Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-6401, USA.
J Biol Chem. 2011 Apr 8;286(14):12702-11. doi: 10.1074/jbc.M110.166538. Epub 2011 Feb 1.
Residues comprising the guanine nucleotide-binding sites of the α subunits of heterotrimeric (large) G-proteins (Gα subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the phosphate-binding loop (P-loop) where both Gα subunits and Ras-related G-proteins have a conserved serine or threonine residue. Substitutions for this residue in Ras and related (small) G-proteins yield nucleotide-depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved serine residue in the P-loop to asparagine, within a chimeric Gα subunit (designated αT*) that is mainly comprised of the α subunit of the retinal G-protein transducin and a limited region from the α subunit of Gi1. The αT*(S43N) mutant exhibits a significantly higher rate of intrinsic GDP-GTP exchange compared with wild-type αT*, with light-activated rhodopsin (R*) causing only a moderate increase in the kinetics of nucleotide exchange on αT*(S43N). The αT*(S43N) mutant, when bound to either GDP or GTP, was able to significantly slow the rate of R*-catalyzed GDP-GTP exchange on wild-type αT*. Thus, GTP-bound αT*(S43N), as well as the GDP-bound mutant, is capable of forming a stable complex with R*. αT*(S43N) activated the cGMP phosphodiesterase (PDE) with a dose-response similar to wild-type αT*. Activation of the PDE by αT*(S43N) was unaffected if either R* or β1γ1 alone was present, whereas it was inhibited when R* and the β1γ1 subunit were added together. Overall, our studies suggest that the S43N substitution on αT* stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a GTP-bound Gα subunit, and the β1γ1 complex.
包含异源三聚体（大）G 蛋白（Gα 亚基）和 Ras 相关（小）G 蛋白的鸟嘌呤核苷酸结合位点的残基高度保守。这种情况尤其适用于磷酸结合环（P 环），其中 Gα 亚基和 Ras 相关 G 蛋白都具有保守的丝氨酸或苏氨酸残基。在 Ras 和相关（小）G 蛋白中，该残基的取代会产生核苷酸耗尽的显性负突变体。在这里，我们研究了将 P 环中保守的丝氨酸残基突变为天冬酰胺对嵌合 Gα 亚基（命名为 αT*）的影响，该亚基主要由视网膜 G 蛋白转导素的α 亚基和 Gi1 的α 亚基的有限区域组成。与野生型 αT相比，αT（S43N）突变体表现出更高的内在 GDP-GTP 交换速率，而光激活的视紫红质（R*）仅导致核苷酸交换动力学的适度增加在 αT*（S43N）上。当与 GDP 或 GTP 结合时，αT*（S43N）突变体能够显著降低野生型 αT催化的 R-依赖性 GDP-GTP 交换速率。因此，GTP 结合的 αT*（S43N），以及 GDP 结合的突变体，能够与 R形成稳定的复合物。αT（S43N）以类似于野生型 αT的剂量反应激活 cGMP 磷酸二酯酶（PDE）。如果仅存在 R或β1γ1，则 αT*（S43N）对 PDE 的激活不受影响，而当添加 R和β1γ1 亚基时，它受到抑制。总体而言，我们的研究表明，αT上的 S43N 取代稳定了 G 蛋白激活途径中的一个中间产物，该中间产物由激活的 G 蛋白偶联受体、GTP 结合的 Gα 亚基和β1γ1 复合物组成。
