Juul KV (2012) The evolutionary origin of the vasopressin/V2-
type receptor/aquaporin axis and the urine-concentrating mecha-
nism. Endocrine
42. Kamsteeg EJ, Heijnen I, van Os CH, Deen PM (2000) The
subcellular localization of an aquaporin-2 tetramer depends on
the stoichiometry of phosphorylated and nonphosphorylated
monomers. J Cell Biol 151(4):919930
43. Kamsteeg EJ, Hendriks G, Boone M, Konings IB, Oorschot V,
van der Sluijs P, Klumperman J, Deen PM (2006) Short-chain
ubiquitination mediates the regulated endocytosis of the
aquaporin-2 water channel. Proc Natl Acad Sci USA 103
(48):1834418349
44. Kamsteeg EJ, Savelkoul PJ, Hendriks G, Konings IB, Nivillac
NM, Lagendijk AK, van der Sluijs P, Deen PM (2008) Missorting
of the aquaporin-2 mutant E258K to multivesicular bodies/lyso-
somes in dominant NDI is associated with its monoubiquitination
and increased phosphorylation by PKC but is due to the loss of
E258. Pflugers Arch 455(6):10411054
45. Katsura T, Gustafson CE, Ausiello DA, Brown D (1997) Protein
kinase A phosphorylation is involved in regulated exocytosis of
Pflugers Arch - Eur J Physiol (2012) 464:133144 141
aquaporin-2 in transfected LLC-PK1 cells. Am J Physiol 272(6 Pt
2):F817822
46. Kim HY, Choi HJ, Lim JS, Park EJ, Jung HJ, Lee YJ, Kim SY,
Kwon TH (2011) Emerging role of Akt substrate protein AS160
in the regulation of AQP2 translocation. Am J Physiol Renal
Physiol 301(1):F151161
47. Klokkers J, Langehanenberg P, Kemper B, Kosmeier S, von Bally
G, Riethmuller C, Wunder F, Sindic A, Pavenstadt H, Schlatter E,
Edemir B (2009) Atrial natriuretic peptide and nitric oxide sig-
naling antagonizes vasopressin-mediated water permeability in
inner medullary collecting duct cells. Am J Physiol Renal Physiol
297(3):F693703
48. Klussmann E, Tamma G, Lorenz D, Wiesner B, Maric K, Hofmann
F, Aktories K, Valenti G, Rosenthal W (2001) An inhibitory role of
rho in the vasopressin-mediated translocation of aquaporin-2 into
cell membranes of renal principal cells. J Biol Chem 276
(23):2045120457
49. Knepper MA, Inoue T (1997) Regulation of aquaporin-2 water
channel trafficking by vasopressin. Curr Opin Cell Biol 9(4):560
564
50. Kortenoeven ML, Trimpert C, Brand MV, Li Y, Wetzels JF, Deen
PM (2012) In mpkCCD cells, long-term regulation of aquaporin-
2 by vasopressin occurs independent of protein kinase A and
CREB, but may involve Epac. Am J Physiol Renal Physiol.
51. Kuwahara M, Fushimi K, Terada Y, Bai L, Marumo F, Sasaki S
(1995) cAMP-dependent phosphorylation stimulates water perme-
ability of aquaporin-collecting duct water channel protein expressed
in Xenopus oocytes. J Biol Chem 270(18):1038410387
52. Kwon TH, Nielsen J, Moller HB, Fenton RA, Nielsen S, Frokiaer
J (2009) Aquaporins in the kidney. Handb Exp Pharmacol
190:95132
53. Lande MB, Jo I, Zeidel ML, Somers M, Harris HW Jr (1996)
Phosphorylation of aquaporin-2 does not alter the membrane
water permeability of rat papillary water channel-containing
vesicles. J Biol Chem 271(10):55525557
54. Lee YJ, Kwon TH (2009) Ubiquitination of aquaporin-2 in the
kidney. Electrolyte Blood Pressure: E & BP 7(1):14
55. Lee YJ, Lee JE, Choi HJ, Lim JS, Jung HJ, Baek MC, Frokiaer J,
Nielsen S, Kwon TH (2011) E3 ubiquitin-protein ligases in rat
kidney collecting duct: response to vasopressin stimulation and
withdrawal. Am J Physiol Renal Physiol 301(4):F883896
56. Li C, Wang W, Rivard CJ, Lanaspa MA, Summer S, Schrier RW
(2011) Molecular mechanisms of angiotensin II stimulation on
aquaporin-2 expression and trafficking. Am J Physiol Renal
Physiol 300(5):F12551261
57. Li W, Zhang Y, Bouley R, Chen Y, Matsuzaki T, Nunes P, Hasler
U, Brown D, Lu HA (2011) Simvastatin enhances aquaporin-2
surface expression and urinary concentration in vasopressin-
deficient Brattleboro rats through modulation of Rho GTPase.
