Conformational changes in human integrin αIIbβ3 after platelet activation, monitored by FRET

Abstract

Integrin α IIb β 3, an abundant heterodimeric receptor at the surface of blood platelets, binds adhesive proteins after platelet activation and plays a primary role in haemostasis. In solution, it has been observed mainly in two conformations: the bent and the extended forms.Based on X-ray crystallography, electron microscopy and immunochemical observations of full-length integrin ectodomains and intact integrins, it has been agreed that unactivated integrins are in the bent conformation, both isolated in solution and in living cells. However,consensus is yet to emerge on the bent or extended conformation of activated integrins and on their mechanism of activation (the switchblade, the deadbolt and the S-S reduction models), which require further experimental tests at the cell level to become established facts.Here, we tested the proposed structural re-arrangements undergone by integrin α IIb β 3 after cell activation, by using Förster-type fluorescence resonance energy-transfer (FRET) and attached fluorescent labels to Fab fragments of monoclonal antibodies directed to the βA-domain of the β 3 subunit (donor, Alexa488-P97 Fab) and to the Calf-2 domain of the α IIb subunit (acceptor, Cy3-M3 Fab or Cy3-M10 Fab). The FRET efficiencies observed after ADP or TRAP platelet activation changed less than 20% from the resting values, showing that the distance between the labeled Fab fragments changes only modestly after platelet activation by physiological agonists. This observation is consistent with a conformational model of the activated integrin in the cell less extended than in the switchblade model.

Three-dimensional Model of Human Platelet Integrin αIIbβ3 in Solution Obtained by Small Angle Neutron Scattering

“…2b) (12). subunit and to the N-terminal domain of the ␤3 subunit (9) and by fluorescence intramolecular energy transfer between Fab fragments bound to these regions (15). Finally, the SANS model is consistent with the bent conformation of ␣V␤3 in resting and Mn 2ϩ -activated K562 cells, observed by intermolecular energy transfer (37).…”

“…In addition, the soluble Mn 2ϩ -bound r-␣V␤3 ectodomain, the complex of this ectodomain with a fibronectin fragment (with type III domains 7-10 and the extradomain B), and Mn 2ϩ -activated ␣IIb␤3 were equally in the bent configuration, as determined by electron microscopy (EM) 2 (13,14). Finally, ␣IIb␤3 in resting and physiologically activated platelets were also in the bent configuration, as found by epitope mapping and competition among monoclonal antibodies directed to the C-terminal region of the ␣IIb subunit and to the N-terminal domain of the ␤3 subunit (9) and by fluorescence intramolecular energy transfer (15). However, all of these results are inconsistent to some extent with the extended conformation found: in whole ␣IIb␤3 observed by cryo-EM (16); in different recombinant ectodomain products of ␣V␤3 in the presence of Mn 2ϩ alone or Mn 2ϩ with an RGD peptide or other ligand-mimetic antagonists, observed by EM (5); and in experiments of fluorescence intermolecular energy transfer between a fluorescent peptide bound to integrin ␣4␤1, expressed at the surface of Mn 2ϩ or chemokine-activated monoblastoid cells, and a fluorescent lipophilic probe incorporated into their membrane (17).…”

“…Finally, ␣IIb␤3 in resting and physiologically activated platelets were also in the bent configuration, as found by epitope mapping and competition among monoclonal antibodies directed to the C-terminal region of the ␣IIb subunit and to the N-terminal domain of the ␤3 subunit (9) and by fluorescence intramolecular energy transfer (15). However, all of these results are inconsistent to some extent with the extended conformation found: in whole ␣IIb␤3 observed by cryo-EM (16); in different recombinant ectodomain products of ␣V␤3 in the presence of Mn 2ϩ alone or Mn 2ϩ with an RGD peptide or other * This work was supported by Grants SAF 2003-042/66 (2003-2007 and Intramural Frontera 200680F-0083 (2007 (to J. G.-R.). □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs.…”

Visualizing and quantifying adhesive signals

“…To date quantification of the activation dynamics of multiple signaling molecules and/or the assembly dynamics of multiple structural components in the same cell has been hindered by the spectral overlap of fluorescent proteins, allowing simultaneous observation of at most 4 -6 colors, but often only 2 -3 colors. Whereas this has provided the possibility to monitor structural interactions among several adhesion proteins [10,13,[37][38][39][40][41][42][43][44][45][46], the imaging of multiple biosensors is not yet as well established. Many of the existing biosensors use fluorophores with similar wavelengths and thus cannot be combined.…”

The Structure of a Full-length Membrane-embedded Integrin Bound to a Physiological Ligand

“…Observations such as a surprising compact, head-up conformation for inactive ␣ IIb ␤ 3 integrin in nanodiscs (17), a stable integrin ␣ L ␤ 2 conformation with intermediate affinity in cells (26), and conflicting inferences on the conformation of different integrin isoforms in the cell membrane drawn from fluorescence studies (25,(27)(28)(29) further complicate this issue.…”