Kashiwagi H, Schwartz M A, Eigenthaler M, Davis K A, Ginsberg M H, Shattil S J
Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Cell Biol. 1997 Jun 16;137(6):1433-43. doi: 10.1083/jcb.137.6.1433.
Platelet agonists increase the affinity state of integrin alphaIIbbeta3, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. beta3-Endonexin is a novel 111-amino acid protein that binds selectively to the beta3 tail. Since beta3-endonexin is present in platelets, we asked whether it can affect alphaIIbbeta3 function. When beta3-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor alphaIIbbeta3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and alphaIIbbeta3 bound little or no PAC1. However, those transfected with GFP/beta3-endonexin and alphaIIbbeta3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/beta3-endonexin did not affect levels of surface expression of alphaIIbbeta3 nor did it modulate the affinity of an alphaIIbbeta3 mutant that is defective in binding to beta3-endonexin. Affinity modulation of alphaIIbbeta3 by GFP/beta3-endonexin was inhibited by coexpression of either a monomeric beta3 cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that beta3-endonexin can modulate the affinity state of alphaIIbbeta3 in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein-protein interactions at the level of the cytoplasmic tails of alphaIIbbeta3.
血小板激动剂可增加整合素αIIbβ3的亲和状态，这是纤维蛋白原结合和血小板聚集的前提条件。该过程可能由一种与整合素细胞质尾巴结合的调节分子触发，从而引起受体的结构变化。β3内毒素是一种新型的111个氨基酸的蛋白质，它选择性地与β3尾巴结合。由于β3内毒素存在于血小板中，我们研究了它是否能影响αIIbβ3的功能。当β3内毒素与绿色荧光蛋白（GFP）融合并转染到CHO细胞中时，它在细胞质和细胞核中均有发现，并且可以在细胞裂解物的蛋白质印迹中检测到。PAC1是一种模拟纤维蛋白原的单克隆抗体，用于通过流式细胞术监测转染细胞中αIIbβ3的亲和状态。转染了GFP和αIIbβ3的细胞几乎不结合或不结合PAC1。然而，转染了GFP/β3内毒素和αIIbβ3的细胞以能量依赖的方式特异性结合PAC1，并且它们发生了纤维蛋白原依赖性聚集。GFP/β3内毒素不影响αIIbβ3的表面表达水平，也不调节与β3内毒素结合有缺陷的αIIbβ3突变体的亲和力。GFP/β3内毒素对αIIbβ3的亲和力调节被单体β3细胞质尾巴嵌合体或活化形式的H-Ras的共表达所抑制。这些结果表明，β3内毒素可以以结构特异性且受代谢调节的方式调节αIIbβ3的亲和状态。类似地，血小板的黏附功能可能通过αIIbβ3细胞质尾巴水平的这种蛋白质-蛋白质相互作用来调节。