Am J Physiol Renal Physiol 301(2):F309318
58. Li YH, Eto K, Horikawa S, Uchida S, Sasaki S, Li XJ, Noda Y
(2009) Aquaporin-2 regulates cell volume recovery via tropomy-
osin. Int J Biochem Cell Biol 41(12):24662476
59. Liebenhoff U, Rosenthal W (1995) Identification of Rab3-,
Rab5a- and synaptobrevin II-like proteins in a preparation of rat
kidney vesicles containing the vasopressin-regulated water chan-
nel. FEBS Lett 365(23):209213
60. Lu H, Sun TX, Bouley R, Blackburn K, McLaughlin M, Brown
D (2004) Inhibition of endocytosis causes phosphorylation
(S256)-independent plasma membrane accumulation of AQP2.
Am J Physiol Renal Physiol 286(2):F233243. doi:10.1152/
ajprenal.00179.2003
61. Lu HA, Sun TX, Matsuzaki T, Yi XH, Eswara J, Bouley R,
McKee M, Brown D (2007) Heat shock protein 70 interacts with
aquaporin-2 (AQP2) and regulates its trafficking. J Biol Chem
282(39):2872128732
62. Lu HJ, Matsuzaki T, Bouley R, Hasler U, Qin QH, Brown D
(2008) The phosphorylation state of serine 256 is dominant over
that of serine 261 in the regulation of AQP2 trafficking in renal
epithelial cells. Am J Physiol Renal Physiol 295(1):F290294
63. Mandon B, Chou CL, Nielsen S, Knepper MA (1996) Syntaxin-4
is localized to the apical plasma membrane of rat renal collecting
duct cells: possible role in aquaporin-2 trafficking. J Clin Invest
98(4):906913
64. Matsumura Y, Uchida S, Rai T, Sasaki S, Marumo F (1997)
Transcriptional regulation of aquaporin-2 water channel gene by
cAMP. J Am Soc Nephrol 8(6):861867
65. McDill BW, Li SZ, Kovach PA, Ding L, Chen F (2006) Congen-
ital progressive hydronephrosis (cph) is caused by an S256L
mutation in aquaporin-2 that affects its phosphorylation and
apical membrane accumulation. Proc Natl Acad Sci USA 103
(18):69526957
66. Mistry AC, Mallick R, Klein JD, Weimbs T, Sands JM, Frohlich O
(2009) Syntaxin specificity of aquaporins in the inner medullary
collecting duct. Am J Physiol Renal Physiol 297(2):F292300
67. Moeller HB, Knepper MA, Fenton RA (2009) Serine 269 phos-
phorylated aquaporin-2 is targeted to the apical membrane of
collecting duct principal cells. Kidney Int 75(3):295303
68. Moeller HB, MacAulay N, Knepper MA, Fenton RA (2009) Role
of multiple phosphorylation sites in the COOH-terminal tail of
aquaporin-2 for water transport: evidence against channel gating.
