Basson C T, Knowles W J, Bell L, Albelda S M, Castronovo V, Liotta L A, Madri J A
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Cell Biol. 1990 Mar;110(3):789-801. doi: 10.1083/jcb.110.3.789.
Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs.
含精氨酸 - 甘氨酸 - 天冬氨酸（RGD）的可溶性肽可抑制牛主动脉内皮细胞（BAEC）与层粘连蛋白、纤连蛋白和纤维蛋白原的附着，并且YGRGDSP活性对可溶性肽或基质蛋白的滴定有反应。为了评估RGD依赖性受体的存在，进行了免疫沉淀和免疫印迹研究，结果表明整合素β1、β3以及相关的α亚基和一种β1前体。接种在层粘连蛋白、纤连蛋白和纤维蛋白原上的BAEC的免疫荧光显示了这些整合素亚类各自不同的基质结合特异性。接种后1小时，层粘连蛋白和纤连蛋白会影响β1整合素形成纤维状条纹的组织，而纤维蛋白原会影响β3整合素的点状组织，并且整合素的空间分布在培养过程中会随时间变化。相比之下，非整合素层粘连蛋白结合蛋白LB69仅在接种数小时后细胞 - 底物接触充分建立后才会形成组织。BAEC的迁移也由整合素和非整合素基质结合蛋白介导。具体而言，BAEC在层粘连蛋白上的迁移对RGD肽抑制非常敏感，并且在其存在下，β1整合素的组织会消散并重新组织成核周小泡。然而，RGD肽在迁移过程中不会改变LB69的线性组织。同样，阻断LB69的试剂，如抗LB69抗体以及YIGSR - NH2肽，不会抑制静止的BAEC与层粘连蛋白的附着。但是，抗LB69和YIGSR - NH2都能抑制晚期黏附事件，如铺展。因此，我们提出在附着过程中，BAEC中的整合素和非整合素细胞外基质结合蛋白组织在时间和空间上都是分离的。与基质的高亲和力非整合素相互作用可能为长期附着创造必要的稳定接触，而较低亲和力的整合素对于初始细胞黏附以及运动性BAEC的瞬时接触可能很重要。
