Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras
Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras
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DOI：
10.1128/mcb.17.3.1396
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发表时间：
1997-03-01
影响因子：
5.3
通讯作者：
Moran, MF
中科院分区：
文献类型：
--
作者：
Fam, NP;Fan, WT;Moran, MF
Ras蛋白转化为能够结合效应蛋白的活化GTP结合状态是由特异性鸟嘌呤核苷酸交换因子催化的，以响应大量的细胞外刺激。在这里，我们报告了小鼠编码Ras-GRF 2的cDNA的分离，Ras-GRF 2是一种含有COOH末端Cdc 25相关结构域的多结构域蛋白，其在体外刺激Ras释放GDP，但不刺激其他GTP酶。Ras-GRF 2特异性结合到缺乏结合核苷酸的固定化Ras上，表明作为催化核苷酸交换的机制的无核苷酸形式的Ras的稳定化。Ras-GRF 2的NH 2-末端区域被预测含有各种信号传导蛋白共有的特征，包括两个普列克底物蛋白同源结构域和一个Db 1同源区域。Ras-GRF 2还含有IQ基序，其与异位表达RaS-GRF 2的上皮细胞中的钙调蛋白表观组成性缔合是必需的。Ras-GRF 2在肾上皮细胞中的瞬时表达刺激Ras与GTP的结合，并增强钙离子载体诱导的依赖于IQ基序的丝裂原活化蛋白激酶（ERK 1）的活化。钙离子内流引起Ras-GRF 2亚细胞定位从胞质到外周的变化，这表明控制Ras-GRF 2与Ras在质膜上相互作用的可能机制。过表达Ras-GRF 2的上皮细胞发生形态学转化，并以无组织的方式生长，细胞间接触最少。北方分析表明，在脑和肺中有一个9-kb的GRF 2转录本，已知p135 Ras-GRF 2在这些部位表达，在几种组织中检测到12 kb和2.2 kb的RNA。因此，具有不同结构域结构的Ras-GRF 2蛋白可以广泛表达，并将不同的细胞外信号与Ras激活偶联。
Conversion of Ras proteins into an activated GTP-bound state able to bind effector proteins is catalyzed by specific guanine nucleotide exchange factors in response to a large number of extracellular stimuli, Here we report the isolation of mouse cDNAs encoding Ras-GRF2, a multidomain 135-kDa protein containing a COOH-terminal Cdc25-related domain that stimulates release of GDP from Ras but not other GTPases in vitro. Ras-GRF2 bound specifically to immobilized Ras lacking bound nucleotides, suggesting stabilization of the nucleotide-free form of Ras as a mechanism of catalyzing nucleotide exchange. The NH2-terminal region of Ras-GRF2 is predicted to contain features common to various signaling proteins including two pleckstrin homology domains and a Dbl homology region, Ras-GRF2 also contains an IQ motif which was required for its apparent constitutive association,vith calmodulin in epithelial cells ectopically expressing RaS-GRF2. Transient expression of Ras-GRF2 in kidney epithelial cells stimulated GTP binding by Ras and potentiated calcium ionophore induced activation of mitogen-activated protein kinase (ERK1) dependent upon the IQ motif. Calcium influx caused Ras-GRF2 subcellular localization to change from cytosolic to peripheral, suggesting a possible mechanism for controlling Ras-GRF2 interactions with Ras at the plasma membrane. Epithelial cells overexpressing Ras-GRF2 are morphologically transformed and grow in a disorganized manner with minimal intercellular contacts. Northern analysis indicated a 9-kb GRF2 transcript in brain and lung, where p135 Ras-GRF2 is known to be expressed, and RNAs of 12 kb and 2.2 kb were detected in several tissues. Thus, Ras-GRF2 proteins with different domain structures may be widely expressed and couple diverse extracellular signals to Ras activation.