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ARF PROTEIN IN INSULIN SIGNALING

胰岛素信号转导中的 ARF 蛋白

基本信息

批准号：
6517393
负责人：
GUILLERMO G ROMERO
金额：
$ 19.91万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-06-01 至 2004-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6517393
关键词：
ADP ribosylation biological signal transduction cell adhesion molecules cell line enzyme activity guanine nucleotide exchange factors insulin insulin receptor phospholipase D platelet derived growth factor protein reconstitution protein tyrosine kinase receptor binding transfection vascular smooth muscle

项目摘要

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.

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，其机制对于不同的受体和细胞类型是相对特异和不同的。特别是，在本研究项目的初始资助期间，我们已经表明，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血管平滑肌细胞系中PDGF和内皮素-1激活PLD至关重要。然而，ARF蛋白的激活可能受胰岛素和其他细胞外信号调节的机制仍然不清楚。由于存在许多RF蛋白（ARF 1-ARF 6）、ARF-GDP交换因子（ARF-GEF;即ARNO、细胞粘连蛋白-1、GRP 1、Gea等）和PLD形式（PLD 1和PLD 2），这一问题特别复杂，这些蛋白和因子表明PLD激活的平行冗余途径。在前一个资助期间，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，在ARF-GEF共免疫沉淀与胰岛素受体的胰岛素依赖性的方式。其他实验表明，ARNO与胰岛素受体相关。我们建议研究ARF-GEFs ARNO和cytohesin-1被细胞表面受体激活的机制，并确定这些GEFs的激活在胰岛素、PDGF和佛波酯调节PLD中的作用。我们建议通过生物化学，分子和成像方法的组合来做到这一点。我们将集中于以下几个方面的研究：a）通过对ARF-GEFs的Sec 7和pleckstrin同源结构域以及ARF的Switch 1区的特异性残基的突变分析，分析ARNO和cytohesin-1在受体介导的ARF和PLD激活中的作用; B）通过检查ARNO和细胞粘连素-1的磷酸化状态来确定它们的调节机制，它们与细胞表面受体的相互作用和它们被磷酸肌醇募集;和c）确定PLD 1和PLD 2在受体依赖性磷脂酶D活性中的相对作用。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The guanine nucleotide exchange factor ARNO mediates the activation of ARF and phospholipase D by insulin.

鸟嘌呤核苷酸交换因子Arno通过胰岛素介导了ARF和磷脂酶D的激活。

DOI：
10.1186/1471-2121-4-13
发表时间：
2003-09-11
期刊：
BMC cell biology
影响因子：
0
作者：
Li HS;Shome K;Rojas R;Rizzo MA;Vasudevan C;Fluharty E;Santy LC;Casanova JE;Romero G
通讯作者：
Romero G

The role of G proteins in insulin signalling.

G 蛋白在胰岛素信号传导中的作用。