Analysis of Laminin - alpha7beta1 Integrin Interactions: Ligand specificity and Binding sites

α 7 β 1整联蛋白-除了α-肌营养不良聚糖-是骨骼肌、心肌和平滑肌中的主要层粘连蛋白受体。主要作用于肌腱接头和神经肌肉突触，已被证明对骨骼肌的功能维持和再生至关重要。因此，在小鼠和人中破坏a7基因itga7会导致进行性肌营养不良和肌病。此外，它已被定位在某些黑色素瘤细胞和胶质母细胞瘤细胞，以及在胚胎滋养外胚层细胞，它是负责胚泡植入。整合素a7b1是一种跨膜蛋白复合物，以选择性剪接的形式存在，影响细胞外和细胞内结构域，这些结构域是组织特异性和发育调节的。然而，对这些剪接变体的具体作用及其与不同层粘连蛋白亚型的相互作用知之甚少。先前我们可以证明两种胞外剪接变体a7X1和a7X2显示与层粘连蛋白同种型层粘连蛋白-8和层粘连蛋白-10相对于层粘连蛋白-1的选择性结合。该项目的目的是通过交换从α v整合素的晶体结构和种间保守序列预测的X1和X2结构域之间的推定结合结构域来确定确切的层粘连蛋白结合位点。进一步的方法包括定点诱变和用相关的α 6 X 1和α 6 X 2结构域交换结构域。将制备针对α 7X1和α 7X2剪接变体的抗体，以将剪接变体相对于其层粘连蛋白同种型配体定位在胚胎和成体组织中。通过这些研究，我们提出了阐明整合素-层粘连蛋白相互作用的机制，并深入了解骨骼肌和其他组织中α 7剪接变体及其相应的层粘连蛋白亚型的差异功能。

Alpha7beta1 integrin is - besides a-dystroglycan - the major laminin receptor in skeletal, cardiac and smooth muscle. Focussed to myotendinous junctions and neuromuscular synapses, is has been shown to be essential for functional maintenance and regeneration of skeletal muscle. Thus, disruption of the a7 gene itga7 in mice and men leads to progressive muscular dystrophy and myopathy. Furthermore, it has been localized on certain melanoma cells and glioblastoma cells, as well as in embryonic trophectoderm cells where it is responsible for blastocyst impantation. Integrin a7b1, a transmembrane protein complex, exists in alternatively spliced forms affecting the extracellular and intracellular domains, which are tissue specifically and developmentally regulated. Little is known, however, on the specific roles of these splice variants and their interaction with the different laminin isoforms. Previously we could demonstrate that the two extracellular splice variants a7X1 and a7X2 show selective binding to the laminin isoforms laminin-8 and -10 versus laminin-1. The aim of this project is to identify the exact laminin binding sites by swapping putative binding domains between the X1 and X2 domains predicted from the crystallographic structure of av integrin and from interspecies conserved sequences. Further approaches include site directed mutagenesis and swapping domains with the related a6X1 and a6X2 domains. Antibodies against the a7X1 and a7X2 splice variants will be prepared in order to localize the splice variants in relation to their laminin isoform ligands in embryonic and adult tissues. With these studies we propose to elucidate the mechanism of integrin-laminin interactions and to gain insight into the differential functions of a7 splice variants and their corresponding laminin isoforms in skeletal muscle and other tissues.