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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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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 × amount of AQP2 protein expected in membrane) − 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).
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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 × amount of AQP2 protein expected in membrane) − 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).

Mentions: From the Pf values and immunoblot signals of oocytes expressing AQP2-S256D-F, which is completely localized in the plasma membrane, the relation between Pf and AQP2 amount in the plasma membrane was obtained (Pf = [3.97 × amount of AQP2 protein expected in the membrane] − 9.7) (Fig. 6). Using this relation and the calculated expected amounts of AQP2 protein expressed in the plasma membrane when zero, one, two, three, or four AQP2-S256D-F monomers would be essential for AQP2 plasma membrane localization, the expected concomitant Pf values for the different SA/SD ratios were calculated (columns AAAA through DDDD in Table , respectively).


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)

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 × amount of AQP2 protein expected in membrane) − 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).
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Related In: Results  -  Collection

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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 × amount of AQP2 protein expected in membrane) − 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).
Mentions: From the Pf values and immunoblot signals of oocytes expressing AQP2-S256D-F, which is completely localized in the plasma membrane, the relation between Pf and AQP2 amount in the plasma membrane was obtained (Pf = [3.97 × amount of AQP2 protein expected in the membrane] − 9.7) (Fig. 6). Using this relation and the calculated expected amounts of AQP2 protein expressed in the plasma membrane when zero, one, two, three, or four AQP2-S256D-F monomers would be essential for AQP2 plasma membrane localization, the expected concomitant Pf values for the different SA/SD ratios were calculated (columns AAAA through DDDD in Table , respectively).

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