Construction of an activated, high-affinity GP IIb/IIIa (alpha(IIb)beta(3)) integrin receptor and its use for the determination of the role of affinity and cytoskeletal anchorage in cell adhesion, cell migration and fibronectin matrix assembly

The platelet fibrinogen receptor GP IIb/IIIa (alpha(IIb)beta(3)) is a prototypic adhesion molecule of the integrin family. These adhesion molecules are functionally regulated either by changes in affinity to the ligand or by changes in their cytoskeletal anchorage. Recombinant GP IIb/IIIa receptors demonstrating various functional states in respect to their affinity and cytoskeletal anchorage would allow the determination of the relative role of both in a variety of integrin-mediated processes. Thus, GP IIb/IIIa mutants were created which demonstrate a fixed high and low-affinity state and a stable or destroyed cytoskeletal anchorage. The high-affinity state was achieved by a mutation of all integrins, highly conserved in a GFFKR region at the N-terminus of the cytoplasmic domain of the or subunit. A loss of the integrin cytoskeletal anchorage could be achieved by the deletion of the cytoplasmic domain of the beta subunit. Using these mutant cell lines, it could be demonstrated that changes in the integrin cytoskeletal anchorage can be sufficient to regulate integrin-mediated cell adhesion without changes in integrin affinity. High-affinity mutants of GP IIb/IIIa severely inhibit cell migration. This finding is consistent with the concept that integrin affinity changes are necessary for cell migration. Furthermore, the use of the GP Ilb/IIIa mutant cell lines allowed definition of the high-affinity integrin receptor as a prerequisite for the assembly of a fibronectin matrix. In conclusion, cell lines expressing recombinant mutant integrins proved to be unique tools to evaluate basic mechanisms in integrin-mediated processes.