Am J Physiol Renal Physiol 296(3):F649657
69. Moeller HB, Olesen ET, Fenton RA (2011) Regulation of the
water channel aquaporin-2 by posttranslational modification. Am
J Physiol Renal Physiol 300(5):F10621073
70. Moeller HB, Praetorius J, Rutzler MR, Fenton RA (2010) Phos-
phorylation of aquaporin-2 regulates its endocytosis and protein-
protein interactions. Proc Natl Acad Sci USA 107(1):424429
71. Murata K, Mitsuoka K, Hirai T, Walz T, Agre P, Heymann JB,
Engel A, Fujiyoshi Y (2000) Structural determinants of water
permeation through aquaporin-1. Nature 407(6804):599605
72. Nedvetsky PI, Tabor V, Tamma G, Beulshausen S, Skroblin P,
Kirschner A, Mutig K, Boltzen M, Petrucci O, Vossenkamper A,
Wiesner B, Bachmann S, Rosenthal W, Klussmann E (2010)
Reciprocal regulation of aquaporin-2 abundance and degradation
by protein kinase A and p38-MAP kinase. J Am Soc Nephrol 21
(10):16451656
73. Nedvetsky PI, Tamma G, Beulshausen S, Valenti G, Rosenthal W,
Klussmann E (2009) Regulation of aquaporin-2 trafficking.
Handb Exp Pharmacol 190:133157
74. Nejsum LN, Zelenina M, Aperia A, Frokiaer J, Nielsen S (2005)
Bidirectional regulation of AQP2 trafficking and recycling: in-
volvement of AQP2-S256 phosphorylation. Am J Physiol Renal
Physiol 288(5):F930938
75. Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, Harris
HW (1993) Cellular and subcellular immunolocalization of
vasopressin-regulated water channel in rat kidney. Proc Natl Acad
Sci USA 90(24):1166311667
76. Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, Knepper
MA (2002) Aquaporins in the kidney: from molecules to medi-
cine. Physiol Rev 82(1):205244
77. Nielsen S, Marples D, Birn H, Mohtashami M, Dalby NO,
Trimble M, Knepper M (1995) Expression of VAMP-2-like pro-
tein in kidney collecting duct intracellular vesicles. Colocaliza-
tion with Aquaporin-2 water channels. J Clin Invest 96(4):1834
1844
78. Nielsen S, Terris J, Smith CP, Hediger MA, Ecelbarger CA,
Knepper MA (1996) Cellular and subcellular localization of the
vasopressin-regulated urea transporter in rat kidney. Proc Natl
Acad Sci USA 93(11):54955500
79. Noda Y, Horikawa S, Furukawa T, Hirai K, Katayama Y, Asai T,
Kuwahara M, Katagiri K, Kinashi T, Hattori M, Minato N, Sasaki
142 Pflugers Arch - Eur J Physiol (2012) 464:133144
S (2004) Aquaporin-2 trafficking is regulated by PDZ-domain
containing protein SPA-1. FEBS Lett 568(13):139145
80. Noda Y, Horikawa S, Kanda E, Yamashita M, Meng H, Eto
K, Li Y, Kuwahara M, Hirai K, Pack C, Kinjo M, Okabe S,
Sasaki S (2008) Reciprocal interaction with G-actin and
tropomyosin is essential for aquaporin-2 trafficking. J Cell
Biol 182(3):587601
81. Noda Y, Horikawa S, Katayama Y, Sasaki S (2004) Water channel
aquaporin-2 directly binds to actin. Biochem Biophys Res Com-
mun 322(3):740745
82. Noda Y, Horikawa S, Katayama Y, Sasaki S (2005) Identification
of a multiprotein motor complex binding to water channel
aquaporin-2. Biochem Biophys Res Commun 330(4):10411047
83. Noda Y, Sasaki S (2008) The role of actin remodeling in the
trafficking of intracellular vesicles, transporters, and channels:
focusing on aquaporin-2. Pflugers Arch 456(4):737745
