Binding and Uptake of RGD-Containing Ligands to Cellular αvβ3 Integrins
Binding and Uptake of RGD-Containing Ligands to Cellular αvβ3 Integrins
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DOI：
10.1007/s10989-008-9163-y
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发表时间：
2009-03-01
影响因子：
2.5
通讯作者：
Cullis, Pieter R.
中科院分区：
文献类型：
--
作者：
Cressman, Sonya;Sun, Ying;Cullis, Pieter R.
环肽 cRGDf[N(me)]V 与 α(v)beta(3) 整联蛋白结合，并可破坏整联蛋白与其在细胞外基质中的天然配体的结合。在这项工作中，表征了含有 RGD 的肽 (cRGDfK-488) 的水溶性荧光标记变体与人脐带血管内皮细胞 (HUVEC) 上的整合素结合并随后进行内吞作用的能力。该信息与 α(v)β(3) 整合素特异性单克隆抗体 LM609X 的结合和摄取特性进行了比较。通过与不结合 alpha(v)beta(3) 整联蛋白 cRADfK-488 的对照肽进行比较，评估含有 RGD 的肽的特异性。使用高纯度构建体，显示 RGD 配体表现出微摩尔范围内的解离常数，而 LM609X 表现出纳摩尔范围内的解离常数。然而，在允许内吞作用的温度下孵育后，RGD 配体显示出更大的摄取。与 4 摄氏度（内吞作用禁止温度）下的情况相比，在 37 摄氏度（内吞作用允许温度）下与 HUVEC 孵育 1 小时后，观察到 RGD 肽的摄取增加了 7.4 倍。相比之下，使用 LM609X 进行类似孵育时，仅观察到细胞相关荧光增加了 1.9 倍。荧光显微镜结果支持这样的观点：与 LM609X 相比，RGD 肽在 37 摄氏度下被快速内吞。这些结果是关于先前的工作进行讨论的，表明 RGD 配体通过整合素独立途径进入细胞。这些研究提供了 RGD 配体如何刺激内吞作用的良好控制措施。这对于抗血管生成应用中配体相关药物的细胞内递送可能具有相当大的意义。
The cyclic peptide, cRGDf[N(me)]V, binds to the alpha(v)beta(3) integrin and can disrupt binding of the integrin to its natural ligands in the extracellular matrix. In this work, the ability of a water-soluble, fluorescently labeled variant of the RGD-containing peptide (cRGDfK-488) to bind to integrins on human umbilical vascular endothelial cells (HUVEC) and subsequently undergo endocytosis was characterized. This information was compared to the binding and uptake properties of an alpha(v)beta(3) integrin-specific monoclonal antibody, LM609X. The specificity of the RGD-containing peptide is assessed by comparison with control peptide that does not bind to the alpha(v)beta(3) integrin, cRADfK-488. Using a high purity construct, it is shown that the RGD ligand exhibits dissociation constants in the micromolar range whereas LM609X exhibits dissociation constants in the nanomolar range. However, the RGD ligand showed greater uptake following incubation at temperatures which permit endocytosis. A 7.4-fold increase in uptake of the RGD peptide was observed following a 1 h incubation with HUVEC at 37 degrees C (an endocytosis permissive temperature), as compared to that at 4 degrees C (an endocytosis prohibitive temperature). In contrast, only a 1.9-fold increase in cell-associated fluorescence was observed for similar incubations with LM609X. Results from fluorescence microscopy supports the notion that the RGD peptide is rapidly endocytosed at 37 degrees C as compared to LM609X. These results are discussed with regard to previous work indicating that RGD ligands enter cells by integrin-independent pathways. These studies provide well-controlled measures of how RGD ligands stimulate endocytosis. This may be of considerable interest for intracellular delivery of ligand-associated drugs in anti-angiogenic applications.