Evaluation of integrin molecules involved in substrate adhesion.
Integrins were cross-linked to their extracellular matrix ligands using non-penetrating chemical cross-linkers. This procedure did not disturb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extracted with ionic detergents. 80-90% of the beta 1 integrin in the cross-linked culture was extracted with RIPA buffer and the remaining 10-20% was recovered following reversal of the cross-linking. This separated two distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of this procedure for cross-linking of integrins involved in substrate adhesion was demonstrated using NIH 3T3 cells which express both alpha 5 beta 1 and alpha 6 beta 1 integrins. alpha 6 was cross-linked only in cells plated on laminin whereas alpha 5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either alpha 5 or beta 1 integrin, it was demonstrated that these domains can be blocked in the intact cell but the blocking can be removed using ionic detergent extraction after chemical cross-linking. The extracellular matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on the plastic dish following cross-linking.
Enomoto-Iwamoto
M
M
Department of Microbiology, University of Pennsylvania, Philadelphia 19104-6076.
Menko
A S
AS
Philp
N
N
Boettiger
D
D
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D002642:Chick Embryo; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins / Q000502:physiology; D005109:Extracellular Matrix / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000502:physiology; D005353:Fibronectins / Q000502:physiology; D005455:Fluorescent Antibody Technique; D039121:Integrin alpha6beta1; D016023:Integrins / Q000235:genetics / Q000502:physiology*; D007797:Laminin / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D017469:Receptors, Fibronectin; D013388:Succinimides
D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D039121:Integrin alpha6beta1; D016023:Integrins; D007797:Laminin; D017469:Receptors, Fibronectin; D013388:Succinimides; C011240:dithiobis(succinimidylpropionate)