Role of Glypicans in HGF Mediated Cell Morphogenesis

磷脂酰肌醇蛋白聚糖在 HGF 介导的细胞形态发生中的作用

• 批准号: 6744097
• 负责人: ANIL K KARIHALOO
• 金额: $ 11.56万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6744097
• 关键词: biological signal transduction; hepatocyte growth factor; mitogen activated protein kinase; molecular cloning; nephrogenesis; phosphatidylinositol 3 kinase; phospholipase C; point mutation; protein structure function; proteoglycan; proteomics; tissue /cell culture

DESCRIPTION (provided by candidate): This application has evolved from some preliminary observation that HGF (that acts as a mitogen, morphogen and a motogen, in addition to its anti-apoptotic properties) does not induce branching morphogenesis in inner medullary collecting duct cells that specifically lack Gpc3-/ and present low levels of Gpc4. The long term goal is to better understand the role of (Gpcs) in HGF mediated epithelial cell morphogenesis (and signaling downstream of the receptor c-met), and eventually in the developing kidney. The application has been divided into two major specific aims. 1) Identify whether total expression of any Gpc or the presence of a specifc Gpc is necessary for HGF mediated branching morphogenesis? 2) Examine which signaling pathways known to be important for HGF induced morphogenesis are dependent on this interaction of HGF with Gpc? The initial experiments will be designed to address specific aim 1. HA tagged WT Gpc3 and Gpc4 cDNAs will be subcloned into retroviral vector and then transduced into Gpc3-/ cells. The expression of the protein on the cell surface will be confirmed by immunostaining for HA, Flow staining of HA and also by Weternblotting the lysates from these cells for anti-HA tag. Following this, cells will be subject to HGF mediated morphogenesis in collagen matrix to determine whether both Gpc3 or Gpc4 expression can rescue the lack of effect observed in the preliminary data. Depending on the results of this specific aim 1 we will then attempt to identify the specific regions of Gpc (core protein and the GAG side chains) that are necessary for their interaction with c-met. After determining the specific aim1 , we will then proceed to examine the signaling pathways known to be involved in HGF mediated morphogenesis. Once again, based on the preliminary data, we will initially focus on MAPK pathway and determine if there is any change in the kinetic of its activation by HGF in presence or absence of Gpc expression. We will then proceed to examine the kinetics of Gpc and c-met interactions in the same setting. Depending upon the results from these two broad experiments we might proceed to examine other pathways such as PI3-K, PLC-gamma etc. These specific aims are designed to hopefully lead to a better understanding of the role of Gpc expression during kidney development vis-a-vis HGF.

描述（由候选人提供）： 本申请是从一些初步观察发展而来的，即HGF（除了其抗凋亡特性之外，还充当有丝分裂原、形态发生原和运动原）不会在特异性缺乏Gpc 3-f并存在低水平Gpc 4的内髓集合管细胞中诱导分支形态发生。长期目标是更好地了解（Gpcs）在HGF介导的上皮细胞形态发生（和受体c-met下游信号传导）中的作用，并最终在发育中的肾脏中的作用。该申请分为两个主要的具体目标。1)确定任何Gpc的总表达或特定Gpc的存在是否是HGF介导的分支形态发生所必需的？2)检查已知对HGF诱导的形态发生重要的信号通路依赖于HGF与Gpc的这种相互作用？初步实验将针对具体目标1进行设计。将HA标记的WT Gpc 3和Gpc 4 cDNA亚克隆到逆转录病毒载体中，然后转导到Gpc 3-/细胞中。蛋白质在细胞表面上的表达将通过HA的免疫染色、HA的流式染色以及来自这些细胞的裂解物的抗HA标签的Weternblotting来确认。在此之后，细胞将在胶原基质中经历HGF介导的形态发生，以确定Gpc 3或Gpc 4表达是否可以挽救在初步数据中观察到的效果缺乏。根据这一特定目标1的结果，我们将尝试鉴定Gpc的特定区域（核心蛋白和GAG侧链），这些区域是它们与c-met相互作用所必需的。在确定了特定的aim 1后，我们将继续研究已知参与HGF介导的形态发生的信号通路。再一次，基于初步数据，我们将首先关注MAPK途径，并确定在存在或不存在Gpc表达的情况下，HGF激活MAPK途径的动力学是否有任何变化。然后，我们将继续研究Gpc和c-met相互作用的动力学在相同的设置。根据这两个广泛的实验的结果，我们可能会继续研究其他途径，如PI 3-K，PLC-γ等，这些具体的目的是希望导致更好地了解Gpc表达的作用，在肾脏发育过程中相对于肝细胞生长因子。