Inoue S, Hoshino S, Kukimoto I, Ui M, Katada T
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo.
J Biochem. 1995 Sep;118(3):650-7. doi: 10.1093/oxfordjournals.jbchem.a124959.
We expressed the Gly203-->Thr (G203T) mutant of Gi2alpha, which was expected to show a dominant-negative phenotype in Gi2-mediated signal transduction, in baculovirus-inefected Sf9 cells and purified the mutant alpha subunit for its characterization. The rate of dissociation of GDP from G203T Gi2alpha was 3- to 4-fold faster than that from wild type Gi2alpha, but their kappacat values for GTP hydrolysis were almost the same. The affinities of the two Gi2alpha proteins for the beta gamma subunits of G proteins to form alpha beta gamma timers, which served as substrates for pertussis toxin-catalyzed ADP-ribosylation, were the same. In marked contrast, G203T Gi2alpha was unable to form a tight complex with a non- hydrolyzable analog (GTP[gammaS) of GTP; bound GTP[gammaS] was readily released from the mutant Gi2alpha even in the presence of a high concentration of Mg2+. Its susceptibility to tryptic digestion also revealed that GTP[gammaS]-bound G203T Gi2alpha formed a conformation apparently different from that of the GTP[gammaS]-bound form of wild-type Gi2alpha. Both the G203T and wild-type Gi2alpha proteins were capable of coupling with membrane-bound alpha2-adrenergic receptors, resulting in the formation of receptor-G protein complexes with high affinity for agonists. However, GTP[gammaS]-dependent uncoupling from high-affinity receptors was markedly attenuated in the case of G203T Gi2alpha. Thus, G203T-mutated Gi2alpha had a unique property in terms of coupling to membrane receptors, in addition to the previously expected defect in the active conformation of the GTP-bound form of Gi2alpha.
我们在杆状病毒感染的Sf9细胞中表达了Gi2α的Gly203→Thr（G203T）突变体，预期该突变体在Gi2介导的信号转导中表现出显性负性表型，并纯化了该突变体α亚基以进行表征。G203T Gi2α中GDP的解离速率比野生型Gi2α快3至4倍，但其GTP水解的kcat值几乎相同。两种Gi2α蛋白与G蛋白的βγ亚基形成αβγ三聚体（百日咳毒素催化的ADP核糖基化的底物）的亲和力相同。形成鲜明对比的是，G203T Gi2α无法与GTP的非水解类似物（GTP[γS]）形成紧密复合物；即使在高浓度Mg2+存在的情况下，结合的GTP[γS]也很容易从突变体Gi2α中释放出来。其对胰蛋白酶消化的敏感性还表明，结合GTP[γS]的G203T Gi2α形成的构象明显不同于结合GTP[γS]的野生型Gi2α。G203T和野生型Gi2α蛋白都能够与膜结合的α2肾上腺素能受体偶联，从而形成对激动剂具有高亲和力的受体 - G蛋白复合物。然而，在G203T Gi2α的情况下，GTP[γS]依赖性从高亲和力受体的解偶联明显减弱。因此，除了先前预期的Gi2α的GTP结合形式的活性构象缺陷外，G203T突变的Gi2α在与膜受体偶联方面具有独特的性质。
