BIOLOGY OF THE STEM CELL FACTOR RECEPTOR

干细胞因子受体的生物学

基本信息

批准号：
2143603
负责人：
VIRGINIA C BROUDY
金额：
$ 20.12万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-30 至 1998-09-29
项目状态：
已结题

项目摘要

Hematopoietic growth factors stimulate cell growth by interacting with specific receptors. Stem Cell Factor (SCF) is a novel growth factor that acts on hematopoietic stem cells and progenitor cells. The receptor for SCF is encoded by the c-kit proto-oncogene. We propose to characterize the c-kit receptor on hematopoietic and non-hematopoietic cells. The broad, long-term objectives of this proposal are to provide a better understanding of the mechanism of action of SCF in normal and neoplastic hematopoiesis and to provide information about the structure and function of the c-kit receptor. The specific aims are as follows. First, we will determine the binding properties, abundance, cellular distribution and size of the c-kit protein on both normal hematopoietic cells, marrow microenvironmental cells, and neoplastic cells. Three approaches will be used to achieve this aim: (a) equilibrium binding studies with 125I-SCF to quantitate receptor numbers and binding affinity on populations of cells plus autoradiography to permit analysis of 125I-SCF binding to individual cells, (b) isolation and characterization of populations of receptor-bearing cells using our anti-c-kit receptor antibody SR-1, and (c) affinity crosslinking and ligand blotting to determine the size and subunit structure of the receptor. We will compare the structure of the c-kit receptor on normal and neoplastic cells. Second, we will generate additional anti-c-kit receptor monoclonal antibodies by immunizing mice with cells expressing high numbers of c-kit receptors or with purified soluble c-kit protein. Our hybridoma screening techniques are designed to identify both neutralizing and nonneutralizing antibodies. We will use the antibodies to map functional domains of the c- kit receptor. Third, we will identify the ligand binding domain of the c-kit receptor and the regions required for receptor dimerization using interspecies human- mouse hybrid c-kit receptors and truncated receptors. The ability of these mutant receptors to bind human and rat 125I-SCF, to dimerize, to autophosphorylate, and to transmit a proliferative signal to the cell will be determined.

造血生长因子通过与特定受体。干细胞因子（SCF）是一个新的生长因素作用于造血干细胞和祖细胞上。受体 SCF由C-KIT原始癌基因编码。我们建议表征造血和非山大放异化细胞上的C-KIT受体。广泛，该建议的长期目标是提供更好的理解 SCF在正常和肿瘤造血的作用机理并提供有关C-KIT结构和功能的信息受体。具体目标如下。首先，我们将确定结合特性，丰度，细胞 c-kit蛋白的分布和大小在正常造血上细胞，骨髓微环境细胞和肿瘤细胞。三方法将用于实现此目标：（a）平衡结合使用125i-SCF的研究来定量受体数量和结合亲和力关于细胞的种群加上放射自显影，以允许分析125i-SCF 与单个细胞结合，（b）隔离和表征使用我们的抗C-KIT受体的受体细胞种群抗体SR-1和（c）亲和力交联和配体印迹至确定受体的大小和亚基结构。我们将比较在正常和肿瘤细胞上C-KIT受体的结构。其次，我们将生成其他抗C-KIT受体单克隆通过用表达高数量C-KIT的细胞免疫小鼠免疫小鼠的抗体受体或纯化的可溶性C-KIT蛋白。我们的杂交瘤筛查技术旨在识别中和和非中和化抗体。我们将使用抗体来绘制C-的功能域套件受体。第三，我们将确定C-KIT受体的配体结合结构域和使用种间人类的受体二聚化所需的区域小鼠杂交C-kit受体和截短的受体。这些能力突变受体结合人和大鼠125i-scf，以二聚，与自磷酸化，并将增殖信号传输到细胞可以确定。