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Studies on granulocyte colony-stimulating factor receptor

粒细胞集落刺激因子受体的研究

基本信息

批准号：
02680164
负责人：
FUKUNAGA Rikiro
金额：
$ 1.47万
依托单位：
Osaka Bioscience Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Granulocyte colony-stimulating factor(G-CSF)is a growth and differentiation factor which works on cells restricted to the neutrophilic granulocyte lineage. To clarify the structure and function of G-CSF receptor(G-CSF-R), we isolated cDNAs for murine and human G-CSF-R. Sequence analysis revealed that G-CSF-R is an 812(mouse)or 813(human)amino acid polypeptide with a single transmembrane domain. The extracellulsr domain consists of an Ig-like domain, a cytokine-receptor-homologous(CRH)domain and three fibronectin type III(FNIII)domains. Mouse G-CSF-R expressed in COS cells was able to bind G-CSF with an affinity(Kd = 290 pM)similar to that of the receptor of NFS-60 cells, indicating that the single polypeptide is sufficient to form the high-affinity binding site for G-CSF. Molecular cloning and chromosomal mapping of the G-CSF-R gene indicated that the human G-CSF-R gene consists of 17 exons and is located on the p35-34.3 region of human chromosome 1. To explore signal transduction mechanism of the G-CSF-R, we expressed the G-CSF-R CDNA in various hematopoietic cell lines. Introduction of G-CSF-R CDNA into IL-3-dependent mouse myeloid cell line FDC-PL and pro-B cell line BAF-BO3 enabled them to proliferate in response to G-CSF. However, IL-2-dependent mouse CTLL-2 cells expressing G-CSF-R could not grow in the presence of G-CSF. Surface expression of some differentiation markers on the FDC-PL transformants was differentially regulated by G-CSF and IL-3. Mutational analysis of the G-CSF-R in FDC-Pl cells indicated that the N-terminal half of the CRH domain was essential for the recognition of G-CSF, but the Ig-like, FNIII and cytoplasmic domains were not. The CRH domain and a 76-amino acids portion of the cytoplasmic domain were indispensable for the transduction of the G-CSF-triggered growth signal.

粒细胞集落刺激因子（G-CSF）是一种生长和分化因子，作用于仅限于嗜中性粒细胞谱系的细胞。为了阐明G-CSF受体（G-CSF-R）的结构和功能，我们分离了小鼠和人G-CSF-R的cDNA。序列分析表明，G-CSF-R是一个812（小鼠）或813（人）氨基酸的多肽，具有一个单一的跨膜结构域。胞外结构域由Ig样结构域、丝氨酸受体同源（CRH）结构域和三个纤连蛋白III型（FNIII）结构域组成。在COS细胞中表达的小鼠G-CSF-R能够以与NFS-60细胞的受体相似的亲和力（Kd = 290 pM）结合G-CSF，表明单个多肽足以形成G-CSF的高亲和力结合位点。G-CSF-R基因的分子克隆和染色体定位表明，人G-CSF-R基因由17个外显子组成，位于人1号染色体的p35-34.3区域。为了探讨G-CSF-R的信号转导机制，我们在多种造血细胞系中表达了G-CSF-R cDNA。将G-CSF-R cDNA导入IL-3依赖的小鼠骨髓细胞系FDC-PL和pro-B细胞系BAF-BO 3中，使它们能够响应G-CSF而增殖。然而，表达G-CSF-R的IL-2依赖性小鼠CTLL-2细胞在G-CSF存在下不能生长。FDC-PL转化体上的一些分化标志物的表面表达受G-CSF和IL-3的差异调节。FDC-P1细胞中G-CSF-R的突变分析表明，CRH结构域的N-末端一半对于G-CSF的识别是必需的，但Ig样结构域、FNIII结构域和胞质结构域对于G-CSF的识别不是必需的。CRH结构域和胞质结构域的76个氨基酸部分对于G-CSF触发的生长信号的转导是不可缺少的。