Disrupting integrin transmembrane domain heterodimerization increases ligand binding affinity, not valency or clustering
Disrupting integrin transmembrane domain heterodimerization increases ligand binding affinity, not valency or clustering
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DOI：
10.1073/pnas.0409440102
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发表时间：
2005-03-08
影响因子：
11.1
通讯作者：
Springer, TA
中科院分区：
文献类型：
--
作者：
Luo, BH;Carman, CV;Springer, TA
整合素α (IIb)-和β(3)-亚基的23个残基跨膜结构域之间相互作用的重要残基通过将每个非Leu残基突变为Leu来鉴定。α (IIb)在G972、G976和T981上的Leu取代，以及β(3)在1693和G708上的Leu取代，增加了配体结合。在α (IIb)G972和β (3)G708上取代其他氨基酸也可以增加配体的结合。该结果与先前通过半胱氨酸扫描和二硫键形成定义的整合素α和β亚基TM结构域之间的螺旋界面一致并扩展了该结构域。我们通过TM结构域突变区分了基于亲和和基于价的激活模式。突变体α (IIb) W967C在(α (IIb)) β(3))(2)四聚体中形成二硫化物连接的α (IIb)-亚基。该四聚体表现为价模式调控的理想模型，因为它与多价而非单价配体的结合明显增加，并且基本保持弯曲构象。相比之下，激活的Leu突变体与单价拟配体PAC-1 Fab的结合增加，与配体诱导结合位点（LIBS）表位的暴露增加，表明它们部分采用了延伸的构象。此外，先前描述的β (3)G708N突变在中国仓鼠卵巢细胞中增强了配体结合亲和力，而不是价态，并且没有改变共聚焦显微镜定义的细胞表面聚类。我们的研究证明，破坏整合素异二聚体TM螺旋-螺旋界面激活配体结合主要是通过增加配体的单体亲和力，而不是受体价，即聚类。
Residues important in the interaction between the 23-residue transmembrane (TM) domains of the integrin alpha(IIb)- and beta(3)-subunits were identified by mutating each non-Leu residue to Leu. Leu substitutions of alpha(IIb) at G972, G976, and T981, and Of beta(3) at 1693 and G708, increased ligand binding. Substitutions with other amino acids at alpha(IIb)G972 and beta(3)G708 could also increase ligand binding. The results are consistent with and extend the helical interface between the integrin alpha- and beta-subunit TM domains previously defined by cysteine scanning and disulfide bond formation. We differentiated between affinity- and valency-based modes of activation by TM domain mutations. The mutant alpha(IIb) W967C forms disulfide-linked alpha(IIb)-Subunits within an (alpha(IIb))beta(3))(2) tetramer. This tetramer behaved as an ideal model for the valency mode of regulation, because it exhibited significantly increased binding to multivalent but not monovalent ligands and basally retained the bent conformation. By contrast, the activating Leu mutants showed increased binding to the monovalent, ligand-mimetic PAC-1 Fab and increased exposure of ligand-induced binding site (LIBS) epitopes, suggesting that they partially adopt an extended conformation. Furthermore, the previously described beta(3)G708N mutation in Chinese hamster ovary cells enhanced ligand binding affinity, not valency, and did not alter cell-surface clustering as defined by confocal microscopy. Our studies provide evidence that disrupting the integrin heterodimeric TM helix-helix interface activates ligand binding mainly by increasing the monomeric affinity for ligand, but not the receptor valency, i.e., clustering.