Biochemical and biological characterization of a human Rac2 GTPase mutant associated with phagocytic immunodeficiency
Biochemical and biological characterization of a human Rac2 GTPase mutant associated with phagocytic immunodeficiency
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DOI：
10.1074/jbc.m010445200
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发表时间：
2001-05-11
影响因子：
4.8
通讯作者：
Williams, DA
中科院分区：
文献类型：
--
作者：
Gu, Y;Jia, BQ;Williams, DA
Rho GT3，Rac 2，仅在造血细胞谱系中表达，表明这些细胞中的特定细胞功能。在小鼠中的遗传靶向研究表明，Rac 2是中性粒细胞趋化性、L-选择素捕获和滚动以及超氧化物产生的重要调节剂。最近，Rac 2的显性失活突变D57 N被报道与人类吞噬细胞免疫缺陷相关。为了进一步了解与该D57 N Rac 2突变体相关的细胞表型，我们检查了其在原代小鼠骨髓细胞中表达时的生化特征和功能效应。当与野生型（WT）Rac 2相比时，D57 N Rac 2显示出类似于10%的GTP结合能力，这是由于GTP解离速率显著增强，并且对鸟嘌呤核苷酸交换因子没有反应。这些结果表明，D57 N Rac 2可能通过螯合内源性鸟嘌呤核苷酸交换因子以显性负性方式在细胞中起作用。当在造血细胞中表达时，D57 N Rac 2不仅降低Rac 2的内源性活性，而且降低Rad的内源性活性，并且由于增加细胞凋亡而在生长因子存在下降低体外细胞扩增。相反，用WT Rac 2和显性活性突变体Q61 L转导的细胞的扩增与显著增加的增殖相关。将转导的骨髓细胞移植到致死性照射的受者中，显示含D57 N的外周血细胞的百分比从1个月时的40%显著降低到10%。
The Rho GTPase, Rac2, is expressed only in hematopoietic cell lineages, suggesting a specific cellular function in these cells. Genetic targeting studies in mice showed that Rac2 is an essential regulator of neutrophil chemotaxis, L-selectin capture and rolling, and superoxide production. Recently, a dominant negative mutation of Rac2, D57N, has been reported to be associated with a human phagocytic immunodeficiency. To understand further the cellular phenotypes associated with this D57N Rac2 mutant we examined its biochemical characteristics and functional effects when expressed in primary murine bone marrow cells. When compared with wild type (WT) Rac2, D57N Rac2 displayed similar to 10% GTP binding ability resulting from a markedly enhanced rate of GTP dissociation and did not respond to the guanine nucleotide exchange factors. These results suggest that D57N Rac2 may act in a dominant negative fashion in cells by sequestering endogenous guanine nucleotide exchange factors, When expressed in hematopoietic cells, D57N Rac2 reduced endogenous activities of not only Rac2, but also Rad and decreased cell expansion in vitro in the presence of growth factors due to increased cell apoptosis, Unexpectedly, D57N expression had no effect on proliferation. In contrast, expansion of cells transduced with WT Rac2 and a dominant active mutant, Q61L, was associated with significantly increased proliferation. Transplantation of transduced bone marrow cells into lethally irradiated recipients showed that the percentage of D57N-containing peripheral blood cells decreased markedly from 40% at 1 month to