biologia celular de la vasopresina.pdf

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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 ordilute 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: [email protected]

H. B. Moeller

e-mail: [email protected]

Pflugers Arch - Eur J Physiol (2012) 464:133144

DOI 10.1007/s00424-012-1129-4


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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 Ca2+ levels [15, 102]. Although the

mechanism for this is not completely clear, calmodulin-

dependent release of Ca2+ 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 Ca2+ for membrane vesicle

fusion cannot be discounted, activation of the exchange

protein directly activated by cAMP (Epac) also triggers

intracellular Ca2+ 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 proteinabundance 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 fourAQP2 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


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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

bee n tak en as a rol e for act in in AQP2 trans loc ati on

Fig. 1 Regulated trafficking events of AQP2.aAdenylate 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 ofPKA. This promotes apical plasma membrane accumu-

lation of tetramericAQP2by increasing exocytosis of subapical AQP2-

bearing vesicles and decreasing AQP2 endocytosis from the plasma

membrane. Upon removal ofAVP, AQP2 is internalized and canbe stored

in subapical vesicles. Upon restimulation, AQP2 can recycle to the

membrane. b Increased intracellularCa2+ aids AQP2 trafficking. AVP

stimulation results in increased intracellular Ca2+ levels via Ca2+ release

from calmodulin-dependent ryanodine-sensitive intracellular stores. Ad-

ditionally, activation of the exchange protein directly activated by cAMP

(Epac) can also trigger Ca2+ mobilization and apical membrane expres-

sion of AQP2. The role of increased Ca2+ in AQP2 trafficking remainsunclear. 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 from F-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 specificv-SNAREs that bind to specific

t-SNAREson 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.fAfter AVP washout,

AQP2 localizes to clathrin-coated pits and undergoes clathrin-mediated

endocytosis. A direct AQP2 interaction with Hsp70/Hsc70 suggestsa 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 inendocytosis-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


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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-actinthrough 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 releaseof 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



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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 ofclathrin-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 surfaceexpression [109].

After internalization, AQP2 is localized to EEA1-

positive early endosom es before it is tra nsferr ed 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 cellmodels, 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



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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 trea tment [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 extracellularloop; 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 COOHterminus are located in the cytoplasm. In the

hourglass model, twoNPAmotifs in the intracellular and extracellular

loops are thought to dock in the membrane and form the water pore



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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 thisquestion 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 recapitulatethe 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 mechanismfor 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-



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deficientAQP2 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 forthe 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.

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