Dyson Jennifer M, Munday Adam D, Kong Anne M, Huysmans Richard D, Matzaris Maria, Layton Meredith J, Nandurkar Harshal H, Berndt Michael C, Mitchell Christina A
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia.
Blood. 2003 Aug 1;102(3):940-8. doi: 10.1182/blood-2002-09-2897. Epub 2003 Apr 3.
The platelet receptor for the von Willebrand factor (VWF) glycoprotein Ib-IX-V (GPIb-IX-V) complex mediates platelet adhesion at sites of vascular injury. The cytoplasmic tail of the GPIbalpha subunit interacts with the actin-binding protein, filamin, anchoring the receptor in the cytoskeleton. In motile cells, the second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3) induces submembraneous actin remodeling. The inositol polyphosphate 5-phosphatase, Src homology 2 domain-containing inositol polyphosphate 5-phosphatase-2 (SHIP-2), hydrolyzes PtdIns(3,4,5)P3 forming phosphatidylinositol 3,4 bisphosphate (PtdIns(3,4)P2) and regulates membrane ruffling via complex formation with filamin. In this study we investigate the intracellular location and association of SHIP-2 with filamin, actin, and the GPIb-IX-V complex in platelets. Immunoprecipitation of SHIP-2 from the Triton-soluble fraction of unstimulated platelets demonstrated association between SHIP-2, filamin, actin, and GPIb-IX-V. SHIP-2 associated with filamin or GPIb-IX-V was active and demonstrated PtdIns(3,4,5)P3 5-phosphatase activity. Following thrombin or VWF-induced platelet activation, detection of the SHIP-2, filamin, and receptor complex decreased in the Triton-soluble fraction, although in control studies the level of SHIP-2, filamin, or GPIb-IX-V immunoprecipitated by their respective antibodies did not change following platelet activation. In activated platelets spreading on a VWF matrix, SHIP-2 localized intensely with actin at the central actin ring and colocalized with actin and filamin at filopodia and lamellipodia. In spread platelets, GPIb-IX-V localized to the center of the platelet and showed little colocalization with filamin at the plasma membrane. These studies demonstrate a functionally active complex between SHIP-2, filamin, actin, and GPIb-IX-V that may orchestrate the localized hydrolysis of PtdIns(3,4,5)P3 and thereby regulate cortical and submembraneous actin.
血管性血友病因子（VWF）糖蛋白Ib-IX-V（GPIb-IX-V）复合物的血小板受体介导血小板在血管损伤部位的黏附。GPIbα亚基的胞质尾与肌动蛋白结合蛋白细丝蛋白相互作用，将受体锚定在细胞骨架中。在运动细胞中，第二信使磷脂酰肌醇3,4,5-三磷酸（PtdIns(3,4,5)P3）诱导膜下肌动蛋白重塑。肌醇多磷酸5-磷酸酶，含Src同源2结构域的肌醇多磷酸5-磷酸酶-2（SHIP-2），水解PtdIns(3,4,5)P3形成磷脂酰肌醇3,4-二磷酸（PtdIns(3,4)P2），并通过与细丝蛋白形成复合物来调节膜皱褶。在本研究中，我们研究了SHIP-2在血小板中的细胞内定位以及与细丝蛋白、肌动蛋白和GPIb-IX-V复合物的关联。从未刺激血小板的Triton可溶性部分免疫沉淀SHIP-2，结果表明SHIP-2、细丝蛋白、肌动蛋白和GPIb-IX-V之间存在关联。与细丝蛋白或GPIb-IX-V相关的SHIP-2具有活性，并表现出PtdIns(3,4,5)P3 5-磷酸酶活性。在凝血酶或VWF诱导的血小板活化后，Triton可溶性部分中SHIP-2、细丝蛋白和受体复合物的检测减少，尽管在对照研究中，各自抗体免疫沉淀的SHIP-2、细丝蛋白或GPIb-IX-V的水平在血小板活化后没有变化。在铺展于VWF基质上的活化血小板中，SHIP-2在中央肌动蛋白环处与肌动蛋白强烈共定位，并在丝状伪足和片状伪足处与肌动蛋白和细丝蛋白共定位。在铺展的血小板中，GPIb-IX-V定位于血小板中心，在质膜处与细丝蛋白几乎没有共定位。这些研究证明了SHIP-2、细丝蛋白、肌动蛋白和GPIb-IX-V之间存在功能活性复合物，该复合物可能协调PtdIns(3,4,5)P3的局部水解，从而调节皮质和膜下肌动蛋白。
