Chatterjee T K, Fisher R A
Department of Pharmacology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
J Biol Chem. 2000 Aug 4;275(31):24013-21. doi: 10.1074/jbc.M002082200.
RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Galpha subunits, thereby accelerating the turn-off mechanism of Galpha and terminating signaling by both Galpha and Gbetagamma subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned.
RGS蛋白构成了一个因其对异源三聚体G蛋白信号传导具有负调控能力而得名的蛋白家族。生化研究表明，该蛋白家族的成员作为某些Gα亚基的GTP酶激活蛋白，从而加速Gα的关闭机制，并终止Gα和Gβγ亚基的信号传导。在本研究中，我们使用共聚焦显微镜检查了几种RGS蛋白在表达RGS-绿色荧光蛋白（GFP）融合蛋白的COS-7细胞以及内源性表达RGS蛋白的细胞中的细胞内分布。RGS2和RGS10在转染了这些蛋白的GFP构建体的COS-7细胞核中积累。相反，RGS4和RGS16在COS-7转染细胞的细胞质中积累。正如在COS-7细胞中观察到的那样，RGS4在小鼠神经母细胞瘤细胞中表现出细胞质定位，而RGS10在人胶质瘤细胞中表现出核定位。RGS4和RGS16中存在的N端亮氨酸重复基序（在HIV Rev和其他蛋白中被鉴定为核输出序列的结构域）的缺失或丙氨酸替代促进了这些蛋白在COS-7细胞中的核定位。与该观察结果一致，用雷帕霉素B处理小鼠神经母细胞瘤细胞以抑制输出蛋白1介导的核蛋白输出导致RGS4在这些细胞核中积累。定位于细胞核的RGS蛋白的RGS结构域的GFP融合蛋白表明，RGS蛋白的核定位是通过RGS结构域序列进行核靶向的结果。RGSZ在其N端区域与RGS-GAIP共享一个富含半胱氨酸的序列，在COS-7细胞中定位于高尔基体复合体。缺失包含半胱氨酸基序的RGSZ的N端结构域促进了RGSZ的核定位。所检测的RGS蛋白均未定位于质膜。这些结果表明，RGS蛋白定位于细胞核、细胞质，或作为核质穿梭蛋白在细胞核和细胞质之间穿梭。由于这些蛋白之间的结构差异，RGS蛋白在细胞内的定位有所不同，而这些差异似乎并非其G蛋白调节活性的重要决定因素。这些发现表明，RGS蛋白参与了比目前设想更为复杂的细胞功能。
