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The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and nonphosphorylated monomers.
Kamsteeg EJ
,
Heijnen I
,
van Os CH
,
Deen PM
.
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In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non-p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes.
Figure 1. Phosphorylation of AQP2 in oocytes. Oocytes, injected with wt-AQP2 or AQP2-S256A cRNA, were labeled with [32P]orthophosphate for 2 d. Subsequently, oocytes were not stimulated (ctrl, AQP2-S256A), or were incubated with 5 Ã 10â4 M 8-Br-cAMP or 10â4 M H89 for the indicated time periods. Total membranes were isolated and divided into two portions. One portion was solubilized, AQP2 was immunoprecipitated and subjected to PAGE, the gel was dried, and labeled AQP2 (p-AQP2) was visualized using autoradiography. The other portion was directly immunoblotted for AQP2 (Total AQP2).
Figure 3. Immunocytochemistry of oocytes expressing wild-type or mutant AQP2 proteins. 2 d after injection, oocytes injected with cRNA encoding wt-AQP2 (wt), AQP2-S256A (SA), or AQP2-S256D-F (SD), and noninjected oocytes (ctrl) were fixed and embedded in paraffin. In 5-μm sections, AQP2 was visualized using rabbit AQP2 antibodies followed by Alexa-594âconjugated antiârabbit IgG. Arrows indicate the plasma membrane.
Figure 5. Semiquantification of the amounts and ratios of AQP2-S256A and AQP2-S256D-F in coinjections. Copy RNAs encoding AQP2-S256D-F and AQP2-S256A were mixed in the ratios 1:3, 1:2, 1:1, 2:1, and 3:1 (from left to right) and a total of 0. 4 ng was injected into oocytes. 2 d later, the Pf values were determined (see Table ) and total membranes were isolated and immunoblotted for AQP2. The individual signals of AQP2-S256A (SA) and AQP2-S256D-F (SD) were scanned and the amounts of expressed protein were semiquantified (in arbitrary units) using the scanned signals of a twofold dilution series of wt-AQP2 as a standard. From these data, the SD/SA ratio and total AQP2 amounts were calculated.
Figure 6. The relation between the expressed amounts and conferred water permeabilities of AQP2-S256D-F. Of oocytes, injected with 0.1, 0.2, 0.3, or 0.4 ng of AQP2-S256D-F cRNA, the water permeability (Pf ± SEM in μm/s) was measured. Total membranes were isolated and immunoblotted for AQP2 (AQP2-S256D-F; insert). The signals were scanned and the amounts of expressed AQP2-S256D-F (AQP2 amount in arbitrary units) were semiquantified as described in the legend to Fig. 5. The linear relation between the amount of AQP2-S256D-F protein and the conferred water permeability was: Pf = (3.97 à amount of AQP2 protein expected in membrane) â 9. 7. For expression levels lower than obtained for 0.1 ng AQP2-S256D-F cRNA, the relation was extrapolated to the Pf level of control oocytes (8 ± 4 μm/s, dotted line).
Bao,
Threonine phosphorylation diverts internalized epidermal growth factor receptors from a degradative pathway to the recycling endosome.
2000, Pubmed
Bao,
Threonine phosphorylation diverts internalized epidermal growth factor receptors from a degradative pathway to the recycling endosome.
2000,
Pubmed
Brown,
Protein trafficking and polarity in kidney epithelium: from cell biology to physiology.
1996,
Pubmed
Brown,
Membrane recycling and epithelial cell function.
1989,
Pubmed
Brown,
Vasopressin stimulates endocytosis in kidney collecting duct principal cells.
1988,
Pubmed
Cheng,
Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.
1998,
Pubmed
Christensen,
Localization and regulation of PKA-phosphorylated AQP2 in response to V(2)-receptor agonist/antagonist treatment.
2000,
Pubmed
Deen,
Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing.
1995,
Pubmed
,
Xenbase
Deen,
Apical and basolateral expression of aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities.
1997,
Pubmed
Deen,
Aquaporin-2 transfection of Madin-Darby canine kidney cells reconstitutes vasopressin-regulated transcellular osmotic water transport.
1997,
Pubmed
Deen,
Physiology and pathophysiology of the aquaporin-2 water channel.
1998,
Pubmed
Deen,
Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine.
1994,
Pubmed
,
Xenbase
Ecelbarger,
Aquaporin-3 water channel localization and regulation in rat kidney.
1995,
Pubmed
Eskandari,
Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy.
1998,
Pubmed
,
Xenbase
Fushimi,
Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel.
1997,
Pubmed
Fushimi,
Cloning and expression of apical membrane water channel of rat kidney collecting tubule.
1993,
Pubmed
,
Xenbase
Ghosh,
An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.
1998,
Pubmed
Glowatzki,
Subunit-dependent assembly of inward-rectifier K+ channels.
1995,
Pubmed
,
Xenbase
Harris,
Current understanding of the cellular biology and molecular structure of the antidiuretic hormone-stimulated water transport pathway.
1991,
Pubmed
Hasler,
Purified lens major intrinsic protein (MIP) forms highly ordered tetragonal two-dimensional arrays by reconstitution.
1998,
Pubmed
Hoffman,
Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes.
1994,
Pubmed
,
Xenbase
Huang,
Constitutive activation of Mek1 by mutation of serine phosphorylation sites.
