Majumdar Sharmistha, Ramachandran Sekar, Cerione Richard A
Department of Chemistry and Chemical Biology.
J Biol Chem. 2006 Apr 7;281(14):9219-26. doi: 10.1074/jbc.M513837200. Epub 2006 Feb 9.
The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.
GTP水解（GTP酶）反应通过大型（异源三聚体）和小型（Ras相关）GTP结合蛋白（G蛋白）终止信号传导。GTP酶活性所需的两个残基是一个精氨酸（通常称为“精氨酸指”），它存在于大型G蛋白α亚基的开关I结构域中，或者由小型G蛋白的GTP酶激活蛋白提供，以及一个在Gα亚基和小型G蛋白的开关II结构域中高度保守的谷氨酰胺。然而，关于GTP酶反应的机制以及开关II谷氨酰胺所起的确切作用仍然存在问题。在这里，我们对突变体的GTP结合和GTP酶活性进行了表征，在一个主要由视网膜转导素α亚基（α(T)）和G(i1)α亚基的开关III区域组成的Gα嵌合体（命名为α(T)）背景下，必需的精氨酸或谷氨酰胺残基已被改变。正如预期的那样，α(T)(R174C)和α(T)*(Q200L)突变体的GTP酶活性都严重受损。在监测开关II中Trp - 207荧光变化时，这两个突变体都不能对氟化铝作出反应，尽管在没有视紫红质和Gβγ的情况下它们都能刺激效应器活性。令人惊讶的是，每个突变体还显示出一些被视紫红质和Gβγ激活以进行GDP - [(35)S]GTPγS交换的能力。突变体与视紫红质偶联的能力与它们只含有结合的GTP这一假设不一致，这促使我们在从大肠杆菌中表达和纯化后检查它们的核苷酸结合状态。事实上，这两个突变体都含有结合的GDP以及GTP，每个突变体中有35 - 45%被分离为GDP - P(i)复合物。总体而言，这些发现表明R174C和Q200L突变揭示了Gα亚基在GTP水解后出现但仍能充分刺激效应器活性的状态。
