Divalent cations and ligands induce conformational changes that are highly divergent among β1 integrins

Here we show striking differences in conformational regulation among beta(1) integrins. Upon manganese stimulation, a beta(1) epitope defined by monoclonal antibody (mAb) 9EG7 was induced strongly (on alpha(4) beta(1)), moderately (on alpha(5) beta(1)), weakly (on alpha(2) beta(1)), or was scarcely detectable (on alpha(6) beta(1) and alpha(3) beta(1)). Comparable results were seen for the beta(1) epitope defined by mAb 15/7. Likewise, soluble ligands caused strong (alpha(4) beta(1)), moderate (alpha(5) beta(1)), weak (alpha(2) beta(1), alpha(6) beta(1)), Or minimal (alpha(3) beta(1)) induction of the 9EG7 epitope. Exchange or deletion of alpha chain cytoplasmic tails did not alter Mn2+-induced 9EG7 epitope levels. Upon removal of calcium by EGTA or EDTA, the hierarchy of 9EG7 epitope induction was similar (alpha(5) beta(1) > alpha(2) beta(1) > alpha(6) beta(1) > alpha(3) beta(1)), except that EGTA reduced rather than induced 9EG7 expression on alpha(4) beta(1). Thus in contrast to other beta(1) integrins, calcium uniquely supports constitutive expression of the 9EG7 epitope on alpha(4) beta(1). Likewise, calcium supported vascular cell adhesion molecule-stimulated 9EG7 appearance on alpha(4) beta(1), whereas calcium inhibited ligand-induced 9EG7 epitope on other integrins. Constitutive expression of 9EG7 on alpha(4) beta(1) was eliminated by a D698E mutation in alpha(4), suggesting that Asp-698 may play a key role in maintaining atypical alpha(4) beta(1) response to calcium. In conclusion, our results (i) demonstrate that mAb such as 9EG7 and 15/7 have limited diagnostic utility as reporters of ligand or Mn2+ occupancy for beta(1) integrins, (ii) indicate pronounced differences in conformational flexibilities (alpha(4) beta(1) > alpha(5) beta(1) > alpha(2) beta(1) > alpha(6) beta(1) > alpha(3) beta(1)), (iii) allow us to hypothesize that beta(1) integrins may differ markedly in conformation-dependent inside-out signaling, and (iv) have uncovered an atypical alpha(4) beta(1) response to calcium that requires alpha(4) Asp-698.