Induction of exocytosis from permeabilized mast cells by the guanosine triphosphatases Rac and Cdc42
Induction of exocytosis from permeabilized mast cells by the guanosine triphosphatases Rac and Cdc42
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DOI：
10.1091/mbc.9.5.1053
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发表时间：
1998-05-01
影响因子：
3.3
通讯作者：
Gomperts, BD
中科院分区：
文献类型：
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作者：
Brown, AM;O'Sullivan, AJ;Gomperts, BD
我们将重组形式的Rho相关小鸟苷三磷酸酶(GTP酶)rac2和CDC42/G25K应用于通透性肥大细胞，以测试它们调节胞吐分泌的能力。肥大细胞可被溶血素-O渗漏(胞浆)蛋白通透5min，并在约20-30min内对钙(2+)和三磷酸鸟苷(GTP)的刺激变得不耐受。这种敏感性的丧失很可能是由于关键调控蛋白的丢失，这些蛋白通常被拴在细胞内的位置。延缓这种对刺激的敏感性丧失的外源蛋白质可能与那些丢失的分泌调节因子相似，如果不是相同的话。重组Rac和Cdc42/G25K被结合GTP-γS预激活，延缓了敏感性的丧失，更重要的是，使分泌能够被Ca(2+)单独刺激。对这两种GTP酶单独作用于通透性细胞的浓度依赖性的研究，以及在存在最佳浓度的rac2的情况下应用Cdc42/G25K的研究，为共同的效应途径和单独激活的第二个效应途径提供了证据。显性负性突变(N17)形式的rac2和CDc42/G25K抑制Ca(2+)和GTP-Gamma S诱导的分泌。我们的数据表明，rac2和CDc42应被认为是G(E)的候选者，G(E)是介导造血细胞胞吐的GTP酶。
We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca(2+) and guanosine triphosphate (GTP)gamma S over about 20-30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTP gamma S, retard the loss of sensitivity (rundown) and, more importantly, enable secretion to be stimulated by Ca(2+) alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca(2+) and GTP gamma S. Our data suggest that Rac2 and Cdc42 should be considered as candidates for G(E), GTPases that mediate exocytosis in cells of hematopoietic origin.