Integrin (alpha) and beta subunit contribution to the kinetic properties of (alpha)2beta1 collagen receptors on human keratinocytes analyzed under hydrodynamic conditions.
The adhesion of keratinocytes to type I collagen or laminin 5 was studied in a laminar flow chamber. These experiments provided an insight into the binding kinetics of integrins in their natural environment and the effects of monoclonal antibodies specific for (alpha) and beta chains. Cells driven by a force too low to alter the natural lifetime of a single bond displayed multiple arrests. Studying the frequency and duration of these arrests yielded fairly direct information on the rate of bond formation (on-rate) and dissociation (off-rate). Off-rate values obtained on collagen or laminin 5 (0.06 seconds-1) were tenfold lower than values determined on selectins. Bond stability was strongly regulated by anti-beta1 chain antibodies since the off-rate was decreased sixfold by activating antibody TS2/16 and increased fivefold by inhibitory antibodies Lia1/2 or P4C10, whereas neutral antibody K20 had no effect on this parameter. Binding frequencies were not significantly changed by all these antibodies. In contrast, both binding frequency and off-rate were altered by antibodies specific for the (alpha)2 chain, suggesting that these antibodies interfered with ligand recognition and also with the ligand-beta1 chain interactions responsible for bond stabilization. The latter hypothesis was supported by the finding that the partial alteration of (alpha)2 chain function by inhibiting antibodies was corrected by anti-beta1 chain antibody TS2/16. These results could not be ascribed to allosteric changes of the functional region of beta1 integrin subunits regulated by TS2/16 since there was no competition between the binding of TS2/16 and anti-(alpha)2 chain antibodies. Interpreted within the framework of current concepts of integrin-ligand binding topology, these data suggest that ligand-alpha chain interactions may be qualitatively important in ligand recognition and also influence the formation of the ligand-beta1 subunit bonding involved in stabilization of the ligand-integrin complex by regulating its dissociation rate.
Masson-Gadais
B
B
Laboratoire d'Immunologie, INSERM U 387, Hôpital de Sainte-Marguerite, BP 29, France.
Pierres
A
A
Benoliel
A M
AM
Bongrand
P
P
Lissitzky
J C
JC
D019138:Antibodies, Blocking; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D011487:Protein Conformation; D038981:Receptors, Collagen
D019138:Antibodies, Blocking; D016023:Integrins; D008024:Ligands; D038981:Receptors, Collagen; D003094:Collagen