INVITED REVIEWThe ins and outs of aquaporin-2 trafficking - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:36 |只看该作者 |倒序浏览 |打印

作者：DennisBrown作者单位：Program in Membrane Biology and Renal Unit, Department ofMedicine, Massachusetts General Hospital, Charlestown 0212 andHarvard Medical School, Boston, Massachusetts 02114



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      【摘要】
   This review outlines recent advancesrelated to the molecular mechanisms and pathways of aquaporin-2 (AQP2)water channel trafficking. AQP2 is a fascinating protein, whose sortingsignals can be interpreted by different cell types to achieve apical or basolateral membrane insertion, in both regulated and constitutive trafficking pathways. In addition to the well-known cAMP-mediated, stimulatory effect of vasopressin on AQP2 membrane insertion, othersignaling and trafficking events can also lead to AQP2 membrane accumulation via cAMP-independent mechanisms. These include 1 ) elevation of cGMP, mediated by sodium nitroprusside (anitric oxide donor), atrial natriuretic factor, and L -arginine (via nitric oxide synthase); 2 )disruption of the actin cytoskeleton; and 3 ) inhibition ofthe clathrin-mediated endocytotic arm of the AQP2 recycling pathway bydominant-negative dynamin expression and by membrane cholesteroldepletion. Recent data also indicate that AQP2 recycles constitutivelyin epithelial cells, it can be inserted into different membrane domainsin different cell types both in vitro and in vivo, and these pathwayscan be modulated by factors including hypertonicity. The roles ofaccessory proteins, including small GTPases and soluble N -ethylmaleimide-sensitive factor attachment proteinreceptor proteins in AQP2 membrane insertion, are also being uncovered.Understanding cAMP-independent mechanisms for membrane insertion ofAQP2 is especially relevant to the therapeutic bypassing of themutated, dysfunctional vasopressin receptor in patients with X-linkednephrogenic diabetes insipidus.
      【关键词】 REVIEWThe aquaporin trafficking2 _7 r) [( Z% }8 S
   INTRODUCTION$ A. J: H, T0 b7 r2 T
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VASOPRESSIN STIMULATION RESULTS in theaccumulation of aquaporin-2 (AQP2) on the plasma membrane of principalcells in the kidney via a membrane trafficking mechanism that involvesthe recycling of AQP2 between intracellular vesicles and the cell surface ( 14, 44, 58 ). The basic paradigm underlying these cell biological events, which are a crucial part of the urinary concentrating mechanism, was worked out many years ago using amphibian bladder and epidermis as experimental models ( 4, 73 ). The so-called "shuttle hypothesis" of vasopressin action was confirmed by direct observations of water channel (aquaporin) recycling whenantibodies against the vasopressin-regulated AQP2 became available( 18, 42, 43, 56 ). However, despite several years ofinvestigation in the postaquaporin era, many basic features ofvasopressin-regulated AQP2 membrane insertion and retrieval remainunclear. This brief review will summarize selected recent advances thathave been made in this active field of research. In some cases, newdata have resolved questions and confirmed hypotheses that were firstraised over 20 years ago. Other results, however, have forced areassessment of older paradigms and indicate that the cell-surfaceexpression of AQP2 can be induced by mechanisms other than thetraditional vasopressin receptor (V2R) and cAMP signal transduction cascade.$ D7 W, l( `2 V  ~$ G7 B2 v" o' z( E, V
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C AMP-INDEPENDENT CELL-SURFACE EXPRESSIONOF AQP2/ R# |/ f  ]$ g# R

