Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization
Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization
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DOI：
10.1074/jbc.m410538200
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发表时间：
2005-04-15
影响因子：
4.8
通讯作者：
Fukami, Y
中科院分区：
文献类型：
--
作者：
Sakakibara, K;Sato, K;Fukami, Y
在这里，我们描述了质谱鉴定、分子克隆和生化表征的脂质/膜筏相关蛋白，酪氨酸磷酸化的爪蟾卵受精。该蛋白与哺乳动物uroplakin III同源，后者是构成哺乳动物尿路上皮组织不对称单元膜的uroplakin家族蛋白（UPs）的成员，因此被称为Xenopus uroplakin III （xUPIII）。xUPIII含有n -连接糖，在爪蟾卵、卵巢、尿道和肾脏中高度表达。在未受精卵中，通过表面生物素化实验和间接免疫荧光研究判断，xUPIII主要定位于脂质/膜筏并暴露在细胞表面。在受精或过氧化氢诱导的卵子活化后，xUPIII在酪氨酸残基-249上迅速磷酸化，酪氨酸残基-249位于分子的胞质尾部羧基端。通过xUPIII和Xenopus c-Src的共表达，可以在HEK293细胞中重建xUPIII的筏定位和酪氨酸磷酸化，Xenopus c-Src是一种酪氨酸激酶，其在卵筏中的受精诱导激活是发育开始所必需的。在哺乳动物中，UPIII正在与四跨蛋白分子uroplakin Ib形成复合物。由于已知另一种四跨蛋白CD9是小鼠精卵融合的关键成分，我们假设xUPIII参与了精卵相互作用。一种针对xUPIII细胞外结构域的抗体阻断了精子与卵子的相互作用，这可以通过卵子激活和第一细胞分裂的发生来判断。因此，xUPIII代表了一种卵筏相关蛋白，可能参与了爪蟾精子-卵子相互作用以及随后的src依赖性卵激活的细胞内事件。
Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.