Peptide ligands can bind to distinct sites in integrin αIIbβ3 and elicit different functional responses
Peptide ligands can bind to distinct sites in integrin αIIbβ3 and elicit different functional responses
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DOI：
10.1074/jbc.274.24.16923
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发表时间：
1999-06-11
影响因子：
4.8
通讯作者：
Plow, EF
中科院分区：
文献类型：
--
作者：
Cierniewski, CS;Byzova, T;Plow, EF
已表征了整合素α（IIb）β（3）（GPIIb-IIIa）中两种环肽（环（S，S）KYGCRGDWPC（cRGD）和环（S，S）KYGCH arGDWPC（cHarGD），RGD和HLGGAKQAGDV肽配体的高亲和力类似物）结合位点之间的空间关系。为此，分别用异硫氰酸荧光素和四甲基罗丹明B-异硫氰酸酯标记cRGD和cHarGD。两种环肽均为纤维蛋白原与α（IIb）β（3）结合的强效抑制剂，特别是在Mn 2+存在下;在Mn 2+存在下，cRGD和cHarGD的IC 50值分别为1和< 0.1 nM。使用纯化受体的直接结合实验和荧光共振能量转移分析表明，两种肽同时与α（IIb）β（3）中的不同位点相互作用。这些位点之间的距离估计为6.1 +/-0.5 nm。尽管cRGD优先结合一个位点，而cHarGD优先结合另一个位点，但这些位点不是完全特异性的，并且每个环肽或其线性对应物可以在一定程度上置换另一个。Mn 2+显著影响cHarGD位点的结合亲和力。cRGD而不是cHarGD以阳离子依赖性方式结合重组β（3）-（95-373），表明cRGD位点完全位于该片段内。对于完整的血小板，c-RGD和cHarGD与α（IIb）β（3）的结合导致受体中不同的构象改变，如配体诱导的结合位点表位的差异暴露所示，并且还诱导膜流动性的相反变化，如使用5-doxylstearic acid作为自旋探针的电子顺磁共振分析所示，这些数据支持这两种肽配体结合到α（IIb）β（3）中的不同位点并在受体本身和血小板内引发不同的功能结果的概念。
The spatial relationship between the binding sites for two cyclic peptides, cyclo(S,S)KYGCRGDWPC (cRGD) and cyclo(S,S)KYGCHarGDWPC (cHarGD), high affinity analogs for the RGD and HLGGAKQAGDV peptide ligands, in integrin alpha(IIb)beta(3) (GPIIb-IIIa) has been characterized. For this purpose, cRGD and cHarGD were labeled with fluorescein isothiocyanate and tetramethylrhodamine B-isothiocyanate, respectively, Both cyclic peptides were potent inhibitors of fibrinogen binding to alpha(IIb)beta(3), particularly in the presence of Mn2+; IC50 values for cRGD and cHarGD were 1 and < 0.1 nM in the presence of Mn2+. Direct binding experiments and fluorescence resonance energy transfer analysis using the purified receptor showed that both peptides interacted simultaneously with distinct sites in alpha(IIb)beta(3). The distance between these sites was estimated to be 6.1 +/- 0.5 nm, Although cRGD bound preferentially to one site and cHarGD to the other, the sites were not fully specific, and each cyclic peptide or its Linear counterpart could displace the other to some extent. The binding affinity of the cHarGD site was dramatically affected by Mn2+. cRGD, but not cHarGD, bound to recombinant beta(3)-(95-373) in a cation-dependent manner, indicating that the cRGD site is located entirely within this fragment. With intact platelets, binding of c-RGD and cHarGD to alpha(IIb)beta(3) resulted in distinct conformational alterations in the receptor as indicated by the differential exposure of Ligand-induced binding site epitopes and also induced the opposite on membrane fluidity as shown by electron paramagnetic resonance analyses using 5-doxylstearic acid as a spin probe, These data support the concept the two peptide Ligands bind to distinct sites in alpha(IIb)beta(3) and initiate different functional consequences within the receptor itself and within platelets.