Integrin IIb3 Reconstituted into Lipid Bilayers I
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- 作者：Eva-Maria Erb ; Kirsten Tangemann ; Bernd Bohrmann ; Beate Mü ; ller ; and Jü ; rgen Engel
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：June 17, 1997
- 年：1997
- 卷：36
- 期：24
- 页码：7395 - 7402
- 全文大小：221K
- 年卷期：v.36,no.24(June 17, 1997)
- ISSN：1520-4995
文摘
Integrin activation, ligand binding, and integrin clustering wereanalyzed using 
IIb
3reconstituted into phospholipid vesicles and into supported planarlipid bilayers. Strong and specificbinding of fibrinogen and the 
-chain dodecapeptide of fibrinogen toIIb3 indicated that the integrinis in an activated state after membrane reconstitution.Cryoelectron and fluorescence microscopy suggesteda nonclustered state of the protein in the vesicle membrane.Supported planar lipid membranes weregenerated by fusion of vesicles in which approximately equal fractionsof integrins were pointing inside-out and outside-in. This distribution led to an immobilization ofabout 40% of the integrin in supportedbilayers due to attachment of the large extracellular domains to thequartz support. Fluorescence recoveryafter photobleaching indicated a diffusion coefficient of D= (0.70 ± 0.06) × 10-8cm2/s, consistent witha nonclustered state of the mobile integrin. Upon fibrinogenbinding, the integrins became immobile,and fluorescence micrographs showed a patchy distribution offibrinogen-integrin complexes consistingof approximately 250 molecules. In addition to the expected dimerformation by bivalent fibrinogen,additionally induced fibrinogen clustering may account for the largesize of the complexes. In contrast,binding of monovalent GRGDS pentapeptide or the -chain dodecapeptideof fibrinogen altered neitherthe mobile fraction nor the association state of IIb3. Ourdata indicate that integrin IIbb3 is activatedwhile monodisperse, and became clustered upon fibrinogen binding,leading to an irreversibly boundstate.
IIb3reconstituted into phospholipid vesicles and into supported planarlipid bilayers. Strong and specificbinding of fibrinogen and the -chain dodecapeptide of fibrinogen toIIb3 indicated that the integrinis in an activated state after membrane reconstitution.Cryoelectron and fluorescence microscopy suggesteda nonclustered state of the protein in the vesicle membrane.Supported planar lipid membranes weregenerated by fusion of vesicles in which approximately equal fractionsof integrins were pointing inside-out and outside-in. This distribution led to an immobilization ofabout 40% of the integrin in supportedbilayers due to attachment of the large extracellular domains to thequartz support. Fluorescence recoveryafter photobleaching indicated a diffusion coefficient of D= (0.70 ± 0.06) × 10-8cm2/s, consistent witha nonclustered state of the mobile integrin. Upon fibrinogenbinding, the integrins became immobile,and fluorescence micrographs showed a patchy distribution offibrinogen-integrin complexes consistingof approximately 250 molecules. In addition to the expected dimerformation by bivalent fibrinogen,additionally induced fibrinogen clustering may account for the largesize of the complexes. In contrast,binding of monovalent GRGDS pentapeptide or the -chain dodecapeptideof fibrinogen altered neitherthe mobile fraction nor the association state of IIb3. Ourdata indicate that integrin IIbb3 is activatedwhile monodisperse, and became clustered upon fibrinogen binding,leading to an irreversibly boundstate.