Signal transduction pathways leading to Ca2+ release in fertilization: focusing on egg membrane-associated protein complexes

以非洲爪蛙为主要模式生物，分析了非洲爪蛙受精的分子机制。通过对一个57 kDa的蛋白酪氨酸激酶的分子同源性和卵细胞质膜相关蛋白复合体的结构和功能的研究，我们发现非洲爪哇的src基因产物(称为xSrc)和一个30 kDa的跨膜蛋白Uroplakin III(称为xUPIII)是精子诱导卵子激活的重要因素，即受精卵内瞬时的钙释放。XUPIII已被初步确定为非洲爪哇受精卵中的主要酪氨酸磷酸化蛋白。对xUPIII的进一步分析表明，它是精子来源的蛋白酶活性的靶标，受精时xUPIII胞外区的部分蛋白降解可能是促进精子诱导的xSrc激活的信号。由于xUPIII和Src主要定位于高胆固醇卵膜微区或“筏”，推测膜微域中的蛋白质复合体是精子-卵膜相互作用和随后受精时依赖于Src的钙信号转导的功能平台。我们还确定uroplakin Ib(称为xUPIb)是UPIII的结合伙伴，并使用HEK293细胞进行的表达研究表明，xUPIII和xUPIb的共同表达对于它们正确定位于膜微域以及保持xSrc酪氨酸激酶活性在非活性状态是必需的。这些结果突出表明，在非洲爪哇，精子诱导的卵子激活涉及到分子机制，这些分子机制作用于细胞外空间的配子相互作用，并将精子信号翻译到细胞内空间，这两者都定位于卵膜微域。

We have analyzed molecular mechanism of fertilization by using African clawed frog Xenopus laevis as a main model organism. By focusing on molecular identity of a 57-kDa protein-tyrosine kinase that is immunochemically related to the Src family kinases and the structure and function of the egg plasma membrane-associated protein complexes, we have found that Xenopus Src gene product (termed xSrc) and a 30-kDa transmembrane protein uroplakin III (termed xUPIII) are important factors for sperm-induced egg activation, namely transient Ca2+ release within the fertilized egg. xUPIII has been initially identified as a major tyrosine-phosphorylated protein in fertilized Xenopus eggs. Further analysis of xUPIII has demonstrated that it is a target of sperm-derived protease activity and that partial proteolysis of the extracellular domain of xUPIII at fertilization may act as a signal to promote sperm-induced xSrc activation. Because both xUPIII and Src localizes predominantly in cholesterol-enriched egg membrane microdomains or "rafts", it is assumed that protein complexes organized in the membrane microdomains serve as a functional platform for sperm-egg membrane interaction and subsequent Src-dependent calcium signaling at fertilization. We have also identified uroplakin Ib (termed xUPIb), as a binding partner for UPIII and expression studies using HEK293 cells show that co-expression of xUPIII and xUPIb is required for their proper localization to the membrane microdomain as well as maintenance of xSrc tyrosine kinase activity in an inactive state. These results highlights the scheme that sperm-induced egg activation in Xenopus involves molecular machinery that act for gamete interaction in the extracellular space and that translates the sperm signal into the intracellular space, both of which are localized in the egg membrane microdomains