84. Novick P, Brennwald P (1993) Friends and family: the role of the
Rab GTPases in vesicular traffic. Cell 75(4):597601
85. Nunes P, Hasler U, McKee M, Lu HA, Bouley R, Brown D
(2008) A fluorimetry-based ssYFP secretion assay to monitor
vasopressin-induced exocytosis in LLC-PK1 cells expressing
aquaporin-2. Am J Physiol Cell Physiol 295(6):C14761487
86. Nuoffer C, Balch WE (1994) GTPases: multifunctional molecular
switches regulating vesicular traffic. Annu Rev Biochem 63:949
990
87. Olesen ET, Rutzler MR, Moeller HB, Praetorius HA, Fenton RA
(2011) Vasopressin-independent targeting of aquaporin-2 by selec-
tive E-prostanoid receptor agonists alleviates nephrogenic diabetes
insipidus. Proc Natl Acad Sci USA 108(31):1294912954
88. Pisitkun T, Shen RF, Knepper MA (2004) Identification and
proteomic profiling of exosomes in human urine. Proc Natl Acad
Sci USA 101(36):1336813373
89. Procino G, Barbieri C, Tamma G, De Benedictis L, Pessin JE,
Svelto M, Valenti G (2008) AQP2 exocytosis in the renal collect-
ing ductinvolvement of SNARE isoforms and the regulatory
role of Munc18b. J Cell Sci 121(Pt 12):20972106
90. Procino G, Carmosino M, Marin O, Brunati AM, Contri A, Pinna
LA, Mannucci R, Nielsen S, Kwon TH, Svelto M, Valenti G
(2003) Ser-256 phosphorylation dynamics of Aquaporin 2 during
maturation from the ER to the vesicular compartment in renal
cells. FASEB J 17(13):18861888
91. Procino G, Mastrofrancesco L, Tamma G, Lasorsa DR,
Ranieri M, Stringini G, Emma F, Svelto M, Valenti G
(2012) Calcium-sensing receptor and aquaporin 2 interplay in
hypercalciuria-associated renal concentrating defect in humans.
An in vivo and in vitro study. PLoS One 7(3):e33145.
doi:10.1371/journal.pone.0033145
92. Rice WL, Zhang Y, Chen Y, Matsuzaki T, Brown D, Lu HA
(2012) Differential, phosphorylation dependent trafficking of
AQP2 in LLC-PK1 Cells. PLoS One 7(2):e32843. doi:10.1371/
journal.pone.0032843
93. Robertson GL (1987) Physiology of ADH secretion. Kidney Int
Suppl 21:S2026
94. Roos KP, Strait KA, Raphael KL, Blount MA, Kohan DE (2012)
Collecting duct-specific knockout of adenylyl cyclase type VI
causes a urinary concentration defect in mice. Am J Physiol
Renal Physiol 302(1):F7884
95. Rothman JE (1994) Mechanisms of intracellular protein trans-
port. Nature 372(6501):5563
96. Russo LM, McKee M, Brown D (2006) Methyl-beta-cyclodextrin
induces vasopressin-independent apical accumulation of
aquaporin-2 in the isolated, perfused rat kidney. Am J Physiol
Renal Physiol 291(1):F246253
97. Sands JM, Naruse M, Baum M, Jo I, Hebert SC, Brown EM,
Harris HW (1997) Apical extracellular calcium/polyvalent cation-
sensing receptor regulates vasopressin-elicited water permeability
in rat kidney inner medullary collecting duct. J Clin Invest 99
(6):13991405
98. Schenk LK, Bolger SJ, Luginbuhl K, Gonzales PA, Rinschen
MM, Yu MJ, Hoffert JD, Pisitkun T, Knepper MA (2012)
Quantitative proteomics identifies vasopressin-responsive nu-
clear proteins in collecting duct cells. J Am Soc Nephrol.
doi:10.1681/ASN.2011070738
99. Simon H, Gao Y, Franki N, Hays RM (1993) Vasopressin depo-
lymerizes apical F-actin in rat inner medullary collecting duct.
