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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 - J. Cell Biol. (2000)

Bottom Line: In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands.

ABSTRACT
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.

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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.
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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.

Mentions: After measuring the Pf values of the coinjected oocytes (see Table ), total membranes were isolated and immunoblotted. The antibodies used had identical avidity for AQP2-S256A and AQP2-S256D-F (not shown). The densities of individual signals were scanned and compared with a parallel blotted dilution series of wt-AQP2 to calculate the amounts of AQP2 (in arbitrary units). These were then used to calculate the total amounts of expressed AQP2 (Fig. 5; Total AQP2, in arbitrary units) and the ratio between the amounts of AQP2-S256D-F/AQP2-S256A proteins (Fig. 5; Ratio SD/SA) in the five coexpressions.


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 - J. Cell Biol. (2000)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169442&req=5

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.
Mentions: After measuring the Pf values of the coinjected oocytes (see Table ), total membranes were isolated and immunoblotted. The antibodies used had identical avidity for AQP2-S256A and AQP2-S256D-F (not shown). The densities of individual signals were scanned and compared with a parallel blotted dilution series of wt-AQP2 to calculate the amounts of AQP2 (in arbitrary units). These were then used to calculate the total amounts of expressed AQP2 (Fig. 5; Total AQP2, in arbitrary units) and the ratio between the amounts of AQP2-S256D-F/AQP2-S256A proteins (Fig. 5; Ratio SD/SA) in the five coexpressions.

Bottom Line: In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus