SIGNALS FOR RENAL ENDOTHELIAL CAPILLARY MORPHOGENESIS

肾内皮毛细血管形态发生的信号

• 批准号: 2905583
• 负责人: TOM DANIEL
• 金额: $ 18.62万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2905583
• 关键词: angiogenesis; biological signal transduction; capillary; cell cell interaction; cell line; cell migration; chimeric proteins; cytoplasm; gene expression; human tissue; integrins; tissue /cell culture; vascular endothelium; yeast two hybrid system

DESCRIPTION (Adapted from Investigator's Abstract): Vasculogenesis and angiogenic remodeling are basic requirements for organogenesis and wound healing. For vasculogenesis to occur, the endothelial cell progenitors must assemble properly. The current proposal will further define the molecular processes that direct renal microvascular endothelial targeting and assembly. The proposal hypothesizes that renal and other microvascular endothelial cells discriminate appropriate partners for vascular structure assembly based on signals they receive upon juxtacrine contact during directed migration. In this regard, previous studies by this investigator have shown that the ELK subclass of Eph family of receptors are critical in cultured human renal microvascular endothelial cells (HRMEC) attachment and assembly behavior. Further, the investigator has shown that the signals of the ELK receptor depend upon juxtacrine presentation of their ligands in variably oligomerized states on the surfaces of the contacting cells. Three aims will be addressed. The experimental approach will involve traditional biochemical affinity studies as well as yeast-two hybrid system and use of dominant negative protein interactions. The first specific aim, to define the molecular basis for LERK-2 oligomerization, will be accomplished using two complementary approaches. The first will employ a yeast two-hybrid screen utilizing the cytoplasmic domain of LERK-2 as bait to screen a murine embryonic library. This study anticipates finding proteins that interact with the cytoplasmic domain of LERK-2. The complementary approach will involve standard cell culture labeling experiments to provide information about the cellular distribution about LERK-2 in stimulated and unstimulated cells. Subsequently, other experiments will utilize a LERK-2 cytoplasmic domain created as a GST fusion protein to allow binding to GSH-beads. This fusion protein will then be used to capture potential interacting proteins from the cytosol of stimulated cells. Finally, a series of chemical cross-linking studies and gel filtration electrophoresis experiments will be employed to investigate the state of oligomerization of LERK-2 following stimulation of endothelial cells. The second specific aim will focus on ELK signaling and, specifically, will investigate the molecular basis for the interaction between ELK and LMP-PTP, which was demonstrated by previous studies in this investigator's laboratory. In this regard, this laboratory has accumulated a wide range of experimental tools to address this interaction and will use the yeast two-hybrid system and baculovirus expression system to address these issues in addition to transient transfection assays and a dominant negative approach to examine these interactions. The third specific aim is a descriptive study that will determine the type of integrin present on cell lines used in this laboratory, using standard FACS analysis. In addition, transfected cell lines will be established to examine the interaction among integrins and signaling by ELK-LERK-2 interactions. This is a proposal which seeks a further 5 years of funding for the study of capillary morphogenesis by renal microvascular endothelial cells. The project is now in its fourth year, and has provided novel insights into cell-cell recognition events. In brief, it was learned that the ELK-LERK-2 ligand-receptor pair is present in renal endothelial cells during development, and that LERK-2, when presented as tetramers to ELK can stimulate precapillary cord formation in vitro. In contrast, dimeric LERK-2 only stimulates cell adhesion and migration in a matrix-specific fashion. Proteins that associate with the cytoplasmic tail of activated ELK have been identified, and while Grb-2 and Grb-10 associate with ELK stimulated by dimeric LERK-2, a tyrosine phosphatase LMW-PTP only associates with the receptor when it binds clustered, tetrameric LERK-2. It is therefore proposed that ELK and LERK-2 are counter-receptors on renal microvascular endothelial cells which cluster during cell-cell contact, and that both, ELK and LERK-2 serve to signal intracellular events which stimulate capillary cord formation. The approaches proposed for next term of the project are to use the yeast two hybrid system extensively, and to study in vitro interactions with GST fusions of ELK and LERK-2 (and mutants thereof) with the aim of identifying proteins that interact with the cytoplasmic tail of LERK-2 (aim 1) and ELK (aim 2). For ELK, this approach has been partially successful and aim 2 therefore seeks to further define signaling events that stimulate cell assembly by studying specific mutants of ELK and its partners, their mutual interactions, and their potential effects on cell assembly. Since dimeric LERK-2 stimulates cell adhesion and migration as well as ELK tyrosine phosphorylation, and since Grb-2, Grb-4, and Grb-10 have been found to associate with ELK upon stimulation with dimeric and tetrameric LERK-2, aim three is designed to assess the influence of LERK-2-ELK on integrin function, the involvement of small G-proteins in stimulating adhesion, cytoskeletal reorganization and migration, and activation of the JNK/SAP kinase pathway.

描述（根据研究者的摘要改编）：血管生成和 血管生成重塑是器官发生和伤口的基本要求 康复。 为了使血管生成发生，内皮细胞祖细胞必须 正确组装。 当前的建议将进一步定义分子 直接肾脏微血管内皮靶向和 集会。 该提议假设肾脏和其他微血管 内皮细胞区分适合血管结构的伴侣 基于他们在近二氨酸接触时收到的信号的组装 定向迁移。 在这方面，该研究者先前的研究 已经表明，EPH受体家族的麋鹿子类对 培养的人肾脏微血管内皮细胞（HRMEC）附着和 组装行为。 此外，调查人员表明 麋鹿受体取决于其配体的并置置 在接触细胞的表面上可变的寡聚状态。 三 目标将被解决。 实验方法将涉及传统 生化亲和力研究以及酵母 - 两种混合系统以及使用 主要的负蛋白相互作用。 定义的第一个特定目标 LERK-2低聚的分子基础将使用 两种互补方法。 第一个将采用酵母双杂交 屏幕利用LERK-2的细胞质结构域作为诱饵来筛选鼠 胚胎库。 这项研究预计发现相互作用的蛋白质 与LERK-2的细胞质结构域。 互补方法将 涉及标准细胞培养标记实验以提供信息 关于刺激和未刺激的LERK-2的细胞分布 细胞。 随后，其他实验将利用LERK-2细胞质 作为GST融合蛋白创建的结构域可与GSH珠子结合。 这 然后，融合蛋白将用于捕获潜在的相互作用蛋白 来自刺激细胞的胞质溶胶。 最后，一系列化学物质 交联研究和凝胶过滤电泳实验将是 用于调查LERK-2的寡聚状态之后 刺激内皮细胞。 第二个特定目标将集中在麋鹿信号传导上，具体来说， 研究麋鹿和LMP-PTP之间相互作用的分子基础， 这是该研究者的先前研究证明的 实验室。 在这方面，该实验室积累了广泛的 解决这种互动的实验工具，并将使用酵母 两个杂交系统和杆状病毒表达系统解决这些问题 除了瞬态转染测定和主要负数 检查这些相互作用的方法。 第三个特定目的是描述性研究，该研究将确定类型 使用标准 FACS分析。 另外，将建立转染的细胞系 检查整联蛋白之间的相互作用和通过ELK-LERK-2信号传导 互动。 这是一项提议，寻求另外5年的资金 为了研究肾脏微血管内皮的毛细血管形态发生 细胞。 该项目现在已进入第四年，并提供了小说 洞悉细胞电池识别事件。 简而言之 ELK-LERK-2配体受体对存在于肾脏内皮细胞中 在开发过程中，当以四聚体为麋鹿时，那个LERK-2可以 刺激体外刺激毛细血管绳形成。 相反，二聚体LERK-2 仅以特定于基质的方式刺激细胞粘附和迁移。 与活化麋鹿的细胞质尾巴相关的蛋白质已经是 确定，而GRB-2和GRB-10与ELK相关 二聚体LERK-2，酪氨酸磷酸酶LMW-PTP仅与 受体结合聚类时，四聚体LERK-2。 因此是 提议麋鹿和LERK-2是肾脏微血管上的反感应器 内皮细胞在细胞细胞接触过程中聚集，并且两者都麋鹿 LERK-2发出信号的细胞内事件，刺激毛细管 绳索形成。 该项目下一期提出的方法是 广泛使用酵母两种杂种系统，并在体外研究 与麋鹿和LERK-2（及其突变体）与GST融合的相互作用与 鉴定与细胞质尾巴相互作用的蛋白质的目的 LERK-2（AIM 1）和麋鹿（AIM 2）。 对于麋鹿，这种方法部分是 因此，成功和目标2旨在进一步定义信号事件 通过研究麋鹿的特定突变体刺激细胞组件及其 伴侣，他们的相互作用及其对细胞的潜在影响 集会。 由于二聚体LERK-2刺激细胞粘附和迁移 以及麋鹿酪氨酸磷酸化，以及GRB-2，GRB-4和GRB-10以来 发现与二聚体刺激后与麋鹿相关 四聚体LERK-2，AIM三旨在评估 LERK-2-ELK在整联蛋白功能上，小G蛋白参与 刺激粘附，细胞骨架重组和迁移，以及 JNK/SAP激酶途径的激活。