1994,
Pubmed
Inoue,
SNAP-23 in rat kidney: colocalization with aquaporin-2 in collecting duct vesicles.
1998,
Pubmed
Jessus,
Ras family proteins: new players involved in the diplotene arrest of Xenopus oocytes.
1998,
Pubmed
,
Xenbase
Jung,
Molecular structure of the water channel through aquaporin CHIP. The hourglass model.
1994,
Pubmed
,
Xenbase
Kamsteeg,
An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus.
1999,
Pubmed
,
Xenbase
Katsura,
Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells.
1997,
Pubmed
Katsura,
Direct demonstration of aquaporin-2 water channel recycling in stably transfected LLC-PK1 epithelial cells.
1996,
Pubmed
Klussmann,
Protein kinase A anchoring proteins are required for vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.
1999,
Pubmed
Kowlessur,
Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase.
1995,
Pubmed
Kuwahara,
cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes.
1995,
Pubmed
,
Xenbase
Lande,
Phosphorylation of aquaporin-2 does not alter the membrane water permeability of rat papillary water channel-containing vesicles.
1996,
Pubmed
Liebenhoff,
Identification of Rab3-, Rab5a- and synaptobrevin II-like proteins in a preparation of rat kidney vesicles containing the vasopressin-regulated water channel.
1995,
Pubmed
Lukacs,
Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation.
1997,
Pubmed
Mandon,
Syntaxin-4 is localized to the apical plasma membrane of rat renal collecting duct cells: possible role in aquaporin-2 trafficking.
1996,
Pubmed
Marples,
Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct.
1998,
Pubmed
Marples,
Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney inner medullary collecting duct.
1995,
Pubmed
Martin,
Role of aquaporin-2 water channels in urinary concentration and dilution defects.
1998,
Pubmed
Martinez,
Dual phosphorylation of the T-loop in cdk7: its role in controlling cyclin H binding and CAK activity.
1997,
Pubmed
,
Xenbase
McDonald,
Adjacent phosphorylation sites on GABAA receptor beta subunits determine regulation by cAMP-dependent protein kinase.
1998,
Pubmed
McLean,
Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy.
1974,
Pubmed
McNicholas,
Regulation of ROMK1 K+ channel activity involves phosphorylation processes.
1994,
Pubmed
,
Xenbase
Molloy,
Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis.
1999,
Pubmed
Morrill,
Endocytosis in the amphibian oocyte. Effect of insulin and progesterone on membrane and fluid internalization during the meiotic divisions.
1984,
Pubmed
Mulders,
An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex.
1998,
Pubmed
,
Xenbase
Neely,
Heterotetrameric composition of aquaporin-4 water channels.
1999,
Pubmed
,
Xenbase
Nielsen,
CHIP28 water channels are localized in constitutively water-permeable segments of the nephron.
1993,
Pubmed
Nielsen,
Renal aquaporins: key roles in water balance and water balance disorders.
1998,
Pubmed
Nielsen,
Physiology and pathophysiology of renal aquaporins.
1999,
Pubmed
Nielsen,
Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels.
1995,
Pubmed
Nielsen,
Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane.
1995,
Pubmed
Nishimoto,
Arginine vasopressin stimulates phosphorylation of aquaporin-2 in rat renal tissue.
1999,
Pubmed
Quick,
Second messengers, trafficking-related proteins, and amino acid residues that contribute to the functional regulation of the rat brain GABA transporter GAT1.
1997,
Pubmed
,
Xenbase
Rich,
Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.
1993,
Pubmed
Rohn,
Bi-directional trafficking between the trans-Golgi network and the endosomal/lysosomal system.
2000,
Pubmed
Sabolić,
The AQP2 water channel: effect of vasopressin treatment, microtubule disruption, and distribution in neonatal rats.
1995,
Pubmed
Schnermann,
Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice.
1998,
Pubmed
Shimkets,
In vivo phosphorylation of the epithelial sodium channel.
1998,
Pubmed
Sönnichsen,
Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.
2000,
Pubmed
Staub,
Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.
1997,
Pubmed
,
Xenbase
Strange,
Apical membrane endocytosis via coated pits is stimulated by removal of antidiuretic hormone from isolated, perfused rabbit cortical collecting tubule.
1988,
Pubmed
Terris,
Distribution of aquaporin-4 water channel expression within rat kidney.
1995,
Pubmed
Valenti,
A heterotrimeric G protein of the Gi family is required for cAMP-triggered trafficking of aquaporin 2 in kidney epithelial cells.
1998,
Pubmed
Valenti,
The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.
2000,
Pubmed
Wade,
ADH action: evidence for a membrane shuttle mechanism.
1981,
Pubmed
Walz,
The three-dimensional structure of aquaporin-1.
1997,
Pubmed
,
Xenbase
Xu,
Phosphorylation of the ATP-sensitive, inwardly rectifying K+ channel, ROMK, by cyclic AMP-dependent protein kinase.
1996,
Pubmed
,
Xenbase
Yang,
Water and glycerol permeabilities of aquaporins 1-5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes.
1997,
Pubmed
,
Xenbase
Zehavi-Feferman,
Control of p62 binding to TGN38/41 by phosphorylation.
1995,
Pubmed
Zhang,
Expression of mRNA coding for kidney and red cell water channels in Xenopus oocytes.
1990,
Pubmed
,
Xenbase
