Banerjee Jayashree, Fischer Christopher C, Wedegaertner Philip B
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Biochemistry. 2009 Aug 25;48(33):8032-43. doi: 10.1021/bi9010013.
PDZ-RhoGEF is a member of the regulator family of G protein signaling (RGS) domain-containing RhoGEFs (RGS-RhoGEFs) that link activated heterotrimeric G protein alpha subunits of the G12 family to activation of the small GTPase RhoA. Unique among the RGS-RhoGEFs, PDZ-RhoGEF contains a short sequence that localizes the protein to the actin cytoskeleton. In this report, we demonstrate that the actin-binding domain, located between amino acids 561 and 585, directly binds to F-actin in vitro. Extensive mutagenesis identifies isoleucine 568, isoleucine 569, phenylalanine 572, and glutamic acid 573 as being necessary for binding to actin and for colocalization with the actin cytoskeleton in cells. These results define a novel actin-binding sequence in PDZ-RhoGEF with a critical amino acid motif of IIxxFE. Moreover, sequence analysis identifies a similar actin-binding motif in the N-terminus of the RhoGEF frabin, and as with PDZ-RhoGEF, mutagenesis and actin interaction experiments demonstrate an LIxxFE motif, consisting of the key amino acids leucine 23, isoleucine 24, phenylalanine 27, and glutamic acid 28. Taken together, results with PDZ-RhoGEF and frabin identify a novel actin-binding sequence. Lastly, inducible dimerization of the actin-binding region of PDZ-RhoGEF revealed a dimerization-dependent actin bundling activity in vitro. PDZ-RhoGEF exists in cells as a dimer, raising the possibility that PDZ-RhoGEF could influence actin structure in a manner independent of its ability to activate RhoA.
PDZ-RhoGEF是含G蛋白信号调节（RGS）结构域的Rho鸟苷酸交换因子（RGS-RhoGEFs）调节家族的成员，它将G12家族的活化异三聚体G蛋白α亚基与小GTP酶RhoA的活化联系起来。在RGS-RhoGEFs中，PDZ-RhoGEF独一无二，它含有一段能将该蛋白定位于肌动蛋白细胞骨架的短序列。在本报告中，我们证明位于氨基酸561和585之间的肌动蛋白结合结构域在体外直接与F-肌动蛋白结合。广泛的诱变确定异亮氨酸568、异亮氨酸569、苯丙氨酸572和谷氨酸573是与肌动蛋白结合以及在细胞中与肌动蛋白细胞骨架共定位所必需的。这些结果确定了PDZ-RhoGEF中一个新的肌动蛋白结合序列，其关键氨基酸基序为IIxxFE。此外，序列分析在Rho鸟苷酸交换因子frabin的N端鉴定出一个相似的肌动蛋白结合基序，与PDZ-RhoGEF一样，诱变和肌动蛋白相互作用实验证明了一个由关键氨基酸亮氨酸23、异亮氨酸24、苯丙氨酸27和谷氨酸28组成的LIxxFE基序。综合起来，PDZ-RhoGEF和frabin的结果确定了一个新的肌动蛋白结合序列。最后，PDZ-RhoGEF肌动蛋白结合区域的诱导二聚化在体外揭示了一种依赖二聚化的肌动蛋白成束活性。PDZ-RhoGEF在细胞中以二聚体形式存在，这增加了PDZ-RhoGEF可能以独立于其激活RhoA能力的方式影响肌动蛋白结构的可能性。
