Conformational changes in human integrin alphaIIbbeta3 after platelet activation, monitored by FRET.
Integrin alpha(IIb)beta(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 rearrangements undergone by integrin alpha(IIb)beta(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 betaA domain of the beta(3) subunit (donor, Alexa488-P97 Fab) and to the Calf-2 domain of the alpha(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.
Coutinho
Ana
A
Departamento de Biofísica, Instituto de Química Física Rocasolano. Consejo Superior de Investigaciones Científicas, Serrano 119. 28006 Madrid, Spain.
García
Carolina
C
González-Rodríguez
José
J
Lillo
M Pilar
MP
D002232:Carbocyanines / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry*; D011487:Protein Conformation
D002232:Carbocyanines; D007140:Immunoglobulin Fab Fragments; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C089187:cyanine dye 3