Integrin alpha and beta subunit contribution to the kinetic properties of alpha 2 beta 1 collagen receptors on human keratinocytes analyzed under hydrodynamic conditions

The adhesion of keratinocytes to type I collagen or laminin 5 was studied i n a laminar flow chamber. These experiments provided an insight into the bi nding 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 mu ltiple arrests. Studying the frequency and duration of these arrests yielde d fairly direct information on the rate of bond formation ton-rate) and dis sociation (off-rate), Off-rate values obtained on collagen or laminin 5 (0. 06 seconds(-1)) were tenfold lower than values determined on selectins, Bon d stability was strongly regulated by anti-beta 1 chain antibodies since th e off-rate was decreased sixfold by activating antibody TS2/16 and increase d fivefold by inhibitory antibodies Lia1/2 or P4C10, whereas neutral antibo dy K20 had no effect on this parameter. Binding frequencies were not signif icantly changed by all these antibodies. In contrast, both binding frequenc y and off-rate were altered by antibodies specific for the alpha 2 chain, s uggesting that these antibodies interfered with ligand recognition and also with the ligand-beta 1 chain interactions responsible for bond stabilizati on. The latter hypothesis was supported by the finding that the partial alt eration of alpha 2 chain function by inhibiting antibodies was corrected by anti-beta 1 chain antibody TS2/16, These results could not be ascribed to allosteric changes of the functional region of beta 1 integrin subunits reg ulated 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 bin ding topology, these data suggest that ligand-alpha chain interactions may be qualitatively important in ligand recognition and also influence the for mation of the ligand-beta 1 subunit bonding involved in stabilization of th e ligand-integrin complex by regulating its dissociation rate.