The C-Terminus of the G Protein α Subunit Controls the Affinity of Nucleotides
The C-Terminus of the G Protein α Subunit Controls the Affinity of Nucleotides
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G 蛋白 α 亚基的 C 末端控制核苷酸的亲和力
DOI：
10.1021/bi201702d
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Yoshinori Shichida
中科院分区：
文献类型：
--
作者：
Naoki Kimata;Takahiro Yamashita;Take Matsuyama;Yasushi Imamoto;Yoshinori Shichida
G蛋白α亚基的C末端在决定与同源G蛋白偶联受体的选择性偶联中起着重要作用。事实上，视紫红质是一种典型的GPCR，它通过与G蛋白相互作用而表现出活性状态[变紫红质II(MII)]的稳定，而稳定的程度受Gα的C端序列的影响。在这里，我们考察了GOα具有不同长度的GOαC末端序列的GI突变体的eMII数量与激活效率之间的关系。结果表明，GI的激活效率和eMII的数量都受到突变的影响，但两者之间没有相关性。这一发现表明，G-α的C-末端区域不仅稳定了MII(活性状态)，而且还影响了G-α的核苷酸结合部位。因此，我们测量了这些突变体在不同浓度的GDP和GTP下的激活效率，并计算了GDP释放、GDP摄取和GTP摄取的速率常数。Gi突变体的这些速率常数与野生型有很大不同，表明C末端氨基酸残基的替换改变了核苷酸的亲和力。Gdp摄取和GTP摄取的速率常数显示出很强的相关性，表明Giα的C末端控制核苷酸结合位点的可及性。因此，我们的结果有力地表明，GIα的C末端与其核苷酸结合部位之间存在着长距离的互连。
The C-terminus of the G protein α subunit has a well-known role in determining the selective coupling with the cognate G protein-coupled receptor (GPCR). In fact, rhodopsin, a prototypical GPCR, exhibits active state [metarhodopsin II (MII)] stabilization by interacting with G protein [extra formation of MII (eMII)], and the extent of stabilization is affected by the C-terminal sequence of Gα. Here we examine the relationship between the amount of eMII and the activation efficiency of Gi mutants whose Giα forms have different lengths of the C-terminal sequence of Goα. The results show that both the activation efficiencies of Gi and the amounts of eMII were affected by mutations; however, there was no correlation between them. This finding suggested that the C-terminal region of Gα not only stabilizes MII (active state) but also affects the nucleotide-binding site of Gα. Therefore, we measured the activation efficiency of these mutants by MII at several concentrations of GDP and GTP and calculated the rate constants of GDP release, GDP uptake, and GTP uptake. These rate constants of the Gi mutants were substantially different from those of the wild type, indicating that the replacement of the amino acid residues in the C-terminus alters the affinity of nucleotides. The rate constants of GDP uptake and GTP uptake showed a strong correlation, suggesting that the C-terminus of Giα controls the accessibility of the nucleotide-binding site. Therefore, our results strongly suggest that there is a long-range interlink between the C-terminus of Giα and its nucleotide-binding site.