AlphaIIb's cytoplasmic domain is not required for ligand-induced clustering of integrin alphaIIbbeta3

Biochim Biophys Acta. 2001 Jul 25;1540(1):82-95. doi: 10.1016/s0167-4889(01)00120-3.

Abstract

The platelet integrin alphaIIbbeta3 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 alphaIIbbeta3 and alphaIIbDelta991beta3, an integrin truncation mutant which lacks one cytoplasmic domain. Lysates of (35)S-labeled human A549 cells expressing either full-length alphaIIbbeta3 or alphaIIbDelta991beta3 were examined by sucrose density gradient sedimentation followed by immunoprecipitation to determine the distributions of integrin protomers and oligomers. Recombinant alphaIIbbeta3 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 alphaIIbDelta991beta3 exhibited a similar pattern with 65% sedimenting as protomers/dimers. Upon ligation with eptifibatide, both full-length alphaIIbbeta3 and alphaIIbDelta991beta3 sedimented mainly at >14 S, indicating 2-3-fold increased oligomerization. Thus we have demonstrated that alphaIIb's cytoplasmic region is not required for integrin clustering, a key event in outside-in signaling.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line
  • Cell Membrane / metabolism
  • Centrifugation, Density Gradient
  • Cloning, Molecular
  • Cytoplasm / chemistry
  • Flow Cytometry
  • Gene Deletion
  • Humans
  • Ligands
  • Mutation
  • Platelet Glycoprotein GPIIb-IIIa Complex / chemistry*
  • Platelet Glycoprotein GPIIb-IIIa Complex / genetics
  • Platelet Glycoprotein GPIIb-IIIa Complex / metabolism
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Signal Transduction
  • Transfection
  • Vaccinia virus / genetics

Substances

  • Ligands
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • Recombinant Proteins