Ma Hongzheng, Peterson Yuri K, Bernard Michael L, Lanier Stephen M, Graber Stephen G
Department of Biochemistry and Molecular Pharmacology, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9142, USA.
Biochemistry. 2003 Jul 8;42(26):8085-93. doi: 10.1021/bi034561p.
Activator of G protein signaling 3 (AGS3) activates the Gbetagamma mating pathway in yeast in a manner that is independent of heptahelical receptors. It competes with Gbetagamma subunits to bind GDP-bound Gi/o(alpha) subunits via four repeated G protein regulatory (GPR) domains in the carboxyl-terminal half of the molecule. However, little is known about the functional role of AGS3 in cellular signaling. Here the effect of AGS3 on receptor-G protein coupling was examined in an Sf9 cell membrane-based reconstitution system. A GST-AGS3-GPR fusion protein containing the four individual AGS3-GPR domains inhibits receptor coupling to Galpha subunits as effectively as native AGS3 and more effectively than GST fusion proteins containing the individual AGS3-GPR domains. While none of the GPR domains distinguished among the three G(i)alpha subunits, both individual and full-length GPR domains interacted more weakly with G(o)alpha than with G(i)alpha. Cytosolic AGS3, but not membrane-associated AGS3, can interact with G(i)alpha subunits and disrupt their receptor coupling. Immunoblotting studies reveal that cytosolic AGS3 can remove G(i)alpha subunits from the membrane and sequester G(i)alpha subunits in the cytosol. These findings suggest that AGS3 may downregulate heterotrimeric G protein signaling by interfering with receptor coupling.
G蛋白信号转导激活因子3（AGS3）以一种独立于七螺旋受体的方式激活酵母中的Gβγ交配途径。它通过分子羧基末端一半的四个重复的G蛋白调节（GPR）结构域与Gβγ亚基竞争，以结合与GDP结合的Gi/o（α）亚基。然而，关于AGS3在细胞信号传导中的功能作用知之甚少。在此，在基于Sf9细胞膜的重组系统中研究了AGS3对受体-G蛋白偶联的影响。含有四个单独的AGS3-GPR结构域的GST-AGS3-GPR融合蛋白抑制受体与Gα亚基的偶联，其效果与天然AGS3一样有效，且比含有单个AGS3-GPR结构域的GST融合蛋白更有效。虽然没有一个GPR结构域能区分三种Giα亚基，但单个和全长GPR结构域与Goα的相互作用比与Giα的相互作用更弱。胞质中的AGS3而非膜相关的AGS3能与Giα亚基相互作用并破坏它们的受体偶联。免疫印迹研究表明，胞质中的AGS3能将Giα亚基从膜上移除并将Giα亚基隔离在胞质中。这些发现表明，AGS3可能通过干扰受体偶联来下调异源三聚体G蛋白信号传导。
