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Moleculan mechanism of IP_3 receptor Ca^<2+> channel and the role of the receptor in signal transduction and growth and development

IP_3受体Ca^2通道的分子机制及其在信号转导和生长发育中的作用

基本信息

批准号：
02101001
负责人：
MIKOSHIBA Katsuhiko
金额：
$ 161.28万
依托单位：
The University of Tokyo (1992-1994)Osaka University (1990-1991)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Specially Promoted Research
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1994
项目状态：
已结题

项目摘要

Inositol 1,4,5-trisphosphate (InsP_3) is a second messenger which induces calcium release from the intracellular store sites. This InsP_3-induced calcium release (IICR) is mediated by an intracellular calcium release channel, InsP_3 receptor (InsP_3R). We have cloned the cDNAs of three different types of InsP_3R (types 1,2and3) so far, and have also found various alternatively-spliced variants of type 1 (neuronal type). The basic structure of all InsP_3R types is composed of three functional domains ; the ligand-binding domain (N-terminal portion) ; the modulatory domain (middle portion) which contains various sites for modulator-binding (ATP,calmodulin, Ca^<2+>, etc.) and phosphorylation (PKA,PKC,CaMKII) ; the channel domain (C-terminal portion) which contains six membrane-spanning segments and one putative "pore" -forming segment as the ion channel superfamily including voltage-sensitive and nucleotide-gated channels. InsP_3R forms a tetramer complex, so that each InsP_3-gated ion channel can have four ligand-binding sites. Our data indicate that each member of the InsP_3R family is differentially expressed in various cell-types, and in some cell-types can form heteromeric InsP_3R channels by assembling with the other receptor types. These results suggest that intracellular calcium signaling mediated by IICR in each cell-type is differently regulated. Thus, we recently have characterized the IICR activity of sole InsP_3R type by using the type-specifically purified receptor reconstituted in liposomes. We found that phosphorylation of type 1 receptor enhances IICR activity. We have also characterized the function role of the type 1 InsP_3R in some cell signalings by using the specific antibody as a channel blocker for the type 1 InsP_3R-mediated IICR.Calcium waves and oscillations in hamster eggs were clearly blocked. Introduction of the antisense nucleotide of the cDNA of Xenopus IP_3R which we cloned, into Xenopus eggs suppressed the egg activation.

1，4，5-三磷酸肌醇(Insp3)是细胞内钙离子释放的第二信使。这种由InSP_3诱导的钙释放(IICR)是由细胞内钙释放通道InSP_3受体(InSP_3R)介导的。到目前为止，我们已经克隆了三种不同类型的Insp3R(类型1、2和3型)的cDNA，并发现了类型1(神经元型)的各种选择性剪接变体。所有InSP_3R类型的基本结构由三个功能结构域组成：配体结合区(N-末端部分)；调节域(中间部分)，它包含各种调节器结合位点(ATP、钙调蛋白、钙离子等)。和磷酸化(PKA，PKC，CaMKII)；通道结构域(C-末端部分)，它包含六个跨膜片段和一个可能的“孔”形成片段，是离子通道超家族，包括电压敏感通道和核苷酸门控通道。InSP_3R形成一个四聚体复合体，使得每个InSP_3门控离子通道可以有四个配体结合位点。我们的数据表明，Insp3R家族的每个成员在不同的细胞类型中都有不同的表达，在某些细胞类型中，可以通过与其他受体类型的组装形成异构体Insp3R通道。这些结果表明，IICR介导的细胞内钙信号在每种细胞类型中受到不同的调控。因此，我们最近利用脂质体中重组的类型特异性纯化的受体来表征单一Insp3R型的IICR活性。我们发现I型受体的磷酸化增强了IICR的活性。我们还利用特异性抗体作为I型Insp3R介导的IICR的通道阻断剂，研究了I型Insp3R在某些细胞信号转导中的作用。仓鼠卵中的钙波和振荡明显被阻断。将我们克隆的非洲爪蛙IP_3R基因的反义核苷酸导入非洲爪哇卵中，可抑制卵子的激活。