The cGMP Pathway( b; f# y2 _" ^* S/ ~/ O3 C9 x

Activation of PKA by cAMP leads to phosphorylation of AQP2 on aserine residue at position 256 on the cytoplasmic COOH terminus. Withthe use of a serine 256-to-alanine (S256A) point mutation of AQP2,expressed in LLC-PK 1 cells, it was clearly shown that phosphorylation of the serine 256 residue by PKA is required for vasopressin-induced translocation of AQP2 from vesicles to the plasmamembrane ( 17, 28 ). Vasopressin also stimulates serine 256 phosphorylation of native AQP2 in collecting duct principal cells insitu ( 46, 77 ). However, studies of alternative (or parallel) trafficking pathways that do not seem to involve cAMP or PKAare being uncovered. Hormones and drugs that increase cGMP levels(sodium nitroprusside, L -arginine, atrial natriureticpeptide) as well as permeable cGMP analogs also induce AQP2 trafficking in cultured cells and in collecting ducts in vitro (Fig. 1, A and B )( 1 ). PKG can phosphorylate the COOH terminus of AQP2 invitro, and cGMP-stimulated membrane accumulation of AQP2 does not occurin cells expressing the S256A mutation of AQP2. Figure 2 shows the potential interactions of thecAMP-dependent and cGMP-dependent signaling pathways on AQP2 membraneinsertion. It is not yet clear whether PKG directlyphosphorylates AQP2 in vivo or whether the cGMP/PKG effect isultimately mediated by activation of PKA. The physiological relevanceof this alternative signaling pathway is undetermined, butinterestingly the effect is most pronounced in the collecting ducts ofthe outer medulla. The cGMP effect is barely detectable in the corticaland inner medullary collecting ducts. It is possible that thiscGMP-mediated AQP2 insertion may at least partially explain theapparently vasopressin-independent urinary concentration that occurs insome animal models and in humans under certain conditions. These issueswere discussed in more detail in a previous report ( 1 ).; b" j. z" k6 P% h8 {# A2 q* A  W

Fig. 1. Pathways of aquaporin-2 (AQP2) insertion in collecting ductprincipal cells in kidney tissue slices incubated in vitro. All imagesshow confocal microscopy of AQP2-labeled tissues. A : AQP2staining in a collecting duct from the inner stripe (IS) of the outermedulla. AQP2 is distributed throughout the cytoplasm under baselineconditions. After addition of sodium nitroprusside (SNP) to the bathfor 10 min ( B ), AQP2 accumulates at the apical membrane ofprincipal cells. Thus SNP has a vasopressin-like effect in inner stripecollecting ducts. C and D : collecting ducts fromthe distal inner medulla (IM; papilla). In C, baseline,cytoplasmic distribution of AQP2 is shown. In D, therelocation of AQP2 mainly to the basolateral membrane of principalcells after acute (15-min) treatment with vasopressin (VP) andforskolin (For) is shown. Thus, in the distal inner medulla, AQP2 canbe inserted into the basolateral plasma membrane of principal cells.Modified from Refs. 1 and 72. Bar = 20 µm.4 T+ O6 L( ~3 m3 [3 m
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Fig. 2. Bottom : vasopressin (AVP) activation ofadenylyl cyclase (A.C.), which increases cAMP levels and inducesPKA-mediated phosphorylation of AQP2 that is necessary for itsregulated membrane insertion via this signaling pathway. Newer datashow that an alternative, cGMP-mediated signaling pathway is present inepithelial cells in some regions of the collecting duct, notably theouter medulla. Membrane insertion of AQP2 also occurs when cGMP levelsare elevated by SNP [a nitric oxide (NO) donor], by L -arginine and nitric oxide synthase (NOS) activity, or byatrial natriuretic factor (ANF). While PKG can phosphorylate AQP2 invitro, it is unclear whether PKG directly phosphorylates AQP2 in intactcells, or whether it activates PKA, which in turn phosphorylates AQP2.G.C., guanylyl cyclase. Adapted from Ref. 1 withpermission.* X, l; C. B- c0 E, g" P& ^# o; Q

The Actin Cytoskeleton and Membrane Accumulation of AQP21 I* t8 R$ Z7 P# t8 M+ J1 O

