STRUCTURE/FUNCTION OF A HEPATIC CEA BINDING PROTEIN

肝脏 CEA 结合蛋白的结构/功能

• 批准号: 6173300
• 负责人: PETER THOMAS
• 金额: $ 24.95万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-15 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6173300
• 关键词: Kupffer's cell; animal genetic material tag; athymic mouse; binding proteins; binding sites; biological signal transduction; carcinoembryonal antigen; cell adhesion molecules; cell line; colorectal neoplasms; cytokine; human genetic material tag; laboratory rat; liver metabolism; metastasis; molecular cloning; protein sequence; protein structure function; site directed mutagenesis

Carcinoembryonic antigen (CEA) is involved in the development of hepatic metastases from colorectal cancers. The major hepatic CEA binding protein is 80kD Kupffer cell surface molecule. The binding site for the 80kD protein has been located to the junction of the N-terminal and first loop domains of CEA and is a pentapeptide (PELPK). Treatment of Kupffer cells with CEA results in the production of IL-1, IL-6 and TNF- alpha and two proteins of 115 and 125kD are rapidly phosphorylated when Kupffer cells are exposed to CEA. In this application we will also study the structure and function of the Kupffer cell 80kD carcinoembryonic antigen (CEA) peptide binding protein by further protein sequencing, production of antibodies and by cloning and expressing its gene from both rat and human Kupffer cell cDNA libraries using the available peptide sequences as the basis for probes. To examine the function of the CEA binding protein in metastasis we will examine the mechanism of activation of hepatic Kupffer cells by CEA and the synthetic ligand for the 80kD binding protein PELPK-albumin by a) Studying the consequences of ligand binding to the 80kD protein on Kupffer cells, on the metastatic proclivity of cancer cells in the hepatic sinusoid and the effects on adhesion to the hepatic endothelium. Using inhibitory antibodies to endothelial cells binding proteins we will identify the adhesion molecules involved. b) We will examine the effects of inhibition of signal transduction (specifically phosphorylation of tyrosine) on the regulation of metastatic potential of human colorectal cancer cell lines by CEA. c) We will use site directed mutagenesis to alter the five amino acid (PELPK) binding site for the 80kD protein to examine its effect on Kupffer cell response to CEA and on hepatic metastasis formation. We will also use a construct that has the first 75 amino acids of the CEA N-domain deleted. This CEA does not form homotypic aggregates but retains the PELPK sequence. This will allow us to determine if CEA produced to enhance metastatic potential. These studies will increase our knowledge of the role of the 80kD binding protein in the liver and its relationship with CEA in the mechanism by which colorectal cancer cells attach and invade in the hepatic sinusoid.

癌腹抗原（CEA）参与了肝的发展 结直肠癌的转移。 主要的肝CEA绑定 蛋白质是80kD kupffer细胞表面分子。 的结合位点 80kD蛋白已定位到N末端的连接处 CEA的第一个环域，是五肽（PELPK）。 处理 具有CEA的库普弗细胞会产生IL-1，IL-6和TNF-的产生 当α和两个115和125KD的蛋白质迅速磷酸化时 Kupffer细胞暴露于CEA。 在此应用程序中，我们也将 研究Kupffer单元80KD的结构和功能 通过进一步的进一步 蛋白质测序，抗体的产生以及克隆和 从大鼠和人库普弗细胞cDNA库中表达其基因 使用可用的肽序列作为探针的基础。 到 检查CEA结合蛋白在转移中的功能，我们将 检查CEA和 A） 研究配体与80KD蛋白在80kd蛋白上的后果 Kupffer细胞，在癌细胞的转移性倾向 肝正弦曲线以及对肝内皮粘附的影响。 使用抑制性抗体来结合蛋白质的内皮细胞 将确定涉及的粘附分子。 b）我们将检查 抑制信号转导的影响（特别是 酪氨酸的磷酸化）对转移电位的调节 CEA的人类结直肠癌细胞系。 c）我们将使用网站 定向诱变改变了五个氨基酸（PELPK）结合位点 为了使80kD蛋白检查其对Kupffer细胞反应的影响 CEA和肝转移形成。 我们还将使用一个构造 它具有删除CEA N域的前75个氨基酸。 这个CEA 不形成同型聚集体，而是保留PELPK序列。这 将使我们能够确定CEA是否生产以增强转移性 潜在的。 这些研究将增加我们对 肝脏中的80kD结合蛋白及其与CEA的关系 结直肠癌细胞附着并入侵的机制 肝正弦。