A Structural Determinant That Renders Gαi Sensitive to Activation by GIV/Girdin Is Required to Promote Cell Migration
A Structural Determinant That Renders Gαi Sensitive to Activation by GIV/Girdin Is Required to Promote Cell Migration
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DOI：
10.1074/jbc.m109.045161
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发表时间：
2010-04-23
影响因子：
4.8
通讯作者：
Farquhar, Marilyn G.
中科院分区：
文献类型：
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作者：
Garcia-Marcos, Mikel;Ghosh, Pradipta;Farquhar, Marilyn G.
尽管已鉴定出异源三聚体 G 蛋白的几种非受体激活剂，但决定其与此类激活剂相互作用的 G 蛋白的结构特征以及随后的生物学效应却知之甚少。在这里，我们研究了 G α i(3) 中 GIV/girdin 调节所必需的结构决定因素，GIV/girdin 是一种激活 G α(i) 亚基的鸟嘌呤核苷酸交换因子 (GEF)。使用 G 蛋白活性和体外 Pulldown 测定，我们证明 G α i(3) 是比高度同源的 G α(o) 更好的 GIV 底物。通过比较 GIV 与 G α(i3)/G alpha(o) 嵌合体的结合，我们将 G α(i) 亚基中的 Trp-258 鉴定为 GIV 结合的新结构决定因素。 Trp-258 突变为 G α(o) 中相应的 Phe 会降低体外和培养细胞中的 GIV 结合，但不会干扰与其他 G α 结合伴侣的相互作用，即 G β γ、AGS3（鸟嘌呤核苷酸解离抑制剂）、GAIP/RGS19（GTP 酶激活蛋白）和 LPAR1（G 蛋白偶联受体）。当 Trp-258 被 Tyr、Leu、Ser、His、Asp 或 Ala 取代时，GIV 对 G α(i3) 的激活也显着降低，这突出表明 Trp 是最大激活所必需的。此外，当突变体 G α(i3) W258F 在 HeLa 细胞中表达时，它们无法进行细胞迁移，也无法在生长因子或 G 蛋白偶联受体刺激后增强 Akt 信号传导。因此，GIV 激活 G α(i3) 对于与 G α(i3) 激活相关的生物学功能至关重要。总之，我们发现了 G α(i) 上的一种新的结构决定因素，它在定义 GIV 对 G α(i) 的 GEF 活性的选择性和效率方面发挥着关键作用，并且代表了设计小分子以破坏 G α(i)-GIV 界面以达到治疗目的的有吸引力的靶位点。
Although several non-receptor activators of heterotrimeric G proteins have been identified, the structural features of G proteins that determine their interaction with such activators and the subsequent biological effects are poorly understood. Here we investigated the structural determinants in G alpha i(3) necessary for its regulation by GIV/girdin, a guanine-nucleotide exchange factor (GEF) that activates G alpha(i) subunits. Using G protein activity and in vitro pulldown assays we demonstrate that G alpha i(3) is a better substrate for GIV than the highly homologous G alpha(o). We identified Trp-258 in the G alpha(i) subunit as a novel structural determinant for GIV binding by comparing GIV binding to G alpha(i3)/G alpha(o) chimeras. Mutation of Trp-258 to the corresponding Phe in G alpha(o) decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other G alpha-binding partners, i.e. G beta gamma, AGS3 (a guanine nucleotide dissociation inhibitor), GAIP/RGS19 (a GTPase-activating protein), and LPAR1 (a G protein-coupled receptor). Activation of G alpha(i3) by GIV was also dramatically reduced when Trp-258 was replaced with Tyr, Leu, Ser, His, Asp, or Ala, highlighting that Trp is required for maximal activation. Moreover, when mutant G alpha(i3) W258F was expressed in HeLa cells they failed to undergo cell migration and to enhance Akt signaling after growth factor or G protein-coupled receptor stimulation. Thus activation of G alpha(i3) by GIV is essential for biological functions associated with G alpha(i3) activation. In conclusion, we have discovered a novel structural determinant on G alpha(i) that plays a key role in defining the selectivity and efficiency of the GEF activity of GIV on G alpha(i) and that represents an attractive target site for designing small molecules to disrupt the G alpha(i)-GIV interface for therapeutic purposes.