A role of the actin cytoskeleton in vasopressin-stimulated waterchannel insertion was also proposed based on work in toad bladder( 15, 50 ). Subsequently, studies of collecting ducts confirmed these observations ( 23 ). However, an unexpectedrecent finding was that AQP2 translocation can be achieved simply by modulating actin polymerization, in the complete absence of hormonal stimulation. The potential role of actin in AQP2 membrane insertion wasdirectly examined in transfected inner medullary collecting duct(IMCD) cells in culture. Exposure of these cells to Clostridium toxin B caused actin depolymerization andaccumulation of AQP2 in the plasma membrane ( 67 ). Thistoxin inhibits RhoGTPases that are involved in regulating thepolymerization state of the actin cytoskeleton ( 52 ). AQP2translocation was also seen in cells treated with Y-27632, thedownstream Rho kinase inhibitor ( 32 ). This occurred in theabsence of any detectable elevation of intracellular cAMP. Conversely,expression of constitutively active RhoA in these cells induced stressfiber formation, indicating actin polymerization, and inhibited thenormal AQP2 translocation response to forskolin. These data providestrong evidence for a major regulatory role of the actin cytoskeletonin the vasopressin-induced recycling of AQP2 between intracellularvesicles and the cell surface. Whether the endocytotic or exocytoticpathways are affected has not yet been determined directly, however(see below). Interestingly, principal cells of both rat and mousekidney contain high levels of the actin-severing protein adseverin,which may be involved in regulating the actin cytoskeleton in thesecells in vivo ( 34 ).* N% W0 O3 d4 U3 p; C! K
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APICAL VS. BASOLATERAL AQP2 EXPRESSION IN EPITHELIAL CELLS6 O) P# y- W+ M- `; V( ?

Since the original studies on AQP2 localization in kidneycollecting ducts, it has been clear that AQP2 can be expressed not onlyon the apical plasma membrane but also at the basolateral surface ofcollecting duct principal cells ( 2, 10, 42 ). Furthermore,in our original study describing trafficking of AQP2 transfected intoLLC-PK 1 cells ( 29 ), AQP2 was inserted into thebasolateral plasma membrane after vasopressin stimulation. In contrast,subsequent studies reported the apical delivery of AQP2 in Madin-Darbycanine kidney (MDCK) cells and rabbit collecting duct cells aftervasopressin stimulation ( 12, 68 ). Remarkably, however, apredominantly basolateral AQP2 expression in response to acutevasopressin stimulation occurred in primary cultures of IMCD cells( 9, 37 ). Thus, while regulated trafficking of AQP2 occursin a variety of transfected cell lines, the polarity signals on AQP2(as well as those on some other proteins) seem to be interpreted indifferent ways by a variety of cell types ( 21, 51 ). AQP2in the vas deferens of the male reproductive tract is insertedconstitutively into the apical plasma membrane of epithelial cells inthis tissue, further underlining the cell specificity of the targetingprocess ( 61 ).0 u, P5 T/ V3 f" a9 K
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What could contribute to this variable polarity of AQP2 expression?Recent data indicate that interstitial osmolality may be at leastpartially responsible for the basolateral targeting of AQP2 in the IMCD( 72 ). In MDCK cells adapted to hypertonic culture medium,basolateral rather than apical targeting of AQP2 is induced byforskolin. Furthermore, kidney slices taken from vasopressin-deficientBrattleboro rats show apical insertion of AQP2 in the inner medullawhen exposed to vasopressin. In contrast, slices from normal rats (witha hypertonic interstitium in vitro) show a marked basolateral insertionof AQP2 when challenged acutely with vasopressin in vitro (Fig. 1, C and D ) ( 72 ). Finally, inner medullary principal cells in kidney slices from Brattleboro rats pretreated with vasopressin for 11 days in vivo also show basolateral AQP2 insertion when exposed to acute vasopressin stimulation in vitro.While hypertonicity cannot be the only factor involved in this changein polarity of AQP2 insertion (because cortical connecting segmentsalso show basolateral AQP2 insertion, and LLC-PK 1 cellswith basolateral AQP2 expression are grown in isotonic culture medium),this observation provides a starting point for the examination of geneexpression patterns that are involved in determining the polarity ofinsertion of AQP2 in epithelial cells.' t: u0 r. {% M* n
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In the case of LLC-PK 1 cells, it was shown that thepolarity of expression of a transfected LDL receptor is apical, rather than basolateral, as in MDCK cells ( 48 ). This change inpolarity was ascribed to the lack of a key sorting adaptor, µ1b,which interacts with some tyrosine-based sorting motifs and is notexpressed in LLC-PK 1 cells. Restoration of the missingµ1b adaptor into LLC-PK 1 cells by cotransfection restoredthe basolateral polarity of the LDL receptor in these cells. However,the absence of this adaptor does not lead to a generic disruption ofapical/basolateral polarity in these cells, and we have shown that AQP2is still inserted basolaterally in LLC-PK 1 cells thatexpress the µ1b protein (Sun T, Bouley R, and Brown D, unpublished observations).5 k" ], T4 }5 T# R3 N) M: v* S* S
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MEMBRANE ACCUMULATION OF AQP2 BY INHIBITION OF CLATHRIN-MEDIATEDENDOCYTOSIS

