Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets
Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets
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DOI：
10.1074/jbc.m603227200
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发表时间：
2006-09-29
影响因子：
4.8
通讯作者：
Horiuchi, Hisanori
中科院分区：
文献类型：
--
作者：
Kondo, Hirokazu;Shirakawa, Ryutaro;Horiuchi, Hisanori
我们先前已经证明Rab 27调节血小板中致密颗粒的分泌。在这里，我们分析了Rab 27的激活状态，使用薄层色谱法分析结合到免疫沉淀Rab 27的核苷酸，并使用下拉法定量Rab 27结合到其特异性效应子Slac 2-B的GTP-Rab 27结合结构域（突触结合蛋白样蛋白（Slp）同源结构域）。我们发现，Rab 27主要存在于GTP结合的形式在未刺激的血小板由于组成GDP/GTP交换活性。GTP结合Rab 27的水平急剧下降，由于颗粒分泌后增强的GTP水解活性。在透化血小板中，Ca 2+浓度的增加诱导致密颗粒分泌，伴随着GTP-Rab 27的减少，而在非水解的GTP类似物GppNHp（β-γ-亚氨基鸟苷5 '-三磷酸）负载的透化血小板中，GTP（GppNHp）-Rab 27水平在Ca 2+诱导的分泌后没有减少。这些数据表明Rab 27的GTP水解不是诱导分泌所必需的。综上所述，Rab 27在未刺激的血小板中保持活性状态，其可以起到使致密颗粒保持在分泌的准备状态的作用。
We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca2+ concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (beta-gamma-imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca2+-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.