Department of Nephrology and Hypertension, University Hospital of Bern, Freiburgstrasse 15, 3010 Bern, Switzerland.
J Biol Chem. 2010 Dec 3;285(49):38293-303. doi: 10.1074/jbc.M110.115089. Epub 2010 Sep 20.
The ubiquitously expressed mammalian Na(+)/H(+) exchanger 1 (NHE1) controls cell volume and pH but is also critically involved in complex biological processes like cell adhesion, cell migration, cell proliferation, and mechanosensation. Pathways controlling NHE1 turnover at the plasma membrane, however, are currently unclear. Here, we demonstrate that NHE1 undergoes ubiquitylation at the plasma membrane by a process that is unprecedented for a mammalian ion transport protein. This process requires the adapter protein β-arrestin-1 that interacts with both the E3 ubiquitin ligase Nedd4-1 and the NHE1 C terminus. Truncation of NHE1 C terminus to amino acid 550 abolishes binding to β-arrestin-1 and NHE1 ubiquitylation. Overexpression of β-arrestin-1 or of wild type but not ligase-dead Nedd4-1 increases NHE1 ubiquitylation. siRNA-mediated knock-down of Nedd4-1 or β-arrestin-1 reduces NHE1 ubiquitylation and endocytosis leading to increased NHE1 surface levels. Fibroblasts derived from β-arrestin-1 and Nedd4-1 knock-out mice show loss of NHE1 ubiquitylation, increased plasmalemmal NHE1 levels and greatly enhanced NHE1 transport compared with wild-type fibroblasts. These findings reveal Nedd4-1 and β-arrestin-1 as key regulators of NHE1 ubiquitylation, endocytosis, and function. Our data suggest a broader role for β-arrestins in the regulation of membrane ion transport proteins than currently known.
哺乳动物普遍表达的 Na(+)/H(+)交换蛋白 1（NHE1）控制细胞体积和 pH 值，但也与细胞黏附、细胞迁移、细胞增殖和机械感觉等复杂的生物学过程密切相关。然而，目前尚不清楚控制质膜上 NHE1 周转率的途径。在这里，我们证明 NHE1 通过一种以前从未在哺乳动物离子转运蛋白中发现的过程在质膜上发生泛素化。这个过程需要衔接蛋白β-arrestin-1，它与 E3 泛素连接酶 Nedd4-1 和 NHE1 C 末端相互作用。NHE1 C 末端截断至 550 个氨基酸会破坏与β-arrestin-1 的结合和 NHE1 的泛素化。β-arrestin-1 的过表达或野生型但不是连接酶失活的 Nedd4-1 的过表达会增加 NHE1 的泛素化。Nedd4-1 或β-arrestin-1 的 siRNA 介导敲低会减少 NHE1 的泛素化和内吞作用，从而导致 NHE1 表面水平增加。与野生型成纤维细胞相比，来自β-arrestin-1 和 Nedd4-1 敲除小鼠的成纤维细胞显示 NHE1 泛素化、质膜上 NHE1 水平增加和 NHE1 转运大大增强的现象。这些发现揭示了 Nedd4-1 和β-arrestin-1 是 NHE1 泛素化、内吞作用和功能的关键调节因子。我们的数据表明，β-arrestin 在调节膜离子转运蛋白方面的作用比目前已知的更为广泛。
