Determination of interaction sites on the small G protein RhoA for phospholipase D
Determination of interaction sites on the small G protein RhoA for phospholipase D
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DOI：
10.1074/jbc.273.19.11596
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发表时间：
1998-05-08
影响因子：
4.8
通讯作者：
Exton, JH
中科院分区：
文献类型：
--
作者：
Bae, CD;Min, DS;Exton, JH
磷脂酶D（PLD）已被鉴定为Rho家族的小G蛋白的靶标。本研究利用RhoA与Ha-Ras或Cdc 42 Hs的嵌合蛋白及点突变，在体外确定RhoA与大鼠脑PLD的相互作用位点，其中RhoA的开关I区是Ras样G蛋白的共同效应域，是PLD的关键作用位点。保守氨基酸（Tyr（34）、Thr（37）、Phe（39））的突变完全消除了PLD激活，而瓦尔（38）或Tyr（42）的突变导致部分丢失。另外两个位点负责RhoA和Cdc 42 Hs之间的差异PLD激活能力。将RhoA开关II区域中的Asp（76）改变为Cdc 42 Hs中的相应氨基酸导致PLD活化的部分丧失。一个嵌合蛋白的N-末端三分之一的Cdc 42 Hs改变RhoA显示增强PLD激活。对其他Rho/Ha-Ras嵌合蛋白和突变的分析表明，Gln（52）邻近开关II区域负责这种功能的获得。总之，本研究表明，RhoA开关I区域的保守氨基酸是主要的PLD相互作用位点，开关II和内部区域的残基负责RhoA和Cdc 42 Hs对PLD的差异激活。
Phospholipase D (PLD) has been identified as a target of small G proteins of the Rho family. The present study was directed at defining the interaction sites of RhoA with rat brain PLD in vitro using chimeric proteins between RhoA and Ha-Ras or Cdc42Hs and point mutations.The switch I region of RhoA, which is the common effector domain of Ras-like G proteins, was a crucial interaction site for PLD. Mutations in conserved amino acids (Tyr(34), Thr(37), Phe(39)) totally abolished PLD activation, while mutations in Val(38) or Tyr(42) caused partial loss. Two additional sites were responsible for the differential PLD activation ability between RhoA and Cdc42Hs. Changing Asp(76) in the switch II region of RhoA to the corresponding amino acid in Cdc42Hs led to partial loss of PLD activation. A chimeric protein with the N-terminal third of Cdc42Hs changed to RhoA showed enhanced PLD activation. Analysis of other Rho/Ha-Ras chimeric proteins and mutations indicated that Gln(52) adjacent to the switch II region is responsible for this gain of function.In conclusion, the present study shows that conserved amino acids in the switch I region of RhoA are major PLD interaction sites and that residues in the switch II and internal regions are responsible for the differential activation of PLD by RhoA and Cdc42Hs.