While the precise pathway leading to AQP exocytosis has not beenidentified, recent data have confirmed a role for clathrin-coated pitsin the endocytotic pathway ( 64, 65 ). A role for clathrin in water channel internalization was first proposed almost two decadesago, based on indirect evidence ( 5, 6, 63 ). New data usinglabel-fracture on AQP2-transfected LLC-PK 1 cells and antibodies against an externally oriented epitope of AQP2 in collecting duct principal cells have shown that AQP2 accumulates inclathrin-coated plasma membrane domains during vasopressinstimulation and washout (Fig. 3 ).Furthermore, when clathrin-mediated endocytosis was inhibited by theexpression of a dominant-negative form of the protein dynamin inLLC-PK 1 cells, AQP2 accumulated on the plasma membrane andwas depleted from cytoplasmic vesicles (Fig. 4 ) ( 65 ). Dynamin is a GTPasethat is involved in the formation and pinching off of clathrin-coatedpits to form clathrin-coated vesicles ( 24, 41, 59 ). Thedominant-negative form has a single point mutation, K44A, that rendersthe protein GTPase deficient and arrests clathrin-mediated endocytosis( 8, 49, 59 ). However, dynamin mutants also inhibitendocytosis via caveolae ( 47 ), and the electronmicroscopic detection of AQP2 in clathrin-coated pits described abovewas, therefore, essential to prove the involvement of clathrin in AQP2 endocytosis.

Fig. 3. Immunogold electron microscopy showing AQP2 antigenicsites (gold particles) concentrated in a clathrin-coated pit (arrow) atthe apical plasma membrane of a collecting duct principal cell. Theantibody was raised against an external epitope of the AQP2 protein;hence, most of the gold labeling is on the extracellular surface of theplasma membrane. The image shows that AQP2 accumulates inclathrin-coated pits in preparation for subsequent internalization.Modified from Ref. 64.

Fig. 4. Blocking clathrin-mediated endocytosis in culturedLLC-PK 1 cells results in accumulation of AQP2 on the plasmamembrane. A : baseline, perinuclear distribution ofAQP2-containing vesicles in transfected LLC-PK 1 cells.Bar = 20 µm. B : after adenoviral-mediated expressionof a dominant-negative mutation of dynamin (K44A dynamin) in thesecells, which prevents clathrin-mediated endocytosis, AQP2 accumulatesat the cell surface even in the absence of hormonal stimulation. C : for comparison, the effect of a 10- to 15-min treatmentwith vasopressin on AQP2 distribution is shown. After hormonetreatment, AQP2 rapidly accumulates at the surface of these cells, aspreviously described ( 29 ).

