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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13 November 2000
Article|
November 13 2000
The Subcellular Localization of an Aquaporin-2 Tetramer Depends on the Stoichiometry of Phosphorylated and Nonphosphorylated Monomers
E.J. Kamsteeg,
E.J. Kamsteeg
aDepartment of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands
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I. Heijnen,
I. Heijnen
aDepartment of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands
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C.H. van Os,
C.H. van Os
aDepartment of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands
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P.M.T. Deen
P.M.T. Deen
aDepartment of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands
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E.J. Kamsteeg
aDepartment of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands
I. Heijnen
aDepartment of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands
C.H. van Os
aDepartment of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands
P.M.T. Deen
aDepartment of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands
Abbreviations used in this paper: AQP, aquaporin; AVP, arginine vasopressin; HbA, homogenization buffer A; MBS, modified Barth's solution; MBSS, MED buffered saline for silica; MSB, membrane solubilization buffer; NDI, nephrogenic diabetes mellitus; p-AQP2, PKA-phosphorylated AQP2; Pf, osmotic water permeability; PKA, protein kinase A; PLP, periodate-lysine-paraformaldehyde; wt-AQP2, wild-type AQP2.
Received:
May 30 2000
Revision Requested:
September 28 2000
Accepted:
October 02 2000
Online ISSN: 1540-8140
Print ISSN: 0021-9525
© 2000 The Rockefeller University Press
2000
The Rockefeller University Press
J Cell Biol (2000) 151 (4): 919–930.
Article history
Received:
May 30 2000
Revision Requested:
September 28 2000
Accepted:
October 02 2000
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Citation
E.J. Kamsteeg, I. Heijnen, C.H. van Os, P.M.T. Deen; The Subcellular Localization of an Aquaporin-2 Tetramer Depends on the Stoichiometry of Phosphorylated and Nonphosphorylated Monomers. J Cell Biol 13 November 2000; 151 (4): 919–930. doi: https://doi.org/10.1083/jcb.151.4.919
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