Banno Asoka, Ginsberg Mark H
Department of Medicine, University of California, San Diego, 9500 Gilman Drive, MC 0726, San Diego, CA 92093-0726, USA.
Biochem Soc Trans. 2008 Apr;36(Pt 2):229-34. doi: 10.1042/BST0360229.
Agonist stimulation of integrin receptors, composed of transmembrane alpha and beta subunits, leads cells to regulate integrin affinity ('activation'), a process that controls cell adhesion and migration, and extracellular matrix assembly. A final step in integrin activation is the binding of talin to integrin beta cytoplasmic domains. We used forward, reverse and synthetic genetics to engineer and order integrin activation pathways of a prototypic integrin, platelet alphaIIbbeta3. PMA activated alphaIIbbeta3 only after expression of both PKCalpha (protein kinase Calpha) and talin at levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas expression of constitutively active Rap1A(G12V) bypassed the requirement for PKCalpha. Overexpression of a Rap effector, RIAM (Rap1-GTP-interacting adaptor molecule), activated alphaIIbbeta3 and bypassed the requirement for PKCalpha and Rap1. In addition, shRNA (short hairpin RNA)-mediated knockdown of RIAM blocked talin interaction with and activation of integrin alphaIIbbeta3. Rap1 activation caused the formation of an 'activation complex' containing talin and RIAM that redistributed to the plasma membrane and activated alphaIIbbeta3. The central finding was that this Rap1-induced formation of an 'integrin activation complex' leads to the unmasking of the integrin-binding site on talin, resulting in integrin activation.
由跨膜α和β亚基组成的整合素受体的激动剂刺激，会使细胞调节整合素亲和力（“激活”），这一过程控制细胞黏附、迁移以及细胞外基质组装。整合素激活的最后一步是踝蛋白与整合素β细胞质结构域的结合。我们运用正向、反向和合成遗传学技术来构建并梳理一种典型整合素——血小板αIIbβ3的整合素激活途径。仅在蛋白激酶Cα（PKCα）和踝蛋白的表达水平接近血小板中的水平时，佛波酯（PMA）才会激活αIIbβ3。抑制Rap1 GTP酶会降低αIIbβ3的激活，而组成型活性Rap1A（G12V）的表达则绕过了对PKCα的需求。一种Rap效应器RIAM（Rap1-GTP相互作用衔接分子）的过表达激活了αIIbβ3，并绕过了对PKCα和Rap1的需求。此外，短发夹RNA（shRNA）介导的RIAM敲低阻断了踝蛋白与整合素αIIbβ3的相互作用及其激活。Rap1激活导致形成一个包含踝蛋白和RIAM的“激活复合物”，该复合物重新分布到质膜并激活αIIbβ3。核心发现是，这种由Rap1诱导形成的“整合素激活复合物”导致踝蛋白上整合素结合位点的暴露，从而引发整合素激活。
