Integrin activation by dithiothreitol or Mn2+ induces a ligand-occupied conformation and exposure of a novel NH2-terminal regulatory site on the beta1 integrin chain.
Integrins can be expressed in at least three functional states (i.e. latent, active, and ligand-occupied). However, the molecular bases for the transitions between these states are unknown. In the present study, changes in the accessibility of several beta1 epitopes (e.g. N29, B44, and B3B11) were used to probe activation-related conformational changes. Dithiothreitol or Mn2+ activation of integrin-mediated adhesion in the human B cell line, IM9, resulted in a marked increase in the exposure of the B44 epitope, while N29 expression levels were most sensitive to dithiothreitol treatment. These results contrasted with the epitope expression patterns of spontaneously adherent K562 cells, where N29 was almost fully accessible and B44 was low. Addition of a soluble ligand resulted in a marked increase in B44 levels, suggesting that this antibody detected a ligand-induced binding site. The N29 epitope was mapped to a cysteine-rich region near the NH2 terminus of the integrin chain, thus defining a novel regulatory site. These studies indicate that the activation of integrin function by different stimuli may involve related but nonidentical conformations. Both Mn2+ and dithiothreitol appear to induce localized conformational changes that mimic a ligand-occupied receptor. This differs from the "physiologically" activated integrins on K562 cells that display a marked increase in overall epitope accessibility without exposure of the ligand-induced binding site epitopes. The increased exposure of the N29 site on K562 cells may indicate a role for this region in the regulation of integrin function.
Ni
H
H
RDU Research Laboratory, University of Manitoba, Winnipeg MB R3A 1M4, Canada.
Li
A
A
Simonsen
N
N
Wilkins
J A
JA
D001402:B-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D004229:Dithiothreitol / Q000494:pharmacology*; D006801:Humans; D019012:Integrin beta1 / Q000737:chemistry* / Q000187:drug effects; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D019012:Integrin beta1; D008024:Ligands; D013439:Sulfhydryl Reagents; D008274:Magnesium; D004229:Dithiothreitol