Kamsteeg E J, Heijnen I, van Os C H, Deen P M
Department of Cell Physiology, University Medical Center, St. Radboud, 6500HB Nijmegen, The Netherlands.
J Cell Biol. 2000 Nov 13;151(4):919-30. doi: 10.1083/jcb.151.4.919.
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.
在肾主细胞中，血管加压素调节水通道蛋白（AQP）2水通道在细胞内囊泡和顶端质膜之间的穿梭。血管加压素通过蛋白激酶A（PKA）诱导AQP2的丝氨酸256（S256）磷酸化对于其在膜中的定位至关重要。然而，在未诱导的主细胞的细胞内囊泡中也检测到了磷酸化的AQP2（p-AQP2）。由于AQP2以同四聚体形式表达，我们推测四聚体中p-AQP2单体的数量可能对其稳态分布至关重要。在卵母细胞中表达时，AQP2-S256D和AQP2-S256A分别模拟了p-AQP2和非p-AQP2，因为AQP2-S256D和野生型AQP2（wt-AQP2）的转运和功能相同，而AQP2-S256A保留在细胞内。在共注射实验中，AQP2-S256A和AQP2-S256D形成了异四聚体。不同比例的AQP2-S256A和AQP2-S256D cRNAs共注射实验表明，AQP2四聚体中至少三个AQP2-S256D单体对于其质膜定位至关重要。因此，我们的结果表明，在主细胞中，每个AQP2四聚体至少三个单体必须被磷酸化才能在顶端膜中实现稳态定位。由于其他多亚基通道也受磷酸化调节，预计其磷酸化和未磷酸化亚基的化学计量可能会微调这些复合物的活性或亚细胞定位。