Why does inhibition of clathrin-mediated endocytosis result in theplasma membrane accumulation of AQP2? Studies in transfected LLC-PK 1 cells have shown that AQP2 can recycleconstitutively between the plasma membrane and intracellular vesicles,even in the absence of increased intracellular cAMP ( 22 ).However, accumulation of AQP2 at the cell surface does notusually occur under baseline conditions presumably because the relativerates of exo- and endocytosis are in equilibrium. The amount of AQP2 onthe plasma membrane could, therefore, be increased either by increasingthe rate of exocytosis, by inhibiting endocytosis, or both. It has beenshown, for example, that the insulin-regulated glucose transporter,GLUT4, also recycles constitutively but the amount at the cell surface is increased by insulin treatment of target cells ( 7, 26, 49 ).

How rapidly is AQP2 accumulated at the cell surface after inhibition ofendocytosis? While expression of dominant-negative dynamin in cellsresults in increased cell-surface AQP2 expression, several hours arerequired before the effect takes place in these transfectionexperiments. More recently, an extensive accumulation of AQP2 at thecell surface has been accomplished within 30 min after inhibition ofendocytosis by treatment with methyl- -cyclodextrin ( 64 ). This drug depletes membranes of cholesterol andresults in a rapid inhibition of endocytosis ( 53 ).Therefore, at least in LLC-PK 1 cells, AQP2 can accumulateat the cell surface with a time course similar to that seen aftervasopressin stimulation, simply by blocking endocytosis. This impliesthat constitutive AQP2 recycling between intracellular vesicles and thecell surface is rapid in this system. It must now be determined whethera similar constitutive process occurs in the collecting duct in situ.When interpreting the results of manipulations that result in a net increase in AQP2 cell-surface expression, one must therefore bear inmind the contribution of an inhibition of endocytosis to the overall effect.$ R( K2 b. l' t! ~& ?
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PATHWAYS OF INTRACELLULAR AQP2 RECYCLING

The development of transfected cell models has greatly facilitatedthe dissection of intracellular AQP2 trafficking pathways. AQP2 isinternalized by a clathrin-mediated mechanism ( 65 ), andinternalized fluid phase markers can be detected in subapical vesicles,as well as in tubular structures in proximity to the Golgi region inboth cultured cells and collecting duct principal cells in situ( 6 ). Several rounds of exo- and endocytosis of AQP2 couldbe followed in LLC-PK 1 cells despite the completeinhibition of de novo AQP2 synthesis ( 27 ), but details ofthe pathway(s) followed by this recycling AQP2 were vague. The natureof this pathway was addressed in cultured cells using manipulationsthat blocked transport at intermediate steps within the cell. Lowering the temperature to 20°C or incubating cells with theH -ATPase inhibitor bafilomycin caused an accumulation ofAQP2 in a perinuclear compartment that partially overlapped withclathrin immunostaining but did not stain for Golgi markers such asgiantin or -coat protein. The 20°C block prevents exit ofproteins from the trans -Golgi ( 40 ), andclathrin-coated vesicles are enriched in this cellular compartment( 20 ). In addition, recycling AQP2 is at least partiallycolocalized with internalized transferrin in recycling endosomes inLLC-PK 1 cells ( 76 ). Motifs in the sixthtransmembrane domain of AQP2, including a dileucine motif, are involvedin regulated trafficking of this water channel ( 75 ). Domain-swap experiments, however, show that while the cytoplasmic COOHterminus of AQP2 is necessary for regulated insertion of AQP2, it isnot sufficient, implying that other domains of the protein play a rolein this process ( 13 ).

