Siebler T, Kiess W, Linder B, Kessler U, Schwarz H P, Nissley S P
Children's Hospital, Ludwig Maximilians University of Munich, Germany.
Regul Pept. 1996 Apr 23;62(2-3):65-71. doi: 10.1016/0167-0115(95)00159-x.
There is controversy as to whether or not a pertussis toxin sensitive G-protein is involved in the signaling pathway of insulin-like growth factor-I. We have used normal rat kidney epithelial (NRKE) cells to ask whether or not a pertussis toxin sensitive G-protein was involved in IGF-I stimulated DNA synthesis. NRKE cells express both IGF and IGF-II/M6P receptors and respond to IGF-I with increased thymidine incorporation into DNA. Under many circumstances incubation of cells/cell membranes with GTP analogues will inhibit binding of ligands that are linked to a G-protein-receptor pathway. However, when NRKE membrane preparations were incubated with 125I-IGF-I or 125I-IGF-II in the presence or absence of GTP gamma S, ATP and GTP, binding of the radioligands was not affected by the GTP-analogue. IGF-I and factors from serum of hypophysectomized rats (HRS) stimulated [3H]thymidine incorporation into DNA of NRKE cells. Under serum-free conditions in the presence of EGF (2 ng/ml) and PDGF (1 ng/ml) pertussis toxin over a wide range of doses had no effect upon IGF-I stimulated [3H]thymidine incorporation into DNA of NRKE cells. In addition, PT at a dose of 100 ng/ml had no effect on IGF-I(0.2-50 ng/ml) stimulated DNA synthesis of NRKE cells. However, PT at doses of 5, 50, 500, 5000 and 50,000 ng/ml was capable to ADP-ribosylate a 40 kDa protein in NRKE cell plasma membrane preparations corresponding to known PT-sensitive G-proteins. We conclude, that (1) PT-sensitive G-proteins and both IGF-I and IGF-II/M6P receptors are present in NRKE cell plasma membrane preparations, and most importantly, that (2) PT-sensitive G-proteins are not involved in the mitogenic signaling pathway of IGF-I in NRKE cells.
关于百日咳毒素敏感的G蛋白是否参与胰岛素样生长因子-I的信号通路存在争议。我们使用正常大鼠肾上皮（NRKE）细胞来研究百日咳毒素敏感的G蛋白是否参与胰岛素样生长因子-I刺激的DNA合成。NRKE细胞同时表达胰岛素样生长因子和胰岛素样生长因子-II/M6P受体，并对胰岛素样生长因子-I作出反应，使胸苷掺入DNA的量增加。在许多情况下，用GTP类似物孵育细胞/细胞膜会抑制与G蛋白受体途径相关的配体结合。然而，当NRKE细胞膜制剂在有或无GTPγS、ATP和GTP的情况下与125I-胰岛素样生长因子-I或125I-胰岛素样生长因子-II孵育时，放射性配体的结合不受GTP类似物的影响。胰岛素样生长因子-I和来自垂体切除大鼠血清（HRS）的因子刺激NRKE细胞的[3H]胸苷掺入DNA。在无血清条件下，存在表皮生长因子（2 ng/ml）和血小板衍生生长因子（1 ng/ml）时，大范围剂量的百日咳毒素对胰岛素样生长因子-I刺激的NRKE细胞[3H]胸苷掺入DNA没有影响。此外，100 ng/ml剂量的百日咳毒素对胰岛素样生长因子-I（0.2 - 50 ng/ml）刺激的NRKE细胞DNA合成没有影响。然而，5、50、500、5000和50000 ng/ml剂量的百日咳毒素能够使NRKE细胞质膜制剂中的一种40 kDa蛋白进行ADP核糖基化，该蛋白与已知的百日咳毒素敏感G蛋白相对应。我们得出结论：（1）百日咳毒素敏感的G蛋白以及胰岛素样生长因子-I和胰岛素样生长因子-II/M6P受体都存在于NRKE细胞质膜制剂中，最重要的是，（2）百日咳毒素敏感的G蛋白不参与NRKE细胞中胰岛素样生长因子-I的促有丝分裂信号通路。
