Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins
Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins
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DOI：
10.1074/jbc.m005291200
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发表时间：
2001-01-12
影响因子：
4.8
通讯作者：
Lanier, SM
中科院分区：
文献类型：
--
作者：
Bernard, ML;Peterson, YK;Lanier, SM
AGS3是在酵母功能筛选异源三聚体G蛋白的受体非依赖性激活物中分离得到的，作为确定AGS3在哺乳动物信号处理中作用的初步方法，我们定义了参与G蛋白相互作用的AGS3亚域，它对G蛋白的选择性，以及它对G蛋白激活状态的影响，用AGS3抗血清免疫印迹分析表明，AGS3在大鼠脑、神经元样细胞系PC12和NG108-15以及平滑肌细胞系DDT1-MF2中表达，免疫荧光研究和共聚焦成像表明AGS3主要分布在细胞微域中。AGS3与Gα(I3)从细胞和组织裂解物中免疫共沉淀，表明AGS3和Gα(1)的亚群在细胞内以复合体的形式存在。AGS3和Gα(I)的免疫共沉淀依赖于Gα(I3)的构象(GDP远大于GTP Gamma(5‘-3-O-(硫代)三磷酸))。AGS3中与Gα(I)结合的区域定位于其羧基末端(Pro(465)-Ser(650))上的4个氨基酸重复序列(G蛋白调节基序(GPR))，每个氨基酸重复序列都能与Gα(I)结合。AGS3-GPR结构域在组织和细胞裂解产物中选择性地与Gα(I)相互作用，并与纯化的Gα/G(Alphat)相互作用。随后对纯化的Gα(I2)和Gα(I3)进行的实验表明，含有四个GPR基序的羧基末端实际上同时结合了不止一个Gα(I)亚单位。AGS3GPR结构域有效地与GP Gamma竞争结合Gα(t(GDP))，并阻断GTP Gamma与Gα(I)的结合。AGS3及其相关蛋白为G蛋白信号通路的协调提供了意想不到的机制。
AGS3 (activator of G-protein Signaling 3) was isolated in a yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins, As an initial approach to define the role of AGS3 in mammalian signal processing, we defined the AGS3 subdomains involved in G-protein interaction, its selectivity for G-proteins, and its influence on the activation state of G-protein, Immunoblot analysis with AGS3 antisera indicated expression in rat brain, the neuronal-like cell lines PC12 and NG108-15, as well as the smooth muscle cell line DDT1-MF2, Immunofluorescence studies and confocal imaging indicated that AGS3 was predominantly cytoplasmic and enriched in microdomains of the cell. AGS3 coimmunoprecipitated with G alpha (i3) from cell and tissue lysates, indicating that a subpopulation of AGS3 and G alpha (1) exist as a complex in the cell. The coimmunoprecipitation of AGS3 and G alpha (i) was dependent upon the conformation of G alpha (i3) (GDP much greater than GTP gammaS (guanosine 5'-3-O-(thio)triphosphate)). The regions of AGS3 that bound G alpha (i) were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro(465)-Ser(650)), each of which were capable of binding G alpha (i). AGS3-GPR domains selectively interacted with G alpha (i) in tissue and cell lysates and with purified G alphai/G(alphat). Subsequent experiments with purified G alpha (i2) and G alpha (i3) indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one G alpha (i) subunit at the same time. The AGS3GPR domains effectively competed with GP gamma for binding to G alpha (t(GDP)) and blocked GTP gammaS binding to G alpha (i). AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways.