Shome K, Vasudevan C, Romero G
Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Curr Biol. 1997 Jun 1;7(6):387-96. doi: 10.1016/s0960-9822(06)00186-2.
ADP-ribosylation factors (ARFs) have been shown to activate phospholipase D (PLD), an enzyme modulated by extracellular signals, including several growth factors and, in particular, insulin. We have tested the hypothesis that ARF proteins are involved specifically in insulin-induced activation of PLD.
We found that in membranes obtained from HIRcB cells, a cell line derived from Rat-1 fibroblasts that overexpresses normal human insulin receptors, binding of the GTP analogue GTPgammaS to purified bovine or recombinant ARF was enhanced in the presence of insulin. Membranes obtained from cells that overexpressed a mutated, nonfunctional insulin receptor failed to stimulate ARF activation. Insulin promoted the association of ARF proteins with membranes in the presence of GTPgammaS in permeabilized cells. Insulin activated PLD in permeabilized HIRcB cells by a process that required GTPgammaS and ARF. Azido-gamma[32P]-GTP labelling of immunoprecipitated receptors revealed the presence of a unique 19 kD band; ARF proteins are approximately this size, and analysis using specific monoclonal antibodies demonstrated that ARF proteins coimmunoprecipitated with the insulin receptor. Coimmunoprecipitation of ARF with the receptor was inhibited by guanine nucleotides and stimulated by insulin. No evidence of the coprecipitation of ARF with mutant receptors could be obtained using azido-gamma[32P]-GTP or anti-ARF antibodies.
The activation of ARF proteins is stimulated by insulin and this process plays an important role in insulin-mediated regulation of PLD.
ADP-核糖基化因子（ARFs）已被证明可激活磷脂酶D（PLD），PLD是一种受细胞外信号调节的酶，这些信号包括多种生长因子，尤其是胰岛素。我们检验了ARF蛋白特异性参与胰岛素诱导的PLD激活这一假说。
我们发现，在从HIRcB细胞获得的膜中，HIRcB细胞系源自过表达正常人胰岛素受体的大鼠-1成纤维细胞，在胰岛素存在的情况下，GTP类似物GTPγS与纯化的牛或重组ARF的结合增强。从过表达突变的、无功能胰岛素受体的细胞获得的膜未能刺激ARF激活。在通透细胞中，胰岛素在GTPγS存在的情况下促进ARF蛋白与膜的结合。胰岛素通过一个需要GTPγS和ARF的过程激活通透的HIRcB细胞中的PLD。免疫沉淀受体的叠氮基-γ[32P]-GTP标记显示存在一条独特的19 kD条带；ARF蛋白大小与此相近，使用特异性单克隆抗体进行的分析表明ARF蛋白与胰岛素受体共免疫沉淀。ARF与受体的共免疫沉淀受到鸟嘌呤核苷酸的抑制，并受到胰岛素的刺激。使用叠氮基-γ[32P]-GTP或抗ARF抗体均未获得ARF与突变受体共沉淀的证据。
胰岛素刺激ARF蛋白的激活，这一过程在胰岛素介导的PLD调节中起重要作用。
