ARF PROTEIN IN INSULIN SIGNALING

磷脂酶D （PLD）被外界信号激活是一个有据可查的事实。我们和其他人已经表明，受体诱导的PLD激活是由小G蛋白介导的，其机制对不同的受体和细胞类型具有相对特异性和不同。特别是，在本研究项目的初始资助阶段，我们已经证明ARF家族的小G蛋白对于HIRcB细胞中胰岛素和PDGF激活PLD以及A10血管平滑细胞系中PDGF和内皮素-1激活PLD至关重要。然而，胰岛素和其他细胞外信号调控ARF蛋白激活的机制尚不清楚。由于存在许多RF蛋白（ARF1-ARF6）、ARF-GDP交换因子（ARF-GEF，即ARNO、cytohesin-1、GRP1、Gea等）和PLD形式（PLD1和PLD2），这一问题尤其复杂，它们表明PLD激活的平行冗多途径。在之前的资助期间，我们已经开始研究胰岛素和其他生长因子可能调节ARF激活状态的机制。我们发现ARF-GEF以胰岛素依赖的方式与胰岛素受体共免疫沉淀。另外的实验表明，ARF-GEF被称为ARNO与胰岛素受体相关。我们拟研究arf - gef的ARNO和cytohesin-1被细胞表面受体激活的机制，并确定这些gef的激活在胰岛素、PDGF和磷酯对PLD的调节中所起的作用。我们建议通过生物化学、分子和成像方法的结合来做到这一点。我们将专注于以下研究：a)通过对ARF- gef的Sec7和pleckstrin同源结构域以及ARF的Switch 1区域的特定残基的突变分析，分析ARNO和cytohesin-1在受体介导的ARF和PLD激活中的作用；b)通过检测ARNO和cytohesin-1的磷酸化状态、它们与细胞表面受体的相互作用以及它们被磷酸肌苷募集来确定它们的调节机制；c)确定PLD1和PLD2在受体依赖性磷脂酶D活性中的相对作用。

The activation of phospholipase D (PLD) by external signals is a well- documented fact. We and others have shown that receptor-induced PLD activation is mediated by small G proteins by mechanisms that are relatively specific and distinct for different receptors and cell types. In particular, during the initial funding period of this research project, we have shown that small G proteins of the ARF family are essential for the activation of PLD by insulin and PDGF in HIRcB cells and for the activation of PLD by PDGF and endothelin-1 in the A10 vascular smooth cell line. However, the mechanisms by which the activation of ARF proteins might be regulated by insulin and other extracellular signals remains obscure. This issue is particularly complicated by the existence of many RF proteins (ARF1-ARF6), ARF-GDP exchange factors (ARF-GEF; namely ARNO, cytohesin-1, GRP1, Gea and others) and PLD forms (PLD1 and PLD2) which suggest parallel redundant pathways for the activation of PLD. During the preceding funding period we have started studying the mechanisms by which insulin and other growth factors may regulate the state of activation of ARF. We showed that on ARF-GEF co-immunoprecipitated with the insulin receptor in an insulin-dependent manner. Additional experiments suggest the ARF-GEF known as ARNO associates to the insulin receptor. We propose to study the mechanisms by which the ARF-GEFs ARNO and cytohesin-1 are activated by cell surface receptors and to determine the role that the activation of these GEFs plays in the regulation of PLD by insulin, PDGF and phorbol esters. We propose to do this by a combination of biochemical, molecular and imaging approaches. We will concentrate on the following studies: a) analysis of the role of ARNO and cytohesin-1 on receptor-mediated ARF and PLD activation, by mutational analysis of the Sec7 and pleckstrin-homology domain of the ARF-GEFs and of specific residues of the Switch 1 region of ARF; b) determining the mechanisms of regulation of ARNO and cytohesin-1 by examination of their state of phosphorylation, their interaction with cell surface receptors and their recruitment by phosphoinositides: and c) determining the relative role of PLD1 and PLD2 in receptor-dependent phospholipase D activity.