Divalent Cations and Ligands Induce Conformational Changes That Are Highly Divergent among β1 Integrins

Abstract

Here we show striking differences in conformational regulation among β₁ integrins. Upon manganese stimulation, a β₁ epitope defined by monoclonal antibody (mAb) 9EG7 was induced strongly (on α₄β₁), moderately (on α₅β₁), weakly (on α₂β₁), or was scarcely detectable (on α₆β₁ and α₃β₁). Comparable results were seen for the β₁ epitope defined by mAb 15/7. Likewise, soluble ligands caused strong (α₄β₁), moderate (α₅β₁), weak (α₂β₁, α₆β₁), or minimal (α₃β₁) induction of the 9EG7 epitope. Exchange or deletion of α 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 (α₅β₁ > α₂β₁ > α₆β₁ > α₃β₁), except that EGTA reduced rather than induced 9EG7 expression on α₄β₁. Thus in contrast to other β₁ integrins, calcium uniquely supports constitutive expression of the 9EG7 epitope on α₄β₁. Likewise, calcium supported vascular cell adhesion molecule-stimulated 9EG7 appearance on α₄β₁, whereas calcium inhibited ligand-induced 9EG7 epitope on other integrins. Constitutive expression of 9EG7 on α₄β₁ was eliminated by a D698E mutation in α₄, suggesting that Asp-698 may play a key role in maintaining atypical α₄β₁ 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 β₁ integrins, (ii) indicate pronounced differences in conformational flexibilities (α₄β₁ > α₅β₁> α₂β₁ > α₆β₁> α₃β₁), (iii) allow us to hypothesize that β₁ integrins may differ markedly in conformation-dependent inside-out signaling, and (iv) have uncovered an atypical α₄β₁ response to calcium that requires α₄ Asp-698.