Helsten Teresa L, Bunch Thomas A, Kato Hisashi, Yamanouchi Jun, Choi Sharon H, Jannuzi Alison L, Féral Chloe C, Ginsberg Mark H, Brower Danny L, Shattil Sanford J
Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Mol Biol Cell. 2008 Aug;19(8):3589-98. doi: 10.1091/mbc.e08-01-0085. Epub 2008 May 28.
Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila alphaPS2 betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2 betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2 betaPS with those of human alphaIIb beta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2 betaPS with those of alphaIIb beta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIb beta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2 betaPS affinity because of structural features inherent in the alphaPS2 betaPS extracellular and/or transmembrane domains.
整合素介导的细胞黏附对于多细胞生物的发育至关重要。在蠕虫、果蝇和脊椎动物中，踝蛋白在整合素细胞质结构域与肌动蛋白细胞骨架之间形成物理连接。整合素或踝蛋白的缺失会导致相似的表型。在脊椎动物中，踝蛋白也是整合素亲和力的关键调节因子。我们使用一种模拟配体的Fab片段TWOW-1来评估踝蛋白在调节果蝇αPS2βPS亲和力中的作用。细胞代谢能量的消耗降低了TWOW-1的结合，这表明αPS2βPS亲和力是一个活跃过程，就像脊椎动物整合素一样。与脊椎动物整合素不同，RNA干扰敲低踝蛋白或过表达踝蛋白头部对TWOW-1结合均无显著影响。此外，用人类αIIbβ3的跨膜或踝蛋白结合细胞质结构域替换αPS2βPS的相应结构域，未能使踝蛋白调节TWOW-1结合。然而，用αIIbβ3的细胞外和跨膜结构域替换αPS2βPS的这些结构域，产生了一种组成型激活的整合素，其亲和力因敲低踝蛋白而降低。此外，野生型αIIbβ3通过过表达果蝇踝蛋白头部结构域而被激活。因此，尽管踝蛋白的整合素/细胞骨架连接功能在进化上具有保守性，但由于αPS2βPS细胞外和/或跨膜结构域固有的结构特征，踝蛋白不足以调节果蝇αPS2βPS的亲和力。
