αIIb’s cytoplasmic domain is not required for ligand-induced clustering of integrin αIIbβ3

Abstract

The platelet integrin αIIbβ3 exhibits bidirectional signaling, in that intracellular messengers enable adhesive macromolecules to bind to its ectodomain, while ligation promotes the association of cytoskeletal proteins with its cytoplasmic domains. In order to understand the linkage between these distant regions, we investigated the effects of receptor occupancy on the solution structure of both full-length recombinant αIIbβ3 and αIIbΔ991β3, an integrin truncation mutant which lacks one cytoplasmic domain. Lysates of 35S-labeled human A549 cells expressing either full-length αIIbβ3 or αIIbΔ991β3 were examined by sucrose density gradient sedimentation followed by immunoprecipitation to determine the distributions of integrin protomers and oligomers. Recombinant αIIbβ3 exhibited a weight-average sedimentation coefficient, S w=11.3±1.4 S with 73% sedimenting as protomers/dimers (9.1±1.0 S) and 27% as oligomers (15.4±0.4 S). Truncation mutant αIIbΔ991β3 exhibited a similar pattern with 65% sedimenting as protomers/dimers. Upon ligation with eptifibatide, both full-length αIIbβ3 and αIIbΔ991β3 sedimented mainly at >14 S, indicating 2–3-fold increased oligomerization. Thus we have demonstrated that αIIb’s cytoplasmic region is not required for integrin clustering, a key event in outside-in signaling.