AQP2 EXOCYTOSIS AND VESICLE FUSION$ V$ G# V5 C! X5 z/ y

While intracellular transport processes (via microtubules, actin,or simple random motion of vesicles) can deliver AQP2-containing vesicles close to the appropriate membrane domain, regulated vesicle fusion with target membranes involves the concerted interaction of manyother accessory proteins. In common with membrane fusion events in manycell types, the final step in the exocytotic fusion of AQP2-containingvesicles with the plasma membrane involves soluble N -ethylmaleimide-sensitive factor attachment proteinreceptor (SNARE) protein complexes ( 19 ) as well asintracellular calcium mobilization ( 9 ). The role ofso-called SNARE proteins in vesicle fusion events was first appreciatedin synaptic vesicle fusion events. Since the original "SNAREhypothesis" was proposed ( 60 ), the precise details ofhow proteins on target membranes and donor membranes interact withsoluble cytosolic proteins to regulate vesicle fusion have beensubjected to intense investigation ( 54, 55, 74 ). Incollecting duct principal cells, vesicle-associated membranepolypeptide (VAMP)-2 is present on AQP2-containing vesicles ( 45 ), and the target (t)-SNAREs syntaxin 3, synataxin 4 ( 35 ), and soluble N -ethylmaleimide-sensitivefactor attachment protein (SNAP)-23 ( 25 ) are alsoexpressed. Treatment of collecting duct cells in culture withtetanus toxin, which cleaves the SNARE protein VAMP-2/synaptobrevin,abolishes vasopressin-induced AQP2 translocation to the plasma membrane( 19 ). However, the precise SNARE proteins that areinvolved in this process in vivo remain unclear. The t-SNAREs syntaxin3 and syntaxin 4 were initially reported to have basolateral and apicallocalizations, respectively, in adult rat kidney ( 3, 35, 36 ). However, a recent study using different antibodies hasreported exactly the opposite polarity of these two SNARE proteins inprincipal cells ( 33 ). Both sets of antibodies were raisedagainst seemingly unique peptide sequences, and thus the reason forthis major discrepancy is unclear. A further problem to be resolved isthat heavy syntaxin 3 staining of the proximal tubule brush border wasdescribed in the most recent study ( 33 ), yet previous workdid not detect syntaxin 3 mRNA by RT-PCR in microdissected proximaltubules ( 36 ). This process becomes even more complex whenone considers that AQP2 can be inserted apically and/or basolaterallyin different cell types and under different physiological conditions(see APICAL VS. BASOLATERAL AQP2 EXPRESSION IN EPITHELIAL CELLS ). Does this also require a reorientationof the vesicle fusion machinery, or can the existing polarized fusion machinery adapt to catalyze fusion of AQP2-containing vesicles witheither the apical or basolateral plasma membrane?1 p+ _5 ^4 i# M# e0 w8 O% c
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In addition to SNARE proteins, other accessory proteins known to beinvolved in the AQP2 shuttling and/or fusion process are heterotrimericGTP binding proteins of the G i family ( 70 ).Treatment of cultured rabbit cortical collecting duct cells withpertussis toxin, or exposure of permeabilized cells to syntheticpeptides derived from the COOH terminus of theG i-3 -subunit, abolished cAMP-induced AQP2 trafficking tothe cell surface. However, it has also been proposed that proteins ofthe G i family inhibit AQP2 accumulation at the cell surfacevia calcium receptor signaling upon an increase in luminal calciumlevels ( 57 ). Furthermore, earlier work demonstrated thatG i-3 exerts a negative regulatory effect on thesecretion of heparan sulfate proteoglycans from cultured cells and thatsecretion is actually increased after pertussis toxin treatment, whichinhibits G i protein activity ( 62 ). Thus theprecise step at which this G protein exerts its effect on AQP2trafficking remains to be determined.3 |) N3 z- S! ]9 H3 A1 x8 Y6 n
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ROLE OF PHOSPHORYLATION IN AQP2 TRAFFICKING

