Distinct regions of Gα13 participate in its regulatory interactions with RGS homology domain-containing RhoGEFs
Distinct regions of Gα13 participate in its regulatory interactions with RGS homology domain-containing RhoGEFs
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DOI：
10.1016/j.cellsig.2007.03.004
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发表时间：
2007-08-01
影响因子：
4.8
通讯作者：
Kozasa, Tohru
中科院分区：
文献类型：
--
作者：
Kreutz, Barry;Hajicek, Nicole;Kozasa, Tohru
Gα12和Gα13通过含有RGS同源(RH)结构域的RhoGEF将信号从G蛋白偶联受体转导到RhoA，如p115 Rhogef或白血病相关Rhogef(LARG)。P115的RH结构域与Gα12和Gα13的活性形式具有高亲和力，并赋予GTP酶激活蛋白(GAP)活性，在Gα13中检测到的GAP反应比在Gα12中检测到的更快。同时，在重建实验中，Gα13而不是Gα12直接刺激p115 RhoEF或非磷酸化LARG的Rhogef活性。为了更好地了解Gα13通过与RH-RhoGEF相互作用调节Rhogef活性的分子机制，我们试图确定Gα13参与GAP反应或Rhogef激活的区域(S)。为此，我们在Gα12和Gα13亚基之间构建了嵌合体，并对它们的生化活性进行了表征。在RhoA激活的细胞和重建分析中，我们发现用Gα13(残基264-377)的羧基末端区域取代Gα12(残基267-379)的羧基末端区域可以赋予所产生的嵌合亚单位Gα12C13功能增益。反向嵌合体Gα13C12表现出与Gα12相似的基础RhoA活性。GAP分析表明，Gα12C13或Gα13C12嵌合体分别以类似于Gα12或Gα13的方式对p115 Rhogef或LARG的GAP活性做出反应。根据这些结果，我们得出结论：Gα13的羧基末端区域(残基264-377)是其Rhogef刺激活性所必需的，而Gα12和Gα13的氨基末端a螺旋和开关区负责它们对RH结构域的差异间隙反应。(C)2007 Elsevier Inc.保留所有权利。
G alpha 12 and G alpha 13 transduce signals from G protein-coupled receptors to RhoA through RhoGEFs containing an RGS homology (RH) domain, such as p115 RhoGEF or leukemia-associated RhoGEF (LARG). The RH domain of p115 RhoGEF or LARG binds with high affinity to active forms of G alpha 12 and G alpha 13 and confers specific GTPase-activating protein (GAP) activity, with faster GAP responses detected in G alpha 13 than in G alpha 12. At the same time, G alpha 13, but not G alpha 12, directly stimulates the RhoGEF activity of p115 RhoGEF or nonphosphorylated LARG in reconstitution assays. In order to better understand the molecular mechanism by which G alpha 13 regulates RhoGEF activity through interaction with RH-RhoGEFs, we sought to identify the region(s) of G alpha 13 involved in either the GAP response or RhoGEF activation. For this purpose, we generated chimeras between G alpha 12 and G alpha 13 subunits and characterized their biochemical activities. In both cell-based and reconstitution assays of RhoA activation, we found that replacing the carboxyl-terminal region of G alpha 12 (residues 267-379) with that of G alpha 13 (residues 264-377) conferred gain-of-function to the resulting chimeric subunit, G alpha 12C13. The inverse chimera, G alpha 13C12, exhibited basal RhoA activation which was similar to G alpha 12. In contrast to GEF assays, GAP assays showed that G alpha 12C13 or G alpha 13C12 chimeras responded to the GAP activity of p115 RhoGEF or LARG in a manner similar to G alpha 12 or G alpha 13, respectively. We conclude from these results that the carboxyl-terminal region of G alpha 13 (residues 264-377) is essential for its RhoGEF stimulating activity, whereas the amino-terminal a helical and switch regions of G alpha 12 and G alpha 13 are responsible for their differential GAP responses to the RH domain. (c) 2007 Elsevier Inc. All rights reserved.