Discovery of protein-to-protein interactions in platelets by a novel methods.

在血小板中寻找可以与CrkL的N-末端SH 3结构域相互作用的蛋白质（使用下拉测定法和质谱法的组合），我们发现人血小板表达DOCK 5，一种推定的Rac鸟嘌呤核苷酸交换剂，作为CrkL结合蛋白。我们克隆了DOCK 5并产生了针对该分子的特异性抗体。我们在COS 7细胞中过表达CrkL、DOCK 5或两者的组合。过表达的DOCK 5呈弥漫性胞浆分布。然而，当与野生型CrkL共表达时，两种DOCK 5在CrkL诱导的局灶性粘连处积累，表明相互作用的功能性implication. Use针对WAVE（WASP [Wiskott-Aldrich综合征蛋白]家族Verprolin同源蛋白，也称为Scar）同种型的特异性抗体，我们证明了人血小板表达所有3种同种型。利用体外下拉技术，胰岛素受体底物p53（IRSp 53）的src同源性3（SH 3）结构域比profilin I更有效地从血小板裂解物中沉淀WAVE 2。WAVE 1的情况正好相反，两者都没有沉淀WAVE 3，这表明WAVE同种型对这些配体具有不同的亲和力，而abl的SH 3结构域结合所有3种同种型。通过质谱分析，我们发现血小板中存在与WAVE/Scars相互作用的蛋白质。因此，我们有效地使用质谱分析来证明血小板中迄今未报道的蛋白质及其相互作用。

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express DOCK5, a putative Rac guanine nucleotide exchanger, as a CrkL-binding protein. We cloned DOCK5 and raised specific antibodies against the molecule. We overexpressed CrkL, DOCK5, or both in combination in COS7 cells. Overexpressed DOCK5 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both DOCK5 accumulated at CrkL-induced focal adhesions, suggesting a functional implication of the interaction.Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolin-homologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. By mass spectrometry, we found that the proteins, which reportedly interact with WAVE/Scars, are present in platelets.Thus, we have effectively employed mass spectrometry to demonstrate hitherto unreported proteins in platelets and their interactions.