NOVEL ERYTHROBLAST/MACROPHAGE CONTACT PROTEIN

新型成红细胞/巨噬细胞接触蛋白

• 批准号: 6490608
• 负责人: Manjit Hanspal
• 金额: $ 20.48万
• 依托单位: STEWARD ST. ELIZABETH'S MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490608
• 关键词: HeLa cells; affinity chromatography; binding proteins; cell adhesion molecules; cell differentiation; cell growth regulation; chemical association; chemical kinetics; complementary DNA; erythrocyte membrane; erythroid stem cell; erythropoiesis; gene expression; genetic library; human tissue; immunoprecipitation; macrophage; membrane proteins; protein biosynthesis; protein metabolism; protein protein interaction; protein sequence; protein structure function; western blottings; yeast two hybrid system

The association of erythroblasts with macrophages plays a central role in the formation of distinct anatomic units called erythroblastic islands. Although these islands have been well characterized morphologically, virtually nothing is about the structural and functional basis of cellular contacts within erythroblastic islands. We have previously shown that physical contact between erythroblasts and macrophages promotes the terminal maturation of erythroblasts, leading to their enucleation in vitro. Importantly, we identified a novel protein termed "medhesin" which mediates the association of erythroblasts with macrophages. The disruption of medhesin's cell adhesion function dramatically inhibited the terminal maturation of erythroid cells. Our working hypothesis is that the medhesin-mediated attachment of erythroblasts to macrophages promotes erythroid maturation and is essential for erythroblast enucleation. We have recently determined the complete amino acid sequence of human medhesin from the macrophage cDNA library. The aim of this proposal is to characterize the molecular basis of medhesin function in erythroblast-macrophage association. Specifically, the following issues will be addressed: (1) Is medhesin-dependent erythroblast-macrophage contact sufficient for erythroblast maturation? We will investigate whether medhesin-mediated cellular contact alone is sufficient to produce erythroblastic terminal maturation or additional macrophage-derived factors are required. (2) How does medhesin-mediated contact between erythroblasts and macrophages promote terminal erythroid maturation? To define the molecular basis of medhesin function, an important requisite will be to identify the molecules with which medhesin associates in erythroblasts. This identification will be accomplished using immunoprecipitation, affinity- chromatography, and the yeast two hybrid assays. (3) Do the kinetics of medhesin synthesis, assembly, and turnover correlate with erythroblast maturation? We propose to quantify the rates of medhesin synthesis, assembly, and turnover in the soluble and particulate fractions of erythroblasts at defined stages of differentiation. The precise definition of temporally-regulated medhesin expression will provide clues to the basis of erythroblast-macrophage contact during erythropoiesis. Together, the proposed studies on medhesin may begin to reveal the molecular basis of erythroblastic island formation during mammalian erythropoiesis.

成红细胞与巨噬细胞的结合发挥着核心作用 在形成称为成红细胞的独特解剖单位时， 群岛 尽管这些岛屿的特征 从形态学上讲，几乎没有什么是关于结构和 成红细胞岛内细胞接触的功能基础。 我们以前已经证明，成红细胞和 巨噬细胞促进成红细胞的终末成熟， 在试管中进行去核。 重要的是，我们发现了一种 一种名为“medhesin”的蛋白质， 幼红细胞和巨噬细胞。 Medhesin细胞的破坏 粘附功能显著抑制了 红系细胞 我们的工作假设是， 成红细胞与巨噬细胞的粘附促进红细胞成熟 并且是成红细胞去核所必需的。 我们最近 确定了人Medhesin的完整氨基酸序列， 巨噬细胞cDNA文库。 本提案的目的是描述 成红细胞-巨噬细胞中Medhesin功能的分子基础 协会具体而言，将处理以下问题：（1） Medhesin依赖性成红细胞-巨噬细胞接触是否足以 成红细胞成熟 我们将研究是否medhesin介导 单独的细胞接触足以产生成红细胞终末 需要成熟或额外的巨噬细胞衍生因子。（二） 成红细胞和巨噬细胞之间的Medhesin介导的接触是如何 促进终末红细胞成熟？ 来定义分子基础 的medhesin功能，一个重要的必要条件是确定 成红细胞中与medhesin相关的分子。 这 鉴定将使用免疫沉淀、亲和- 层析和酵母双杂交测定。(3)做的动力学 Medhesin合成、装配和周转与成红细胞相关 成熟？ 我们建议量化Medhesin合成的速率， 组装和周转的可溶性和颗粒部分， 在确定的分化阶段的成红细胞。 的精确 时间调节的Medhesin表达的定义将提供 成红细胞-巨噬细胞接触的基础线索， 红细胞生成 总之，拟议中的关于medhesin的研究可能会开始 揭示成红细胞岛形成的分子基础， 哺乳动物红细胞生成