cDNAs and Antibodies for Small GTP-binding Proteins

小 GTP 结合蛋白的 cDNA 和抗体

• 批准号: 01870017
• 负责人: TAKAI Yoshimi
• 金额: $ 7.23万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B).
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01870017/
• 关键词: ras p21; small GTP-binding proteins; prenylation; GDP; GTP exchange protein; GTPase activating protein; Tumor markers; <ras>___ー p21; GTP結合蛋白質; smgp25; smgp21; GDI

There is a superfamily of ras p21/ras p21-like small GTP-binding proteins (small G proteins) with GTPase activity. Over forty members have been reported, and all of them with Mrs between 20,000 and 41,000 are composed of a single respective polypeptide. Evidence is accumulating that small G proteins are involved in the regulation of cell growth and differentiation. In this research project, we have investigated the C-terminal structures of small G proteins, purified the regulatory proteins for small G proteins, and made monoclonal antibodies against small G proteins and their regulatory proteins. Most of small G proteins have the unique consensus C-terminal motifs containing at least one cysteine residue. We have found that the C-terminal systeine residues of smg p21B, rhoA p21, and smg p25A are geranylgeranylated, and that these prenylation are essential for each small G protein to bind to membranes. We have partially purified and characterized geranyl-geranyl transferase for rhoA p21 … More from bovine brain. Small G proteins have two interconvertible forms, GDP-bound inactive and GTP-bound active forms. The conversion from the GDP-bound to GTP-bound form and the reverse conversion are induced by GDP/GTP exchange and GTPase reactions, respectively. We have purified and characterized several GDP/GTP exchange proteins (GDP dissociation stimulator (GDS) and GDP dissociation inhibitor (GDI)) and GTPase activating Proteins (GAP) for small G proteins. Among these regulatory proteins, we have cloned the cDNAs of smg p21 GDS, smg p25A GDI, and rho GDI, and made monoclonal antibodies against them. We have found that the levels of smg p21 mRNA increase in hematopoietic leukemiae cells after their differentiation into hematopoietic cells. We have also found that smg p25A is mainly detected inneural tissues by use of its specific monoclonal antibody, and that the levels of smg p25A mRNA increase in PC12 cells after their differentiation into neuron-like cells. These results suggest that smg p21 and smg p25A can be used as potential tumor markers for tumors derived from hematopoietic and neural tissues, respectively. Key Words : ras p21, small G proteins, Prenylation, GDP/GTP exchange protein, GTPase activating protein, Tumor markers. Less

有一个超家族的ras p21/ras p21样小GTP结合蛋白(小G蛋白)具有GTP酶活性。已经报道了40多个成员，它们的MRS在20,000到41,000之间，都是由一个单独的多肽组成的。越来越多的证据表明，小G蛋白参与了细胞生长和分化的调节。在本研究项目中，我们研究了小G蛋白的C-末端结构，纯化了小G蛋白的调控蛋白，并制备了抗小G蛋白及其调控蛋白的单抗。大多数小G蛋白都有独特的共同C-末端基序，其中至少含有一个半胱氨酸残基。我们发现SMG p21B、RhoA p21和SMG p25A的C-末端的Systeine残基是香叶基香叶化的，这些预烯基化是每个小G蛋白与膜结合所必需的。我们部分纯化并鉴定了RhoA p21…的香叶基-香叶基转移酶更多来自牛脑的消息。小G蛋白有两种相互转换的形式，即GDP结合的非活性形式和GTP结合的活性形式。GDP结合形式到GTP结合形式的转换和反向转换分别由GDP/GTP交换和GTP酶反应诱导。我们已经纯化并鉴定了几种GDP/GTP交换蛋白(GDP解离刺激因子(GDS)和GDP解离抑制因子(GDI))和针对小G蛋白的GTP酶激活蛋白(GAP)。在这些调控蛋白中，我们克隆了SMG p21 GDS、SMG p25A GDI和Rho GDI的cDNA，并制备了抗它们的单抗。我们发现，在造血白血病细胞分化为造血细胞后，SMG p21基因的表达水平升高。我们还发现，SMG p25A主要通过其特异性的单抗在神经组织中检测到，并且在PC12细胞分化为神经元样细胞后，SMG p25A的mRNA水平增加。提示SMG p21和SMG p25A可分别作为造血和神经来源肿瘤的潜在肿瘤标志物。关键词：RAS p21，小G蛋白，预基化，GDP/GTP交换蛋白，GTP酶激活蛋白，肿瘤标记物。较少

项目成果

Kaibuchi,K.: "Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C." J.Biol.Chem.264. 13489-13496 (1989)

Kaibuchi,K.：“蛋白激酶 C 调节和催化域的分子遗传学分析。”

Yamamoto,J.: "A GTPaseーactivating protein for rhoB p20,a ras p21ーlike GTPーbinding proteinーpartial purification,characterization and subcellular distribution in rat brain." Mol.Brain Res.8. 105-111 (1990)

Yamamoto, J.：“rhoB p20 的 GTP 激活蛋白，一种 ras p21 样 GTP 结合蛋白 - 大鼠脑中的部分纯化、表征和亚细胞分布。”Mol.Brain Res.8（1990）。

ohmori, T.: "Small Molecular Weight GTP-Binding Proteins in Human Platelet Membranes Purification and Characterization of a Novel GTP-Binding Protein with a Molecular Weight of 22, 000." J. Biol. Chem. 264. 1877-1881 (1989)

ohmori, T.：“人血小板膜中的小分子量 GTP 结合蛋白的纯化和分子量为 22, 000 的新型 GTP 结合蛋白的表征。”

Ohga,N.: "Rabbit intestine contains a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to rhoB p20,a ras p21-like GTP-binding protein." Biochem.Biophys.Res.Commun.163. 1523-1533 (1989)

Ohga,N.：“兔肠中含有一种蛋白质，可以抑制 GDP 与 rhoB p20（一种类似 ras p21 的 GTP 结合蛋白）的解离以及随后 GTP 与 rhoB p20 的结合。”

Sasaki,T.: "A mammalian inhibitory GDP/GTP exchange protein (GDI) for smg p25A is active on the yeast SEC4 protein." Mol.Cell.Biol.(1991)

Sasaki,T.：“smg p25A 的哺乳动物抑制性 GDP/GTP 交换蛋白 (GDI) 对酵母 SEC4 蛋白具有活性。”