Identification of signaling molecules associated with β3 integrins using random mutagenesis

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We established cell line expressing chimeric integrin αIIbα6Bβ3 energy-dependently activated. To obtain signaling defective mutant cells that express inactivated αIIbα6Bβ3, the cell line was treated with a chemical mutagen, EMS. Next, we performed expression cloning in which the mutant clone was transfected with cDNA library, and integrin-linked kinase(ILK) was isolated as a molecule induced activation of inactivated αIIbα6Bβ3 in the mutant. Imunoblot analysis showed that the mutant lacked ILK protein. ILK gene in the mutant contained two different nonsense mutations at a compound heterozygous state. ILK knockdown in a parental cell not treated with EMS significantly suppressed integrin activation.