Shirakawa Ryutaro, Higashi Tomohito, Tabuchi Arata, Yoshioka Akira, Nishioka Hiroaki, Fukuda Mitsunori, Kita Toru, Horiuchi Hisanori
Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
J Biol Chem. 2004 Mar 12;279(11):10730-7. doi: 10.1074/jbc.M309426200. Epub 2003 Dec 29.
Platelets store self-agonists such as ADP and serotonin in dense core granules. Although exocytosis of these granules is crucial for hemostasis and thrombosis, the underlying mechanism is not fully understood. Here, we show that incubation of permeabilized platelets with unprenylated active mutant Rab27A-Q78L, wild type Rab27A, and Rab27B inhibited the secretion, whereas inactive mutant Rab27A-T23N and other GTPases had no effects. Furthermore, we affinity-purified a GTP-Rab27A-binding protein in platelets and identified it as Munc13-4, a homologue of Munc13-1 known as a priming factor for neurotransmitter release. Recombinant Munc13-4 directly bound to GTP-Rab27A and -Rab27B in vitro, but not other GTPases, and enhanced secretion in an in vitro assay. The inhibition of secretion by unprenylated Rab27A was rescued by the addition of Munc13-4, suggesting that Munc13-4 mediates the function of GTP-Rab27. Thus, Rab27 regulates the dense core granule secretion in platelets by employing its binding protein, Munc13-4.
血小板在致密核心颗粒中储存自身激动剂，如二磷酸腺苷（ADP）和5-羟色胺。虽然这些颗粒的胞吐作用对止血和血栓形成至关重要，但其潜在机制尚未完全阐明。在此，我们发现用未异戊二烯化的活性突变体Rab27A-Q78L、野生型Rab27A和Rab27B孵育透化血小板会抑制分泌，而无活性的突变体Rab27A-T23N和其他GTP酶则无此作用。此外，我们通过亲和纯化在血小板中得到一种GTP-Rab27A结合蛋白，并将其鉴定为Munc13-4，它是Munc13-1的同源物，而Munc13-1是已知的神经递质释放的启动因子。重组Munc13-4在体外直接与GTP-Rab27A和-Rab27B结合，但不与其他GTP酶结合，并在体外试验中增强了分泌。添加Munc13-4可挽救未异戊二烯化的Rab27A对分泌的抑制作用，这表明Munc13-4介导了GTP-Rab27的功能。因此，Rab27通过其结合蛋白Munc13-4调节血小板中致密核心颗粒的分泌。
