Zhou J, Moroi K, Nishiyama M, Usui H, Seki N, Ishida J, Fukamizu A, Kimura S
Department of Biochemistry and Molecular Pharmacology, Chiba University Graduate School of Medicine, Japan.
Life Sci. 2001 Feb 16;68(13):1457-69. doi: 10.1016/s0024-3205(01)00939-0.
RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.
RGS蛋白（G蛋白信号调节剂）作为Gα亚基的GTP酶激活蛋白（GAPs），对G蛋白偶联受体信号传导起负调控作用。在本研究中，我们对RGS5及其N端（1-33）缺失突变体（deltaN-RGS5）的生化特性进行了表征。在GDP/AIF4-存在的情况下，RGS5与Gα（i1）、Gα（i2）、Gα（i3）、Gα（o）和Gα（q）结合，但不与Gα（s）和Gα13结合，并加速了Gα（i3）亚基的GTP水解催化速率。当在稳定表达血管紧张素（Ang）AT1a受体的293T细胞（AT1a-293T细胞）中表达时，RGS5抑制了Ang II和内皮素（ET）-1诱导的细胞内Ca2+瞬变。RGS5的作用具有浓度依赖性，浓度-反应关系曲线的斜率表明，RGS5量增加10倍会导致Ca2+信号减少约20-25%。此外，对三组具有不同AT1a受体表达水平的293T细胞进行的比较研究表明，在AT1a受体密度较低的293T细胞中，RGS5更有效地抑制了Ang II诱导的反应，这表明RGS蛋白抑制的程度反映了激动剂刺激受体后RGS蛋白与活化Gα亚基量的比例。当在AT1a-293T细胞中表达时，deltaN-RGS5几乎完全定位于胞质部分，并与存在于膜和胞质部分的RGS5一样有效地发挥抑制作用。对RGS5和deltaN-RGS5亚细胞定位与抑制作用之间关系的研究表明，RGS5的N端（1-33）在将该蛋白靶向到膜上起作用，但RGS5的N端区域对发挥活性不是必需的。
