Orlando R A, Cheresh D A
Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1991 Oct 15;266(29):19543-50.
Cell adhesion is characterized by an integrin-mediated ligand binding event followed by reorganization of the actin-cytoskeleton leading to cell spreading and/or migration. In this report we examine the role of integrin alpha v beta 3 in mediating cell attachment to vitronectin or a RGD-containing peptide in the presence of cytochalasin B to prevent actin polymerization. Under these conditions cell attachment to a RGD-containing peptide can be dissociated by excess soluble ligand whereas cells attached to vitronectin cannot. These results suggest that alpha v beta 3-mediated cell attachment to vitronectin results in a highly stabilized interaction that is independent of the actin-cytoskeleton. To investigate the molecular nature of this interaction alpha v beta 3 was purified to homogeneity, and its binding properties toward various ligands were measured in a solid-phase receptor assay. The data indicate that alpha v beta 3 binds to vitronectin or fibronectin in a nondissociable manner whereas a RGD-containing peptide derived from vitronectin binds specifically but is completely dissociable with a Kd of 9.4 x 10(-7) M. Moreover, chemical modification of alpha v beta 3 with limited glutaraldehyde treatment allowed vitronectin to bind in a RGD-dependent and dissociable manner, suggesting that receptor conformational changes or specific amino acid residues proximal to the ligand binding site(s) are involved in the stabilization event. Thus, in the absence of cytoskeletal proteins or other cellular components, integrin alpha v beta 3-ligand binding involves recognition of the RGD sequence leading to a highly stabilized protein-protein association.
细胞黏附的特征是整合素介导的配体结合事件，随后是肌动蛋白细胞骨架的重组，导致细胞铺展和/或迁移。在本报告中，我们研究了整合素αvβ3在细胞松弛素B存在下介导细胞与玻连蛋白或含RGD肽结合的作用，以防止肌动蛋白聚合。在这些条件下，细胞与含RGD肽的结合可被过量的可溶性配体解离，而与玻连蛋白结合的细胞则不能。这些结果表明，αvβ3介导的细胞与玻连蛋白的结合导致了一种高度稳定的相互作用，该相互作用独立于肌动蛋白细胞骨架。为了研究这种相互作用的分子本质，将αvβ3纯化至同质，并在固相受体分析中测量其对各种配体的结合特性。数据表明，αvβ3以不可解离的方式与玻连蛋白或纤连蛋白结合，而源自玻连蛋白的含RGD肽则特异性结合，但可完全解离，解离常数为9.4×10^(-7)M。此外，用有限的戊二醛处理对αvβ3进行化学修饰，使玻连蛋白能够以RGD依赖且可解离的方式结合，这表明受体构象变化或配体结合位点附近的特定氨基酸残基参与了稳定事件。因此，在没有细胞骨架蛋白或其他细胞成分的情况下，整合素αvβ3-配体结合涉及对RGD序列的识别，导致高度稳定的蛋白质-蛋白质结合。
