Miller B A, Bell L, Hansen C A, Robishaw J D, Linder M E, Cheung J Y
Department of Pediatrics, The Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey 17033, USA.
J Clin Invest. 1996 Oct 15;98(8):1728-36. doi: 10.1172/JCI118971.
Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2',7'-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPgammaS or GDPbetaS, which maintain the alpha subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPgammaS. However, injection of GDPbetaS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, alpha or betagamma transducin subunits were purified and microinjected as a sink for betagamma or alpha subunits in the erythroblast, respectively. Transducin betagamma, but not alpha, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the alpha subunit. Microinjected antibodies to Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Gi(alpha)2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G-proteins in hematopoietic cells and show that Gi(alpha)2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.
促红细胞生成素可诱导人成红细胞中胞质钙剂量依赖性增加，该过程由一种电压非依赖性Ca2+通道介导。百日咳毒素对促红细胞生成素这种反应的抑制作用提示鸟嘌呤核苷酸结合调节蛋白（G蛋白）参与其中。本文通过向人红系前体细胞显微注射G蛋白调节剂或亚基，阐明了G蛋白在促红细胞生成素调节的Ca2+通道中的作用。这是关于利用显微注射研究正常前体细胞中促红细胞生成素信号转导的首篇报道。用Fura-2负载的第10天爆式形成单位-红系来源的成红细胞进行显微注射，并用数字视频成像测量细胞内游离钙浓度（[Ca(i)]）。显微注射液中加入了BCECF（1,2',7'-双（2-羧乙基）-5-（及-6-）-羧基荧光素），BCECF荧光增强是成功显微注射的证据。用GTP的非水解类似物GTPγS或GDPβS对细胞进行显微注射，它们分别使α亚基保持激活或失活状态。显微注射GTPγS后，[Ca(i)]以剂量依赖性方式显著增加。然而，注射GDPβS可阻断促红细胞生成素诱导的钙增加，这提供了直接证据表明需要激活G蛋白。为了阐明涉及哪些G蛋白亚基，分别纯化了α或βγ转导蛋白亚基并作为红细胞中βγ或α亚基的汇聚点进行显微注射。转导蛋白βγ亚基而非α亚基消除了对促红细胞生成素的钙反应，证明了α亚基的主要作用。显微注射针对Gi(α)2而非Gi(α)1或Gi(α)3的抗体可阻断促红细胞生成素刺激的[Ca(i)]升高，确定Gi(α)2为相关亚基。显微注射重组肉豆蔻酰化的Gi(α)2而非Gi(α)1或Gi(α)3亚基能够恢复百日咳毒素处理的成红细胞对促红细胞生成素的反应，这证实了上述结果。这些数据直接证明了G蛋白在造血细胞中的生理功能，并表明Gi(α)2是促红细胞生成素通过钙通道内流调节[Ca(i)]所必需的。
