WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome
WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome
复制标题
DOI：
10.1002/j.1460-2075.1996.tb00593.x
复制
发表时间：
1996-05-15
期刊：
影响因子：
11.4
通讯作者：
Rotin, D
中科院分区：
文献类型：
--
作者：
Staub, O;Dho, S;Rotin, D
阿米洛利敏感性上皮钠通道（ENaC）在肾脏和其他上皮细胞的钠转运以及血压调节中起主要作用。该通道由三个亚单位（α β γ）组成，每个亚单位在其C末端含有两个富含脯氨酸的序列（P1和P2）。人类β和γ ENaC中的P2区域与大鼠β γ rENaC相同，最近显示在Liddle综合征（一种遗传性高血压）患者中缺失，导致通道过度激活。使用酵母双杂交筛选，我们现在已经确定了大鼠同源Nedd 4（rNedd 4）作为β和γ rENaC的P2区的结合伴侣。rNedd 4含有一个Ca 2+脂质结合（CaLB或C2）结构域、三个WW结构域和一个泛素连接酶（Hect）结构域。我们的酵母双杂交和体外结合研究表明，rNedd 4-WW结构域通过与P2区结合来介导这种结合，P2区包括β rENaC（PPPNY）或γ rENaC（PPPRY）的PY蛾（XPPXY）。SH 3结构域不能结合这些序列。此外，最近在Liddle患者中描述的β rENaC P2序列内的Pro616或Tyr 618突变为Ala（分别为PPANY或PPPNA）导致rNedd 4-WW结合的废除。Nedd 4-WW结构域还结合到α rENaC的富含脯氨酸的C-末端（含有序列PPPAY），并且内源性Nedd 4与MDCK细胞中表达的α rENaC共免疫沉淀。这些结果表明，rNedd 4的WW结构域结合到从Liddle综合征患者的β或γ ENaC中缺失的PY蛾，并表明Nedd 4可能是上皮Na+通道的调节剂（抑制剂）。
The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure. The channel is composed of three submits (alpha beta gamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus, The P2 regions in human beta and gamma ENaC, identical to the rat beta gamma rENaC, mere recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel. Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gamma rENaC. rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain, Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY moths (XPPXY) of either beta rENaC (PPPNY) or gamma rENaC (PPPRY). SH3 domains were unable to bind these sequences. Moreover, mutations to Ala of Pro616 or Tyr618 within the beta rENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding. Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpha rENaC, and endogenous Nedd4 co-immunoprecipitated with alpha rENaC expressed in MDCK cells. These results demonstrate that the WW domains of rNedd4 bind to the PY moths deleted from beta or gamma ENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.