Role of membrane microdomains in PTH-mediated down-regulation of NaPi-IIa in opossum kidney cells
Role of membrane microdomains in PTH-mediated down-regulation of NaPi-IIa in opossum kidney cells
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DOI：
10.1111/j.1523-1755.2005.00505.x
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发表时间：
2005-09-01
影响因子：
19.6
通讯作者：
Takeda, E
中科院分区：
文献类型：
--
作者：
Nashiki, K;Taketani, Y;Takeda, E
背景甲状旁腺激素（PTH）通过内吞途径快速下调IIa型钠依赖性磷酸盐转运蛋白（NaPi-IIa）。由于甲状旁腺素信号和NaPi-IIa内吞作用之间的关系尚未探讨，我们研究了膜微区在这一过程中的作用。我们检测了NaPi-IIa在稳定表达人NaPi-IIa的负鼠肾（OK-N2）细胞中的膜下定位，并利用针对蛋白激酶磷酸化底物的特异性抗体进行免疫印迹，寻找PTH诱导的特异性磷酸化底物。我们发现，NaPi-IIa主要定位于质膜的低密度膜（LDM）域; PTH降低了这些域中免疫反应性NaPi-IIa的水平。此外，PTH激活蛋白激酶A（PKA）和蛋白激酶C α（PKC α），并增加250 kD和80 kD底物的磷酸化;后者底物被鉴定为ezrin，它是ezrin-radixin-moesin（ERM）蛋白家族的成员。在对PTH的反应中，ezrin被PKA和PKC磷酸化。显性负性ezrin可阻断PTH诱导的LDM区NaPi-IIa表达的降低。这些数据表明，NaPi-IIa和PTH诱导的磷酸化蛋白，包括ezrin在LDM微区室化。这种区室化可能在通过内吞作用下调NaPi-IIa中发挥重要作用。
Background. Parathyroid hormone (PTH) rapidly down-regulates type IIa sodium-dependent phosphate transporter (NaPi-IIa) via an endocytic pathway. Since the relationship between PTH signaling and NaPi-IIa endocytosis has not been explored, we investigated the role of membrane microdomains in this process.Methods. We examined the submembrane localization of NaPi-IIa in opossum kidney (OK-N2) cells that stably expressed human NaPi-IIa, and searched for a PTH-induced specific phosphorylating substrate on their membrane microdomains by immunoblotting with specific antibody against phospho substrates of protein kinases.Results. We found that NaPi-IIa was primarily localized in low-density membrane (LDM) domains of the plasma membrane; PTH reduced the levels of immunoreactive NaPi-IIa in these domains. Furthermore, PTH activated both protein kinase A (PKA) and protein kinase C alpha (PKCa) and increased the phosphorylation of 250 kD and 80 kD substrates; this latter substrate was identified as ezrin, which a member of the ezrin-radixin-moesin (ERM) protein family. In response to PTH, ezrin was phosphorylated by both PKA and PKC. Dominant negative ezrin blocked the reduction in NaPi-IIa expression in the LDM domains that was induced by PTH.Conclusion. These data suggest that NaPi-IIa and PTH-induced phosphorylated proteins that include ezrin are compartmentalized in LDM microdomains. This compartmentalization may play an important role in the down-regulation of NaPi-IIa via endocytosis.