Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2
Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2
复制标题
DOI：
10.1152/ajprenal.00179.2003
复制
发表时间：
2004-02-01
影响因子：
4.2
通讯作者：
Brown, D
中科院分区：
文献类型：
--
作者：
Lu, H;Sun, TX;Brown, D
抑制网格蛋白介导的内吞作用表达的GTP酶缺陷的发动蛋白突变体（发动蛋白-2/K44A）为16小时的结果在积累的质膜水通道蛋白-2（AQP 2）稳定转染野生型AQP 2的上皮细胞。我们现在显示了类似的效果K44 A发动蛋白在LLC-PK 1细胞转染S256磷酸化缺陷的AQP 2突变体，AQP 2（S256 A），并在AQP 2转染内髓集合管（IMCD）细胞。用胆固醇消耗剂甲基-β-环糊精（mbetaCD; 10 mM）对这些细胞内吞作用的更急性阻断导致野生型AQP 2和AQP 2（S256 A）在治疗后15分钟内在细胞表面快速和广泛蓄积。这种效应与用加压素处理细胞所诱导的效应相似。使用FITC-葡聚糖摄取的定量分析证实了mbetaCD对内吞作用的阻断，并通过细胞表面生物素化验证了AQP 2膜插入。这些数据表明，在LLC-PK 1和IMCD细胞中，AQP 2在细胞内储存和细胞表面之间组成性地且快速地粘附。组成性运输过程不依赖于AQP 2的丝氨酸-256残基的磷酸化，然而，这是调节加压素/cAMP介导的AQP 2易位的重要步骤。我们的数据表明，快速和广泛的质膜积累的AQP 2可以发生在一个加压素受体（V2 R）和磷酸化的独立方式，指出一个潜在的手段绕过突变的V2 R在X-连锁肾性尿崩症，以实现细胞表面表达的AQP 2。
Inhibition of clathrin-mediated endocytosis by expression of a GTPase-deficient dynamin mutant (dynamin-2/K44A) for 16 h results in an accumulation of plasma membrane aquaporin-2 (AQP2) in epithelial cells stably transfected with wild-type AQP2. We now show a similar effect of K44A dynamin in LLC-PK1 cells transfected with an S256 phosphorylation-deficient AQP2 mutant, AQP2(S256A), and in AQP2-transfected inner medullary collecting duct (IMCD) cells. More acute blockade of endocytosis in these cells with the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD; 10 mM) resulted in a rapid and extensive cell-surface accumulation of both wild-type AQP2 and AQP2 (S256A) within 15 min after treatment. This effect was similar to that induced by treatment of the cells with vasopressin. Blockade of endocytosis by mbetaCD was confirmed using quantitative analysis of FITC-dextran uptake and AQP2 membrane insertion was verified by cell-surface biotinylation. These data indicate that AQP2 recycles constitutively and rapidly between intracellular stores and the cell surface in LLC-PK1 and IMCD cells. The constitutive trafficking process is not dependent on phosphorylation of the serine-256 residue of AQP2, which is, however, an essential step for regulated vasopressin/ cAMP-mediated translocation of AQP2. Our data show that rapid and extensive plasma membrane accumulation of AQP2 can occur in a vasopressin receptor (V2R)- and phosphorylation-independent manner, pointing to a potential means of bypassing the mutated V2R in X-linked nephrogenic diabetes insipidus to achieve cell surface expression of AQP2.