Regulatory mechanisms of ligand binding and signaling events of laminin-binding integrins

CD151是Tetraspanins的一种，它与细胞表面主要的层粘连蛋白受体整合素α3beta1形成稳定的复合体。我们发现，通过筛选与整合素α3beta1-CD151复合物反应而获得的抗CD151单抗8C3能够将CD151从整合素α3beta1中解离出来，从而使我们能够从纯化的整合素α3beta1-CD151复合物中去除CD151。由此获得的不含CD151的整合素α3beta1与层粘连蛋白-10/11的结合能力显著降低，层粘连蛋白-10/11是整合素α3beta1的高亲和力配体，同时其与识别活化的含有β1的整合素的单抗AG89的反应性也随之降低。这些结果表明，整合素α3beta1与CD151的结合可能通过维持其激活构象来增强整合素的配体结合活性。为了支持这种可能性，当不含CD151的整合素α3beta1与纯化的CD151重新结合时，配体结合活性被恢复。FRET成像显示，8C3诱导的CD151与整合素α3beta1在活细胞表面的解离，伴随着细胞与层粘连蛋白-10/11包被底物的粘附性降低。RNAi下调CD151也可降低细胞的黏附活性。综上所述，这些结果表明，CD151结合不仅通过稳定整合素α3beta1与纯化蛋白的激活构象，而且在生理环境中调节整合素α3beta1的配体结合活性。

CD151, one of the tetraspanins, forms a stable complex with integrin alpha3beta1, the major laminin receptor on the cell surface. We found that 8C3, an anti-CD151 mAb obtained by screening for reactivity with integrin alpha3beta1-CD151 complexes, was capable of dissociating CD151 from integrin alpha3beta1, thereby allowing us to deplete CD151 from purified integrin alpha3beta1-CD151 complexes. The CD151-free integrin alpha3beta1 thus obtained showed a significant reduction in its ability to bind to laminin-10/11, a high-affinity ligand for integrin alpha3beta1, with a concomitant reduction in its reactivity with mAb AG89, which recognizes activated beta1-containing integrins. These results raised the possibility that the association of integrin alpha3beta1 with CD151 potentiates the ligand-binding activity of the integrin through sustaining its activated conformation. In support of this possibility, the ligand-binding activity was restored when CD151-free integrin alpha3beta1 was re-associated with purified CD151. 8C3-induced dissociation of CD151 from integrin alpha3beta1 was also demonstrated on the surface of living cells by FRET imaging, accompanied by a concomitant reduction in the cell adhesion to laminin-10/11-coated substrates. CD151 knock-down by RNAi also resulted in a reduction in the adhesive activity of the cells. Taken together, these results indicate that CD151 association modulates the ligand-binding activity of integrin alpha3beta1 through stabilizing its activated conformation not only with purified proteins but also in a physiological context.