Platko J V, Leonard D A, Adra C N, Shaw R J, Cerione R A, Lim B
Department of Pharmacology, Cornell University, Ithaca, NY 14850, USA.
Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2974-8. doi: 10.1073/pnas.92.7.2974.
The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins for the Rho- and Rab-subtype GTP-binding proteins. As a first step toward identifying the key functional domain(s) on the Rho-subtype GDI, truncations of the amino and carboxyl termini were performed. Deletion of the final four amino acids from the carboxyl terminus of Rho GDI or the removal of 25 amino acids from the amino terminus had no significant effect on the ability of the GDI to inhibit GDP dissociation from the Rho-like protein Cdc42Hs or on its ability to release Cdc42Hs from membrane bilayers. However, the deletion of 8 amino acids from the carboxyl terminus of Rho GDI eliminated both activities. To further test the importance of the carboxyl-terminal domain of the Rho GDI molecule, chimeras were constructed between this GDI and a related protein designated LD4, which is 67% identical to Rho GDI but is less potent by a factor of 10-20 than Rho GDI in functional assays with the Cdc42Hs protein. Two sets of chimeras were constructed that together indicated that as few as 6 amino acids near the carboxyl terminus of Rho GDI could impart full GDP dissociation inhibition and membrane dissociation activities on the LD4 molecule. Further analysis of this region by site-directed mutagenesis showed that a single change at residue 174 of LD4 to the corresponding residue of Rho GDI (i.e., Asp-174-->Ile) could impart nearly full (70%) Rho GDI activity on the LD4 molecule.
GDP解离抑制剂（GDIs）是Rho和Rab亚型GTP结合蛋白的一类重要调节蛋白。作为鉴定Rho亚型GDI上关键功能域的第一步，对其氨基末端和羧基末端进行了截短。从Rho GDI的羧基末端删除最后四个氨基酸或从氨基末端去除25个氨基酸，对GDI抑制GDP从Rho样蛋白Cdc42Hs解离的能力或其从膜双层释放Cdc42Hs的能力没有显著影响。然而，从Rho GDI的羧基末端删除8个氨基酸则消除了这两种活性。为了进一步测试Rho GDI分子羧基末端结构域的重要性，构建了该GDI与一种名为LD4的相关蛋白之间的嵌合体，LD4与Rho GDI有67%的同源性，但在与Cdc42Hs蛋白的功能测定中，其效力比Rho GDI低10 - 20倍。构建了两组嵌合体，结果共同表明，Rho GDI羧基末端附近少至6个氨基酸就能赋予LD4分子完全的GDP解离抑制和膜解离活性。通过定点诱变对该区域进行进一步分析表明，将LD4第174位残基的单个氨基酸替换为Rho GDI的相应残基（即Asp - 174→Ile），可使LD4分子具有近完全（70%）的Rho GDI活性。
