Thibault G
Laboratoire de Biologie Cellulaire de l'Hypertension, Institut de Recherches Cliniques de Montréal and Université de Montréal, Montréal, Quebec, Canada.
Mol Pharmacol. 2000 Nov;58(5):1137-45. doi: 10.1124/mol.58.5.1137.
This study shows that disintegrins, echistatin as a model, can be used as a radiolabeled probe to simultaneously detect the presence of individual RGD-dependent integrins on cardiac fibroblasts. Binding of (125)I-echistatin to fibroblasts was proportional to cell number, time dependent, reversible, saturable, specific, and membrane bound. SDS-polyacrylamide gel electrophoresis and autoradiograms revealed that (125)I-echistatin was associated with three radioactive protein bands of 180, 210, and 220 kDa that were identified by RGD affinity chromatography, immunoblotting, and immunoneutralization as alpha(v)beta(3), alpha(3)beta(1)/alpha(5)beta(1)/alpha(v)beta(1), and alpha(8)beta(1) heterodimeric integrins, respectively. These results suggest that echistatin binds to RGD-dependent integrins, forming SDS-stable complexes in the absence of chemical cross-linkers, reducing conditions and heating. As assessed by radioligand-binding filtration, disintegrins displayed binding characteristics with an IC(50) ranging from 0.044 to 1.1 nM, but with slope factors lower than 1, indicating the presence of several binding sites. Resolved by SDS-polyacrylamide gel electrophoresis to reveal echistatin-integrin complexes, disintegrins and RGD peptides displayed different binding affinities to individual RGD-dependent integrins present on cardiac fibroblasts. Elegantin and flavostatin demonstrated the highest affinity toward integrins, whereas flavoridin and acPenRGDC had a greater specificity toward alpha(v)beta(3)-integrin. In summary, echistatin forms SDS-stable complexes with RGD-dependent integrins. This model offers a novel way to visualize RGD-dependent integrins, to investigate their activation state, and to determine the integrin specificity of RGD peptides.
本研究表明，以echistatin为模型的去整合素可作为放射性标记探针，用于同时检测心脏成纤维细胞上单个RGD依赖性整合素的存在。(125)I-echistatin与成纤维细胞的结合与细胞数量成正比，具有时间依赖性、可逆性、饱和性、特异性且为膜结合型。SDS-聚丙烯酰胺凝胶电泳和放射自显影片显示，(125)I-echistatin与180、210和220 kDa的三条放射性蛋白带相关，通过RGD亲和色谱、免疫印迹和免疫中和分别鉴定为α(v)β(3)、α(3)β(1)/α(5)β(1)/α(v)β(1)和α(8)β(1)异二聚体整合素。这些结果表明，echistatin与RGD依赖性整合素结合，在不存在化学交联剂、还原条件和加热的情况下形成SDS稳定复合物。通过放射性配体结合过滤评估，去整合素显示出结合特性，IC(50)范围为0.044至1.1 nM，但斜率因子低于1，表明存在多个结合位点。通过SDS-聚丙烯酰胺凝胶电泳分离以揭示echistatin-整合素复合物，去整合素和RGD肽对心脏成纤维细胞上存在的单个RGD依赖性整合素表现出不同的结合亲和力。Elegantin和flavostatin对整合素表现出最高亲和力，而flavoridin和acPenRGDC对α(v)β(3)-整合素具有更高特异性。总之，echistatin与RGD依赖性整合素形成SDS稳定复合物。该模型提供了一种可视化RGD依赖性整合素、研究其激活状态以及确定RGD肽整合素特异性的新方法。
