A new functional role of the fibrinogen RGD motif as the molecular switch that selectively triggers integrin αIIbβ3-dependent RhoA activation during cell spreading
A new functional role of the fibrinogen RGD motif as the molecular switch that selectively triggers integrin αIIbβ3-dependent RhoA activation during cell spreading
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DOI：
10.1074/jbc.m500146200
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发表时间：
2005-09-30
影响因子：
4.8
通讯作者：
Kieffer, N
中科院分区：
文献类型：
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作者：
Salsmann, A;Schaffner-Reckinger, E;Kieffer, N
许多RGD型整合素除了依赖典型的RGD位点外，还依赖一个协同位点来进行配体结合和信号传递，尽管目前尚不清楚这两个识别位点是独立发挥作用，还是协同作用，还是相互竞争。实验证据表明，纤维蛋白原与RGD型整合素αIIbβ3的结合仅通过协同伽马(400-411)序列发生，从而质疑RGD识别位点的功能作用。在这里，我们研究了纤维蛋白原γ(400-411)序列和RGD基序在导致配体诱导的依赖于αIIbβ3的中国仓鼠卵巢(CHO)细胞或血小板扩散的分子事件中的各自作用，方法是使用完整的纤维蛋白原和具有良好特性的纤溶酶生成的纤维蛋白原片段，该片段包含RGD基序(片段C)或伽马(400-411)序列(片段D)，以及表达静止的野生型(αIIbβ3wt)、结构性活性(αIIbβ3T562N)或无功能的(αIIbβ3D119Y)受体的CHO细胞。我们的数据提供了证据，证明伽马(400-411)位点本身能够启动αIIbβ3聚集和细胞内蛋白质向早期焦点复合体的募集，介导细胞附着、FAK磷酸化和rac1激活，而RGD基序随后作为β3亚单位上的分子开关来触发细胞扩散。更重要的是，我们发现RGD位点的主要功能作用不是加强细胞附着，而是在β3亚基上印记构象变化，从而在CHO细胞和血小板中最大限度地激活RhoA和肌动蛋白细胞骨架组织。最后，依赖于αIIbβ3的RhoA刺激和细胞扩散，而不是细胞附着，是依赖于Src的，不依赖于磷脂酰肌醇3-激酶，并被Src拮抗剂PP2抑制。
A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alpha IIb beta 3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alpha IIb beta 3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif ( fragment C) or the gamma(400-411) sequence ( fragment D), and CHO cells expressing resting wild type (alpha IIb beta 3wt), constitutively active (alpha IIb beta 3T562N), or non-functional (alpha IIb beta 3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alpha IIb beta 3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta 3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta 3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alpha IIb beta 3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2.