Li Hai-Sheng, Shome Kuntala, Rojas Raúl, Rizzo Megan A, Vasudevan Chandrasekaran, Fluharty Eric, Santy Lorraine C, Casanova James E, Romero Guillermo
Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
BMC Cell Biol. 2003 Sep 11;4:13. doi: 10.1186/1471-2121-4-13.
Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.
Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: DeltaCC-ARNO and CC-ARNO were partially translocated to the membranes while DeltaPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin.
Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor.
磷脂酶D（PLD）参与多种信号通路。在大多数系统中，PLD的活性主要受GTP酶的ADP核糖基化因子（ARF）家族成员调控，但细胞外信号激活PLD和ARF的机制尚未完全明确。在此，我们验证了细胞粘附素/ARNO家族的ARF-鸟嘌呤核苷酸交换因子（ARF-GEFs）介导胰岛素对ARF和PLD激活作用的假说。
在HIRcB细胞中瞬时转染的野生型ARNO以胰岛素依赖的方式转位至质膜，并促进ARF转位至膜上。ARNO突变体：DeltaCC-ARNO和CC-ARNO部分转位至膜上，而DeltaPH-ARNO和PH-ARNO不能转位至膜上。ARNO的Sec7结构域突变体不促进ARF转位。野生型ARNO的过表达显著增加胰岛素刺激的PLD活性，Sec7和PH结构域的突变或PH或CC结构域的缺失抑制胰岛素的作用。
细胞粘附素/ARNO家族的小ARF-GEFs介导胰岛素受体对ARF和PLD的激活。
