Bazzoni G, Ma L, Blue M L, Hemler M E
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1998 Mar 20;273(12):6670-8. doi: 10.1074/jbc.273.12.6670.
Here we show striking differences in conformational regulation among beta1 integrins. Upon manganese stimulation, a beta1 epitope defined by monoclonal antibody (mAb) 9EG7 was induced strongly (on alpha4beta1), moderately (on alpha5beta1), weakly (on alpha2beta1), or was scarcely detectable (on alpha6beta1 and alpha3beta1). Comparable results were seen for the beta1 epitope defined by mAb 15/7. Likewise, soluble ligands caused strong (alpha4beta1), moderate (alpha5beta1), weak (alpha2beta1, alpha6beta1), or minimal (alpha3beta1) 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 (alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), except that EGTA reduced rather than induced 9EG7 expression on alpha4beta1. Thus in contrast to other beta1 integrins, calcium uniquely supports constitutive expression of the 9EG7 epitope on alpha4beta1. Likewise, calcium supported vascular cell adhesion molecule-stimulated 9EG7 appearance on alpha4beta1, whereas calcium inhibited ligand-induced 9EG7 epitope on other integrins. Constitutive expression of 9EG7 on alpha4beta1 was eliminated by a D698E mutation in alpha4, suggesting that Asp-698 may play a key role in maintaining atypical alpha4beta1 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 beta1 integrins, (ii) indicate pronounced differences in conformational flexibilities (alpha4beta1 > alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), (iii) allow us to hypothesize that beta1 integrins may differ markedly in conformation-dependent inside-out signaling, and (iv) have uncovered an atypical alpha4beta1 response to calcium that requires alpha4 Asp-698.
我们在此展示了β1整合素之间在构象调节方面的显著差异。在锰刺激下，由单克隆抗体（mAb）9EG7定义的β1表位被强烈诱导（在α4β1上）、中度诱导（在α5β1上）、微弱诱导（在α2β1上）或几乎检测不到（在α6β1和α3β1上）。对于由mAb 15/7定义的β1表位，也得到了类似的结果。同样，可溶性配体对9EG7表位的诱导作用也呈现出强烈（α4β1）、中度（α5β1）、微弱（α2β1、α6β1）或最小（α3β1）的差异。α链胞质尾部的交换或缺失并未改变锰离子诱导的9EG7表位水平。用乙二醇双四乙酸（EGTA）或乙二胺四乙酸（EDTA）去除钙离子后，9EG7表位诱导的等级顺序相似（α5β1 > α2β1 > α6β1 > α3β1），只是EGTA降低而非诱导α4β1上的9EG7表达。因此，与其他β1整合素不同，钙离子独特地支持α4β1上9EG7表位的组成性表达。同样，钙离子支持血管细胞黏附分子刺激后α4β1上9EG7表位的出现，而钙离子抑制其他整合素上配体诱导的9EG7表位。α4上的D698E突变消除了α4β1上9EG7的组成性表达，这表明天冬氨酸698可能在维持α4β1对钙离子的非典型反应中起关键作用。总之，我们的结果（i）表明，诸如9EG7和15/7之类的单克隆抗体作为β1整合素配体或锰离子占据情况的报告分子，其诊断效用有限；（ii）表明构象灵活性存在明显差异（α4β1 > α5β1 > α2β1 > α6β1 > α3β1）；（iii）使我们能够推测β1整合素在构象依赖性外向内信号传导方面可能存在显著差异；（iv）揭示了α4β1对钙离子的非典型反应，该反应需要α4天冬氨酸698。
