Integrin alphaIIb beta3 reconstituted into lipid bilayers is nonclustered in its activated state but clusters after fibrinogen binding.
Integrin activation, ligand binding, and integrin clustering were analyzed using alphaIIb beta3 reconstituted into phospholipid vesicles and into supported planar lipid bilayers. Strong and specific binding of fibrinogen and the gamma-chain dodecapeptide of fibrinogen to alphaIIb beta3 indicated that the integrin is in an activated state after membrane reconstitution. Cryoelectron and fluorescence microscopy suggested a nonclustered state of the protein in the vesicle membrane. Supported planar lipid membranes were generated by fusion of vesicles in which approximately equal fractions of integrins were pointing inside-out and outside-in. This distribution led to an immobilization of about 40% of the integrin in supported bilayers due to attachment of the large extracellular domains to the quartz support. Fluorescence recovery after photobleaching indicated a diffusion coefficient of D = (0.70 +/- 0.06) x 10(-8) cm2/s, consistent with a nonclustered state of the mobile integrin. Upon fibrinogen binding, the integrins became immobile, and fluorescence micrographs showed a patchy distribution of fibrinogen-integrin complexes consisting of approximately 250 molecules. In addition to the expected dimer formation by bivalent fibrinogen, additionally induced fibrinogen clustering may account for the large size of the complexes. In contrast, binding of monovalent GRGDS pentapeptide or the gamma-chain dodecapeptide of fibrinogen altered neither the mobile fraction nor the association state of alphaIIb beta3. Our data indicate that integrin alphaIIbb3 is activated while monodisperse, and became clustered upon fibrinogen binding, leading to an irreversibly bound state.
Erb
E M
EM
Department of Biophysical Chemistry, Biozentrum of the University of Basel, Switzerland.
Tangemann
K
K
Bohrmann
B
B
Müller
B
B
Engel
J
J
D000595:Amino Acid Sequence; D019281:Dimerization; D004134:Dimyristoylphosphatidylcholine; D005340:Fibrinogen / Q000378:metabolism*; D005615:Freezing; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008854:Microscopy, Electron; D010715:Phosphatidylglycerols; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D008051:Lipid Bilayers; D008081:Liposomes; D010715:Phosphatidylglycerols; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen; C002773:dimyristoylphosphatidylglycerol; D004134:Dimyristoylphosphatidylcholine