Bernasconi Francesca, Malgaroli Antonio, Vallar Lucia
Department of Pharmacology, University of Milan, Milan, Italy.
Neurosignals. 2006;15(4):180-9. doi: 10.1159/000096734. Epub 2006 Nov 2.
Receptors coupled to G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) cascade. The intracellular pathways linking the alpha chains of these G proteins to MAPK activation are not completely understood. One of the signaling molecules which has been suggested to act downstream of Galpha(i/o) is the small G protein Rap1. We investigated the role of Rap1 in MAPK stimulation by Galpha(o) in Chinese hamster ovary (CHO) cells. Our previous results have shown that in this cell system activated Galpha(o) strongly potentiates the MAPK response to the epidermal growth factor (EGF) receptor. Rap1 regulation was examined in cells transfected with Rap1 and wild-type Galpha(o) or the activated mutant Galpha(o)-Q205L. Immunocytochemical analysis detected both Rap1 and the Galpha(o) subunit at the plasma membrane as well as on perinuclear cytoplasmic vesicles. Expression of wild-type Galpha(o) had no significant effect on the levels of activated Rap1. In contrast, Galpha(o)-Q205L virtually abolished the activation of Rap1 induced by EGF. Further experiments showed that MAPK stimulation by EGF was greatly inhibited by expression of activated Rap1, suggesting that Rap1 inhibition could mediate the effect of Galpha(o) on the MAPK cascade. However, Galpha(o)-Q205L efficiently inhibited the activation of Rap1 induced by fibroblast growth factor (FGF). We have previously found that the ability of FGF to activate MAPK is not modified by Galpha(o). In addition, expression of the GAP protein RAP1GAPII blocked Rap1 activation without affecting EGF- or FGF-dependent MAPK stimulation. These findings provide evidence for independent regulation of Rap1 and MAPK by the G(o )alpha chain.
与G(i/o)蛋白偶联的受体可刺激丝裂原活化蛋白激酶（MAPK）级联反应。将这些G蛋白的α链与MAPK激活相联系的细胞内信号通路尚未完全明确。一种被认为在Gα(i/o)下游起作用的信号分子是小G蛋白Rap1。我们研究了Rap1在中国仓鼠卵巢（CHO）细胞中Gα(o)刺激MAPK过程中的作用。我们之前的研究结果表明，在这个细胞系统中，活化的Gα(o)能强烈增强MAPK对表皮生长因子（EGF）受体的反应。在用Rap1和野生型Gα(o)或活化突变体Gα(o)-Q205L转染的细胞中检测了Rap1的调控情况。免疫细胞化学分析在质膜以及核周细胞质囊泡上检测到了Rap1和Gα(o)亚基。野生型Gα(o)的表达对活化Rap1的水平没有显著影响。相比之下，Gα(o)-Q205L几乎完全消除了EGF诱导的Rap1激活。进一步的实验表明，活化Rap1的表达极大地抑制了EGF对MAPK的刺激，这表明Rap1抑制可能介导了Gα(o)对MAPK级联反应的作用。然而，Gα(o)-Q205L有效地抑制了成纤维细胞生长因子（FGF）诱导的Rap1激活。我们之前发现FGF激活MAPK的能力不受Gα(o)的影响。此外，GAP蛋白RAP1GAPII的表达阻断了Rap1的激活，而不影响EGF或FGF依赖的MAPK刺激。这些发现为G(o)α链对Rap1和MAPK的独立调控提供了证据。
