Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.
Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.
复制标题
毒蕈碱受体通过 Ras-GTPase 激活蛋白 SH3 结构域抑制的 Ras 依赖性信号通路转化 NIH 3T3 细胞。
DOI：
10.1128/mcb.14.12.7943-7952.1994
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发表时间：
1994
影响因子：
5.3
通讯作者：
Macara,IG
中科院分区：
文献类型：
--
作者：
Mattingly,RR;Sorisky,A;Brann,MR;Macara,IG
NIH 3T3 细胞中人毒蕈碱受体的某些亚型的表达提供了通过响应卡巴胆碱形成病灶而进行的细胞转化的激动剂依赖性模型。尽管病灶形成与毒蕈碱受体激活磷脂酶 C 的能力相关，但实际的有丝分裂信号转导途径尚不清楚。通过共转染实验和测量天然 Ras 蛋白和表位标记 Ras 蛋白的激活状态，确定了 Ras 和 Ras GTP 酶激活蛋白 (Ras-GAP) 对毒蕈碱受体依赖性转化的贡献。转化毒蕈碱受体能够激活 Ras，并且这种激活是转化所必需的，因为具有显性失活突变的 Ras 或包含催化结构域的 Ras-GAP 构建体的共表达会抑制焦点形成。 GAP 的 N 端区域或其分离的 SH3（Src 同源性 3）结构域（但不是其 SH2 结构域）的共表达也足以抑制毒蕈碱受体依赖性病灶形成。 Ras-GAP SH3 结构域中保守残基的点突变逆转了其作用，导致卡巴胆碱依赖性转化增加。 Ras-GAP SH3 结构域表达的抑制作用发生在 Ras 激活附近，并且对卡巴胆碱激活的有丝分裂途径具有选择性，因为 v-Ras 或trk4/神经生长因子的细胞转化不受影响。
Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonistdependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase- activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras ortrk4/nerve growth factor is unaffected.