Several putative phosphorylation sites for kinases are present inthe AQP2 sequence. These include PKA and PKG, PKC, and casein kinase IIsites. Most work has focused on the role of PKA-induced phosphorylationin the vasopressin-induced signaling cascade, as described above. SomePKA anchoring proteins (AKAPs) are enriched in AQP2-immunopurifiedvesicles from IMCD cells. Inhibition of forskolin-induced AQP2translocation with a peptide that prevents PKA-AKAP interactiondemonstrated that, besides its enzymatic activity, tethering of PKA tosubcellular compartments is essential for AQP2 translocation ( 30, 31 ). While available data consistently show that serine 256 phosphorylation is required for the vasopressin-induced cell-surfaceaccumulation of AQP2 ( 17, 28 ), dephosphorylation of AQP2is probably not necessary for its subsequent internalization. Prostaglandin E 2 stimulates removal of AQP2 from thesurface of principal cells when added after AVP treatment but does notalter the phosphorylated state of AQP2 ( 77 ). In support ofthis, it was shown in cell cultures that PKC-mediated endocytosis ofAQP2 is also independent of the phosphorylation state of this water channel at residue serine 256 ( 71 ).  v" A( V! t* n* y) }- d. o
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A major unresolved issue is how phosphorylation of AQP2 at serine 256 increases the cell-surface expression of this water channel.Phosphorylation could modify the interaction of AQP2 cargo vesicleswith the cytoskeleton, via microtubule and/or microtubule motors oraccessory cross-linking proteins. Phosphorylation-dependent protein-protein interactions may be necessary to augment AQP2 trafficking to the cell surface, although, as previously discussed, constitutive membrane insertion occurs rapidly without vasopressin stimulation or an elevation of intracellular cAMP. Alternatively, phosphorylation could inhibit the endocytotic step of AQP2 recycling, leading to accumulation at the cell surface. However, some data (discussed above) show that serine 256-phosphorylated AQP2 can still beinternalized. Preventing dephosphorylation of AQP2 with the phosphataseinhibitor okadeic acid has the expected effect of increasingcell-surface accumulation of AQP2 in cultured cells, but, surprisingly,the same effect of okadeic acid was observed in the presence of the PKAinhibitor H-89. The authors concluded that okadeic acid stimulates themembrane translocation of AQP2 in a phosphorylation-independent manner( 69 ). As expected, an AQP2 mutant containing a serine256-to-aspartic acid amino acid switch was constitutively expressed atthe cell surface, presumably because the negatively charged asparticacid residue mimics the phosphorylated serine 256 residue of thewild-type protein ( 71 ). As yet, no effect ofphosphorylation on the interaction of accessory proteins with AQP2 hasbeen reported. This is an area toward which future efforts willcertainly be directed.; k* M) h; \1 e  P! R
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NEPHROGENIC DIABETES INSIPIDUS AND AQP2 TRAFFICKING3 W8 L9 u2 X$ V6 M
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Most cases of hereditary nephrogenic diabetes insipidus resultfrom mutations in V2R, although autosomal forms in which AQP2 isdefective have provided a great deal of important information about thepathophysiology of the disease, and about AQP2 trafficking andoligomerization ( 11, 14, 38 ). In the absence of afunctional V2R at the cell surface in nephrogenic diabetes insipiduspatients, it is critical to develop strategies to bypass theV2R-dependent signaling cascade that normally results in anaccumulation of AQP2 on principal cell plasma membranes. Many of theobservations related to the cell biology of AQP2 trafficking outlinedabove could be relevant in this regard. For example, it is now known that AQP2 is trafficked in response to an elevation of cGMP, in addition to cAMP, in some regions of the collecting duct (Figs. 1 and 2 ). Modulation of the actin cytoskeleton alone can cause cell-surfaceaccumulation of AQP2. Finally, AQP2 can move to the cell surface in aconstitutive pathway, so that inhibition of endocytosis in thisconstitutive pathway can be envisaged as a means of accumulating AQP2on the plasma membrane (Fig. 4 ).) ?& ^2 x8 t# P4 g# |( w

In addition, many of the V2R and AQP2 mutations described innephrogenic diabetes insipidus patients result in a misfolded proteinthat is blocked at some point [often the rough endoplasmic reticulum(RER)] inside the cell. Some of these proteins would befunctional if they could reach the cell surface. It has been shown thatsome chemical chaperones (e.g., glycerol, DMSO) can help misfolded AQP2to reach its final cell-surface destination ( 39, 66 ), butthese agents may be difficult or impossible to apply in vivo. In aninteresting recent development, it has been shown that relatively brieftreatment of cultured cells and transgenic mice with the Ca-ATPaseinhibitor thapsigargin can increase the cell-surface expression of F508 CFTR ( 16 ). This effect is probably a result of thedepletion of calcium stores from the RER and the subsequentinactivation of calcium-dependent chaperones that would otherwise bindto the misfolded F508 CFTR protein and direct it into degradativepathways. While this approach is not, of course, without potentialcaveats related to the long-term depletion of calcium stores, theauthors propose that it could be applicable to a variety of geneticdiseases resulting from the defective trafficking and RER retention ofmisfolded proteins.
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SUMMARY