Am J Physiol 265(3 Pt 1):C757762
100. Sokac AM, Bement WM (2006) Kiss-and-coat and compartment
mixing: coupling exocytosis to signal generation and local actin
assembly. Mol Biol Cell 17(4):14951502
101. Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H,
Geromanos S, Tempst P, Rothman JE (1993) SNAP receptors im-
plicated in vesicle targeting and fusion. Nature 362(6418):318324
102. Star RA, Nonoguchi H, Balaban R, Knepper MA (1988) Calcium
and cyclic adenosine monophosphate as second messengers for
vasopressin in the rat inner medullary collecting duct. J Clin
Invest 81(6):18791888
103. Strange K, Willingham MC, Handler JS, Harris HW Jr (1988)
Apical membrane endocytosis via coated pits is stimulated by
removal of antidiuretic hormone from isolated, perfused rabbit
cortical collecting tubule. J Membr Biol 103(1):1728
104. Sun TX, Van Hoek A, Huang Y, Bouley R, McLaughlin M,
Brown D (2002) Aquaporin-2 localization in clathrin-coated pits:
inhibition of endocytosis by dominant-negative dynamin. Am J
Physiol Renal Physiol 282(6):F9981011
105. Tajika Y, Matsuzaki T, Suzuki T, Ablimit A, Aoki T, Hagiwara H,
Kuwahara M, Sasaki S, Takata K (2005) Differential regulation
of AQP2 trafficking in endosomes by microtubules and actin
filaments. Histochem Cell Biol 124(1):112
106. Tajika Y, Matsuzaki T, Suzuki T, Aoki T, Hagiwara H, Kuwahara
M, Sasaki S, Takata K (2004) Aquaporin-2 is retrieved to the
apical storage compartment via early endosomes and phosphati-
dylinositol 3-kinase-dependent pathway. Endocrinology 145
(9):43754383
107. Tamma G, Carmosino M, Svelto M, Valenti G (2005) Bradykinin
signaling counteracts cAMP-elicited aquaporin 2 translocation in
renal cells. J Am Soc Nephrol 16(10):28812889
108. Tamma G, Klussmann E, Maric K, Aktories K, Svelto M, Rosenthal
W, Valenti G (2001) Rho inhibits cAMP-induced translocation of
aquaporin-2 into the apical membrane of renal cells. Am J Physiol
Renal Physiol 281(6):F10921101
109. Tamma G, Klussmann E, Oehlke J, Krause E, Rosenthal W,
Svelto M, Valenti G (2005) Actin remodeling requires ERM
function to facilitate AQP2 apical targeting. J Cell Sci 118(Pt
16):36233630
110. Tamma G, Klussmann E, Procino G, Svelto M, Rosenthal W,
Valenti G (2003) cAMP-induced AQP2 translocation is associat-
ed with RhoA inhibition through RhoA phosphorylation and
interaction with RhoGDI. J Cell Sci 116(Pt 8):15191525
111. Tamma G, Lasorsa D, Ranieri M, Mastrofrancesco L, Valenti G,
Svelto M (2011) Integrin signaling modulates AQP2 trafficking
via Arg-Gly-Asp (RGD) motif. Cell Physiol Biochem: Interna-
tional Journal of Experimental Cellular Physiology, Biochemistry,
and Pharmacology 27(6):739748
112. Tamma G, Procino G, Mola MG, Svelto M, Valenti G (2008)
Functional involvement of Annexin-2 in cAMP induced AQP2
trafficking. Pflugers Arch 456(4):729736
113. Tamma G, Procino G, Strafino A, Bononi E, Meyer G, Paulmichl
M, Formoso V, Svelto M, Valenti G (2007) Hypotonicity induces
aquaporin-2 internalization and cytosol-to-membrane transloca-
tion of ICln in renal cells. Endocrinology 148(3):11181130
114. Tamma G, Robben JH, Trimpert C, Boone M, Deen PM
(2011) Regulation of AQP2 localization by S256 and S261
Pflugers Arch - Eur J Physiol (2012) 464:133144 143
phosphorylation and ubiquitination. Am J Physiol Cell Phys-
iol 300(3):C636646
115. Tamma G, Wiesner B, Furkert J, Hahm D, Oksche A, Schaefer M,
Valenti G, Rosenthal W, Klussmann E (2003) The prostaglandin
E2 analogue sulprostone antagonizes vasopressin-induced anti-
diuresis through activation of Rho. J Cell Sci 116(Pt 16):3285
3294
116. Terris J, Ecelbarger CA, Nielsen S, Knepper MA (1996) Long-
