Natochin M, Lester B, Peterson Y K, Bernard M L, Lanier S M, Artemyev N O
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
J Biol Chem. 2000 Dec 29;275(52):40981-5. doi: 10.1074/jbc.M006478200.
A number of recently discovered proteins that interact with the alpha subunits of G(i)-like G proteins contain homologous repeated sequences named G protein regulatory (GPR) motifs. Activator of G protein signaling 3 (AGS3), identified as an activator of the yeast pheromone pathway in the absence of the pheromone receptor, has a domain with four such repeats. To elucidate the potential mechanisms of regulation of G protein signaling by proteins containing GPR motifs, we examined the effects of the AGS3 GPR domain on the kinetics of guanine nucleotide exchange and GTP hydrolysis by G(i)alpha(1) and transducin-alpha (G(t)alpha). The AGS3 GPR domain markedly inhibited the rates of spontaneous guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to G(i)alpha and rhodopsin-stimulated GTPgammaS binding to G(t)alpha. The full-length AGS3 GPR domain, AGS3-(463-650), was approximately 30-fold more potent than AGS3-(572-629), containing two AGS3 GPR motifs. The IC(50) values for the AGS3-(463-650) inhibitory effects on G(i)alpha and transducin were 0.12 and 0.15 microm, respectively. Furthermore, AGS3-(463-650) and AGS3-(572-629) effectively blocked the GDP release from G(i)alpha and rhodopsin-induced dissociation of GDP from G(t)alpha. The potencies of AGS3-(572-629) and AGS3-(463-650) to suppress the GDP dissociation rates correlated with their ability to inhibit the rates of GTPgammaS binding. Consistent with the inhibition of nucleotide exchange, the AGS3 GPR domain slowed the rate of steady-state GTP hydrolysis by G(i)alpha. The catalytic rate of G(t)alpha GTP hydrolysis, measured under single turnover conditions, remained unchanged with the addition of AGS3-(463-650). Altogether, our results suggest that proteins containing GPR motifs, in addition to their potential role as G protein-coupled receptor-independent activators of Gbetagamma signaling pathways, act as GDP dissociation inhibitors and negatively regulate the activation of a G protein by a G protein-coupled receptor.
最近发现的一些与G(i)样G蛋白的α亚基相互作用的蛋白质含有名为G蛋白调节（GPR）基序的同源重复序列。G蛋白信号激活因子3（AGS3）在没有信息素受体的情况下被鉴定为酵母信息素途径的激活剂，它有一个含有四个此类重复序列的结构域。为了阐明含GPR基序的蛋白质对G蛋白信号调节的潜在机制，我们研究了AGS3 GPR结构域对G(i)α(1)和转导蛋白α（G(t)α）的鸟嘌呤核苷酸交换动力学和GTP水解的影响。AGS3 GPR结构域显著抑制了鸟苷5'-O-(3-硫代三磷酸)（GTPγS）与G(i)α的自发结合速率以及视紫红质刺激的GTPγS与G(t)α的结合速率。全长AGS3 GPR结构域AGS3-(463-650)的效力比含有两个AGS3 GPR基序的AGS3-(572-629)强约30倍。AGS3-(463-650)对G(i)α和转导蛋白的抑制作用的IC(50)值分别为0.12和0.15微摩尔。此外，AGS3-(463-650)和AGS3-(572-629)有效地阻断了GDP从G(i)α的释放以及视紫红质诱导的GDP从G(t)α的解离。AGS3-(572-629)和AGS3-(463-650)抑制GDP解离速率的效力与其抑制GTPγS结合速率的能力相关。与核苷酸交换的抑制一致，AGS3 GPR结构域减慢了G(i)α的稳态GTP水解速率。在单周转条件下测量的G(t)α GTP水解的催化速率在添加AGS3-(463-650)后保持不变。总之，我们的结果表明，含GPR基序的蛋白质除了作为G蛋白偶联受体非依赖性Gβγ信号通路激活剂的潜在作用外，还作为GDP解离抑制剂，并对G蛋白偶联受体激活G蛋白起负调节作用。
