Zhang J H, Barr V A, Mo Y, Rojkova A M, Liu S, Simonds W F
Metabolic Diseases Branch and the Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2001 Mar 30;276(13):10284-9. doi: 10.1074/jbc.M009247200. Epub 2001 Jan 4.
The role that Gbeta(5) regulator of G protein signaling (RGS) complexes play in signal transduction in brain remains unknown. The subcellular localization of Gbeta(5) and RGS7 was examined in rat PC12 pheochromocytoma cells and mouse brain. Both nuclear and cytosolic localization of Gbeta(5) and RGS7 was evident in PC12 cells by immunocytochemical staining. Subcellular fractionation of PC12 cells demonstrated Gbeta(5) immunoreactivity in the membrane, cytosolic, and nuclear fractions. Analysis by limited proteolysis confirmed the identity of Gbeta(5) in the nuclear fraction. Subcellular fractionation of mouse brain demonstrated Gbeta(5) and RGS7 but not Ggamma(2/3) immunoreactivity in the nuclear fraction. RGS7 and Gbeta(5) were tightly complexed in the brain nuclear extract as evidenced by their coimmunoprecipitation with anti-RGS7 antibodies. Chimeric protein constructs containing green fluorescent protein fused to wild-type Gbeta(5) but not green fluorescent fusion proteins with Gbeta(1) or a mutant Gbeta(5) impaired in its ability to bind to RGS7 demonstrated nuclear localization in transfected PC12 cells. These findings suggest that Gbeta(5) undergoes nuclear translocation in neurons via an RGS-dependent mechanism. The novel intracellular distribution of Gbeta(5).RGS protein complexes suggests a potential role in neurons communicating between classical heterotrimeric G protein subunits and/or their effectors at the plasma membrane and the cell nucleus.
G蛋白信号调节因子(RGS)复合物中的Gβ(5)在大脑信号转导中所起的作用尚不清楚。我们在大鼠嗜铬细胞瘤PC12细胞和小鼠大脑中检测了Gβ(5)和RGS7的亚细胞定位。通过免疫细胞化学染色发现,PC12细胞中Gβ(5)和RGS7在细胞核和细胞质中均有明显定位。PC12细胞的亚细胞分级分离显示,Gβ(5)免疫反应性存在于膜、细胞质和细胞核分级分离物中。有限蛋白酶解分析证实了细胞核分级分离物中Gβ(5)的身份。小鼠大脑的亚细胞分级分离显示,细胞核分级分离物中有Gβ(5)和RGS7的免疫反应性,但没有Gγ(2/3)的免疫反应性。RGS7和Gβ(5)在大脑核提取物中紧密结合,这可通过它们与抗RGS7抗体的共免疫沉淀来证明。含有与野生型Gβ(5)融合的绿色荧光蛋白的嵌合蛋白构建体,而不是与Gβ(1)或与RGS7结合能力受损的突变型Gβ(5)的绿色荧光融合蛋白,在转染的PC12细胞中显示出核定位。这些发现表明,Gβ(5)通过一种RGS依赖的机制在神经元中发生核转位。Gβ(5).RGS蛋白复合物新的细胞内分布表明,其在神经元中可能在经典异源三聚体G蛋白亚基和/或其在质膜和细胞核的效应器之间进行通讯方面发挥作用。