term regulation of four renal aquaporins in rats. Am J Physiol 271
(2 Pt 2):F414422
117. Tornroth-Horsefield S, Wang Y, Hedfalk K, Johanson U, Karlsson
M, Tajkhorshid E, Neutze R, Kjellbom P (2006) Structural mecha-
nism of plant aquaporin gating. Nature 439(7077):688694
118. Valenti G, Procino G, Liebenhoff U, Frigeri A, Benedetti PA,
Ahnert-Hilger G, Nurnberg B, Svelto M, Rosenthal W (1998) A
heterotrimeric G protein of the Gi family is required for cAMP-
triggered trafficking of aquaporin 2 in kidney epithelial cells. J
Biol Chem 273(35):2262722634
119. van Balkom BW, Boone M, Hendriks G, Kamsteeg EJ, Robben
JH, Stronks HC, van der Voorde A, van Herp F, van der Sluijs P,
Deen PM (2009) LIP5 interacts with aquaporin 2 and facilitates
its lysosomal degradation. J Am Soc Nephrol 20(5):9901001
120. van Balkom BW, Savelkoul PJ, Markovich D, Hofman E, Nielsen
S, van der Sluijs P, Deen PM (2002) The role of putative phos-
phorylation sites in the targeting and shuttling of the aquaporin-2
water channel. J Biol Chem 277(44):4147341479
121. van Balkom BW, van Raak M, Breton S, Pastor-Soler N, Bouley
R, van der Sluijs P, Brown D, Deen PM (2003) Hypertonicity is
involved in redirecting the aquaporin-2 water channel into the
basolateral, instead of the apical, plasma membrane of renal
epithelial cells. J Biol Chem 278(2):11011107
122. Wade JB, Stetson DL, Lewis SA(1981) ADHaction: evidence for a
membrane shuttle mechanism. Ann N YAcad Sci 372:106117
123. Wu W, Kitamura S, Truong DM, Rieg T, Vallon V, Sakurai
H, Bush KT, Vera DR, Ross RS, Nigam SK (2009) Beta1-
integrin is required for kidney collecting duct morphogenesis
and maintenance of renal function. Am J Physiol Renal
Physiol 297(1):F210217
124. Xie L, Hoffert JD, Chou CL, Yu MJ, Pisitkun T, Knepper MA,
Fenton RA (2010) Quantitative analysis of aquaporin-2 phos-
phorylation. Am J Physiol Renal Physiol 298(4):F10181023
125. Yamamoto T, Sasaki S, Fushimi K, Ishibashi K, Yaoita E, Kawasaki
K, Marumo F, Kihara I (1995) Vasopressin increases AQP-CD
water channel in apical membrane of collecting duct cells in Brattle-
boro rats. Am J Physiol 268(6 Pt 1):C15461551
126. Yasui M, Zelenin SM, Celsi G, Aperia A (1997) Adenylate
cyclase-coupled vasopressin receptor activates AQP2 promoter
via a dual effect on CRE and AP1 elements. Am J Physiol 272(4
Pt 2):F443450
127. Ying Chen WR, Wei Li, Yawei Kong, Robert A. Fenton, Jian Li,
Victor Hsu, Dennis Brown, Hua Ann Jenny Lu (2010) AQP2
Affects Renal Epithelial Cell Adhesion, Migration and Tubule
Formation by Interacting with Integrin 1 Via an External RGD
Motif. In: ASN 2010, Denver, Colorado, US, 2010.
128. Yip KP (2006) Epac mediated Ca2+ mobilization and exocytosis
in inner medullary collecting duct. Am J Physiol Renal Physiol
291(4):F882890
129. Yui NL, Hua J, Bouley R, Brown D (2011) AQP2 is necessary for
vasopressin- and forskolin-mediated filamentous actin depoly-
merization in renal epithelial cells. Biology Open. doi:10.1242/
bio.2011042
130. Zelenina M, Christensen BM, Palmer J, Nairn AC, Nielsen S,
Aperia A (2000) Prostaglandin E(2) interaction with AVP: effects
on AQP2 phosphorylation and distribution. Am J Physiol Renal
Physiol 278(3):F388394
131. Zelenina M, Zelenin S, Bondar AA, Brismar H, Aperia A (2002)
Water permeability of aquaporin-4 is decreased by protein kinase
C and dopamine. Am J Physiol Renal Physiol 283(2):F309318
132. Zwang NA, Hoffert JD, Pisitkun T, Moeller HB, Fenton RA,
Knepper MA (2009) Identification of phosphorylation-
dependent binding partners of aquaporin-2 using protein mass
spectrometry. J Proteome Res 8(3):15401554
144 Pflugers Arch - Eur J Physiol (2012) 464:133144