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Integrin clustering mechanisms explored with a soluble αIIbβ3 ectodomain construct

Authors
Journal
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
1570-9639
Publisher
Elsevier
Publication Date
Volume
1700
Issue
1
Identifiers
DOI: 10.1016/j.bbapap.2004.03.011
Keywords
  • Integrin
  • Activation
  • Oligomerization
  • Clustering
  • Ligand Binding
  • Signaling
Disciplines
  • Biology

Abstract

Abstract The purpose of this study was to test the hypothesis that residues critical for ligand- and temperature-induced clustering of integrin αIIbβ3 are present on its extracellular domain. Sucrose density gradient sedimentation was used to examine the effects of ligand-mimetic peptides and physiological temperature on the oligomeric state of a soluble recombinant ectodomain variant of the αIIbβ3 integrin, αIIbΔ962β3Δ692, and its full-length counterpart. Both the ectodomain construct, isolated from High Five insect cell culture supernatants, and αIIbβ3, isolated from human blood platelets, exhibited similar weight-average sedimentation coefficients at 23 °C, in the absence and presence of the ligand-mimetic peptide eptifibatide. These observations indicate that αIIbβ3's ectodomain exhibits a similar extended conformation in both its free and ligand-bound states. Oligomerization was examined by incubation of both αIIbΔ962β3Δ692 and full-length receptors at 37 °C, in the presence or absence of ligand-mimetic. Minimal oligomerization was observed with αIIbΔ962β3Δ692. In contrast, full-length αIIbβ3 exhibited substantial temperature-induced increases in its distribution of sedimenting species, indicative of thermal aggregation. These observations suggest that optimum oligomerization requires the participation of the integrin's transmembrane and cytoplasmic regions. In vivo, clustering of ligand-bound integrins may enhance signaling by increasing the local concentration of intracellular integrin-associated proteins.

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