Fernando K C, Gregory R B, Katsis F, Kemp B E, Barritt G J
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, G.P.O. Box 2100, Adelaide, South Australia, 5001, Australia.
Biochem J. 1997 Dec 1;328 ( Pt 2)(Pt 2):463-71. doi: 10.1042/bj3280463.
The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 microM intracellular) of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 microM intracellular) of GTP-S- or guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 microM) prevented the inhibition of Ca2+ inflow by GTP-S- and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4(-) (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5))3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.
利用新鲜分离的大鼠肝细胞和大鼠肝微粒体，研究了单体GTP结合调节蛋白在大鼠肝实质细胞中激活储存激活的质膜Ca2+通道以及从光滑内质网（SER）释放Ca2+的作用。低浓度（细胞内约130 microM）的鸟苷5'-[γ-硫代]三磷酸（GTP[S]）在无激动剂的情况下激活完整肝细胞中的Ca2+内流，而高浓度（细胞内约530 microM）的GTP-S或鸟苷5'-[βγ-亚氨基]三磷酸（p[NH]ppG）抑制内质网Ca2+-ATP酶（SERCA）活性抑制剂和血管加压素诱导的Ca2+内流。GTP（530 microM）可防止GTP-S和p[NH]ppG对Ca2+内流的抑制。布雷菲德菌素A和抑制ADP核糖基化因子（Arf）蛋白多种功能的人Arf-1-(2-17)肽，抑制SERCA抑制剂和血管加压素诱导的Ca2+内流，并改变SER释放Ca2+的模式。在与抑制其他系统中Arf蛋白功能的浓度相当的布雷菲德菌素A和Arf-1-(2-17)浓度下观察到了这些效应。琥珀酰化的Arf-1-(2-17)对Ca2+内流的影响可忽略不计。GTP[S]和Arf-1-(2-17)完全抑制了GTP和Ins(1,4,5)P3从负载45Ca2+的大鼠肝微粒体中释放45Ca2+的协同作用。AlF4(-)（在预期激活三聚体G蛋白的条件下）和琥珀酰化的Arf-1-(2-17)对GTP/Ins(1,4,5))3诱导的45Ca2+释放无影响，而一种马斯托帕兰类似物引起部分抑制。Arf-1-(2-17)不抑制毒胡萝卜素或离子霉素诱导的45Ca2+释放。结论是，一种低分子量G蛋白，很可能是Arf蛋白家族的成员，是大鼠肝细胞中储存激活的Ca2+内流所必需的。简要讨论了这种G蛋白的作用是将SER的一个区域维持在正确的细胞内位置的观点。
