PROTEIN 4.1 EXPRESSION DURING ERYTHROID DIFFERENTIATION

4.1 红细胞分化过程中的蛋白质表达

• 批准号: 6564217
• 负责人: JOEL A CHASIS
• 金额: $ 14.33万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564217
• 关键词: band 3 protein; bone marrow; calmodulin; cell adhesion; cell differentiation; erythrocyte membrane; erythropoiesis; extracellular matrix; gene expression; gene rearrangement; glycophorin; human genetic material tag; human tissue; membrane biogenesis; membrane proteins; polymerase chain reaction; protein 4.1; protein sequence; protein structure function; site directed mutagenesis; tissue /cell culture

The long term objective of this proposal is to develop a detailed understanding of membrane assembly and remodeling during erythroid differentiation and to characterize the interactions between extracellular matrix, stromal cells and erythroid precursors which trigger these differentiation-associated processes. To achieve our stated objective we propose the following series of studies: 1) Perform detailed structure-function analysis of the 80 kD erythroid 4. 1 N- terminal 30 kD ~membrane binding~ domain to map the binding site(s) for glycophorin C, p55, band 3 and calmodulin and identify proteins which interact with the highly conserved C-terminal 22-24 kD domain. 2) Analyze plasma membrane-associated protein 4.1 isoform function during terminal erythroid differentiation to explore the hypothesis that assembly of 4.1 isoforms of varying structure onto the plasma membrane during erythropoiesis results in dynamic reorganization of protein interactions and changes in membrane biophysical properties. 3) Define how protein 4.1 isoforms contribute to nuclear and centrosomal architecture and function. We will analyze protein 4.1 localization relative to well characterized nuclear and centrosomal proteins at various stages of the cell cycle and identify 4.1 binding partners. We will explore the functional importance of nuclear 4.1 by manipulating expression levels of transfected normal constructs or putative dominant negative derivatives and investigate centrosomal 4.1 function by testing the effects of 4.1 depletion in centrosome reconstitution assays. 4) Determine whether adhesive interactions between erythroblasts and bone marrow extracellular matrix and stromal cells regulate 4.1 gene expression. We will test our hypothesis that liganding the erythroblast adhesion molecule alpha4beta1 integrin to VCAM-1 or fibronectin alters its intracellular association with the cytoskeleton thereby initiating a signaling cascade to the nucleus. We will also ascertain whether alpha4beta1 -mediated adhesion of cultured erythroblasts to fibronectin peptides, endothelial cells, or bone marrow macrophages influences differentiation-associated changes in 4.1 gene expression. We anticipate that the successful accomplishment of these proposed aims will provide fundamental insights into membrane assembly and remodeling during erythroid differentiation and the role of adhesive interactions in triggering these differentiation-associated processes. This, in turn, should enable a better mechanistic understanding of the pathophysiology of hereditary spherocytosis and hereditary elliptocytosis and provide import insights into the role of the bone marrow microenvironment in regulating differentiation of donor hematopoietic cells following transplantation.

该提案的长期目标是制定详细的 了解红细胞期间的膜组装和重塑 区分并表征之间的相互作用 细胞外基质、基质细胞和红系前体细胞 触发这些分化相关的过程。 为了实现我们的 为了实现既定目标，我们建议进行以下一系列研究：1）执行 80 kD 红细胞的详细结构功能分析 4. 1 N- 末端 30 kD ~膜结合~ 域，用于映射结合位点 血型糖蛋白 C、p55、带 3 和钙调蛋白，并鉴定其蛋白质 与高度保守的 C 端 22-24 kD 结构域相互作用。 2） 分析质膜相关蛋白 4.1 亚型功能 终末红细胞分化以探索以下假设： 4.1 不同结构的异构体在质膜上的组装 在红细胞生成过程中导致蛋白质的动态重组 膜生物物理特性的相互作用和变化。 3）定义 蛋白质 4.1 同工型如何促进核和中心体 架构和功能。 我们将分析蛋白质4.1定位 相对于已充分表征的核蛋白和中心体蛋白 细胞周期的各个阶段并确定 4.1 结合伴侣。 我们 将通过操纵来探索核 4.1 的功能重要性 转染正常构建体或推定显性构建体的表达水平 负导数并通过测试研究中心体 4.1 功能 4.1 中心体重建测定中耗竭的影响。 4） 确定成红细胞和骨之间是否存在粘附相互作用 4.1 骨髓细胞外基质和基质细胞调节基因 表达。 我们将检验我们的假设：配体成红细胞 粘附分子 α4β1 整合素对 VCAM-1 或纤连蛋白的改变 它与细胞骨架的细胞内联系，从而启动 信号级联传递至细胞核。 我们还将确定是否 α4β1 介导的培养成红细胞与纤连蛋白的粘附 肽、内皮细胞或骨髓巨噬细胞的影响 4.1 基因表达的分化相关变化。 我们 预计这些拟议目标的成功实现 将为膜组装和重塑提供基本见解 红细胞分化过程中和粘附相互作用的作用 触发这些分化相关过程。 这，在 转，应该能够更好地理解 遗传性球形红细胞增多症的病理生理学和遗传性 椭圆形红细胞增多症并为骨的作用提供重要的见解 骨髓微环境调节供体分化 移植后的造血细胞。