Specific cysteines in β3 are involved in disulfide bond exchange-dependent and -independent activation of αIIbβ3
Specific cysteines in β3 are involved in disulfide bond exchange-dependent and -independent activation of αIIbβ3
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DOI：
10.1074/jbc.m802399200
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发表时间：
2008-07-11
影响因子：
4.8
通讯作者：
Seligsohn, Uri
中科院分区：
文献类型：
--
作者：
Mor-Cohen, Ronit;Rosenberg, Nurit;Seligsohn, Uri
为了研究特定的β3半胱氨酸在αIIbβ3表达和激活中的作用，我们分别用P2或PAC-1抗体，用流式细胞术检测了EGF-3、EGF-4和β-尾区9个二硫键的单双半胱氨酸取代的正常αIIb和β3在正常αIIb和β3细胞中的表达。大多数突变体表面αIIbβ3的表达降低。EGF-3中二硫键的破坏产生了结构性活性的αIIbβ3，这意味着这些键稳定了非活性的αIIbβ3构象。EGF-4中Cys-567-Cys-581键的突变体即使在暴露于αIIbβ3激活抗体后也是无效的，这表明该键是激活αIIbβ3所必需的。干扰EGF-3/EGF-4中的Cys-560-Cys-583或β-尾段中的Cys-608-Cys-655只在每对Cys-560或Cys-655发生突变时才会导致αIIbβ3的激活，而当它们的伴侣(Cys-583，Cys-608)或两个半胱氨酸都发生突变时，则不会引起αIIbβ3的激活。提示Cys-583和Cys-608的游离基通过二硫键交换机制参与了αIIbβ3的活化。在抗LIBS6抗体诱导的野生型αIIbβ3的激活中，二硫代双硝基苯甲酸抑制了70%，而对突变体的影响较小，提示在αIIbβ3的激活中存在二硫键交换依赖和非依赖的机制。这些数据表明，在β3、EGF和β-尾区中，不同的二硫键在αIIbβ3中发挥着不同的结构和调节作用。
Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of beta 3 was suggested to be involved in activation of alpha IIb beta 3. To investigate the role of specific beta 3 cysteines in alpha IIb beta 3 expression and activation, we expressed in baby hamster kidney cells normal alpha IIb with normal beta 3 or beta 3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and beta-tail domains and assessed alpha IIb beta 3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of alpha IIb beta 3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active alpha IIb beta 3, implying that these bonds stabilize the inactive alpha IIb beta 3 conformer. Mutants of the Cys-567-Cys-581 bond in EGF-4 were inactive even after exposure to alpha IIb beta 3-activating antibodies, indicating that this bond is necessary for activating alpha IIb beta 3. Disrupting Cys-560-Cys-583 in the EGF-3/EGF-4 or Cys-608-Cys-655 in beta-tail domain resulted in alpha IIb beta 3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in alpha IIb beta 3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type alpha IIb beta 3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in alpha IIb beta 3 activation. These data suggest that different disulfide bonds in beta 3 EGF and beta-tail domains play variable structural and regulatory roles in alpha IIb beta 3.