As outlined above, considerable progress has been made inunderstanding several aspects of AQP2 trafficking over the past fewyears. In large part, this has resulted from the development of modelin vitro systems in which regulated apical and basolateral AQP2trafficking has been reconstituted. The trafficking of AQP2 betweenintracellular vesicles and the cell surface involves a complex seriesof membrane fission and fusion steps at various points along therecycling pathway and involves passage through a trans -Golgiand/or recycling endosome-like compartment. AQP2 vesicles also interactwith the actin cytoskeleton and the microtubule network of the cellduring the trafficking process, and calcium mobilization in addition tocyclic nucleotide elevation in the cytosol contributes to theexocytotic insertion of AQP2. These events are regulated byhormone-induced protein phosphorylation in ways that remainundetermined and involve the interaction of GTP binding proteins.Additional features of AQP2 trafficking that have emerged recently arethat AQP2 can recycle in a constitutive pathway, in addition to avasopressin-regulated pathway, and that the polarity of membraneinsertion of AQP2 can be different (apical vs. basolateral) in avariety of cell types in both cultured cells and different regions ofthe collecting duct. While cell culture systems will continue to play amajor role in dissecting the cell biology of vasopressin action, it isalso important to continue to relate these in vitro findings to wholeorgan and whole animal physiology. Thus alternative experimentalsystems from isolated collecting ducts, tissue slices, perfusedkidneys, and transgenic mouse models are expected to play an increasingrole in aquaporin research over the next few years. Genomic andproteomic approaches promise to reveal a wealth of new informationabout the molecular mechanisms of AQP2 trafficking in the collectingduct and about the many as yet undiscovered binding partners andaccessory proteins that regulate the vesicular transport and membraneinsertion of this protein. From both the physiological and cellbiological points of view, AQP2 is a fascinating protein that willcontinue to occupy the efforts of many laboratories for years to come.
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ACKNOWLEDGEMENTS
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I thank all of my colleagues who have made contributions to thesestudies, as well as those who work in the aquaporin field, for makingthis such a stimulating and rewarding area of research.
      【参考文献】. k: z# E( M+ W/ V8 f
1. Bouley, R,Breton S,Sun T,McLaughlin M,Nsumu NN,Lin HY,Ausiello DA,andBrown D. Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane insertion of aquaporin 2 in renal epithelial cells. J Clin Invest 106:1115-1126,2000  .

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2. Breton, S,andBrown D. Cold-induced microtubule disruption and relocalization of membrane proteins in kidney epithelial cells. J Am Soc Nephrol 9:155-166,1998 .& u- f! @+ a" T8 U( w4 X) W
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3. Breton, S,Inoue T,Knepper MA,andBrown D. Antigen retrieval reveals widespread basolateral expression of syntaxin 3 in renal epithelia. Am J Physiol Renal Physiol 282:F523-F529,2002 .$ K: W- Y$ P9 k1 Y3 z7 }" O

4. Brown, D,Grosso A,andDeSousa RC. Membrane architecture and water transport in epithelial cell membranes.In: Advances in Membrane Fluidity. Membrane Transport and Information Storage, edited by Aloia RC.. New York: Liss, 1990, vol. 4, p. 103-132.3 D% f8 P0 k% Z  ?5 F. `
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5. Brown, D,andOrci L. Vasopressin stimulates formation of coated pits in rat kidney collecting ducts. Nature 302:253-255,1983 .6 ?3 j4 g: {( G2 j+ K8 m
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