Chen L L, Whitty A, Lobb R R, Adams S P, Pepinsky R B
Biogen, Inc., Cambridge, Massachusetts 02142, USA.
J Biol Chem. 1999 May 7;274(19):13167-75. doi: 10.1074/jbc.274.19.13167.
We have used the highly specific alpha4beta1 inhibitor 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study alpha4beta1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of alpha4beta1. Observed KD values for BIO1211 binding ranged from a value of 20-40 nM in the non-activated state of the integrin that exists in 1 mM Mg2+, 1 mM Ca2+ to 100 pM in the activated state seen in 2 mM Mn2+ to 18 pM when binding was measured after co-activation by 2 mM Mn2+ plus 10 microgram/ml of the integrin-activating monoclonal antibody TS2/16. The large range in KD values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 x 10(-4) s-1 to >140 x 10(-4) s-1. Association rate constants varied only slightly under the same conditions, all falling in the narrow range from 0.9 to 2.7 x 10(6) M-1 s-1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on alpha4beta1 that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule-1 and CS1 to alpha4beta1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.
我们使用了高度特异性的α4β1抑制剂4-((N'-2-甲基苯基)脲基)-苯基乙酰基-亮氨酸-天冬氨酸-缬氨酸-脯氨酸(BIO1211)作为含LDV的配体模型，以研究在影响α4β1激活状态的不同条件下，Jurkat细胞上α4β1整合素与配体的相互作用。观察到的BIO1211结合的KD值范围从整合素非激活状态下（存在于1 mM Mg2+、1 mM Ca2+中）的20 - 40 nM到激活状态下（在2 mM Mn2+中观察到）的100 pM，当通过2 mM Mn2+加10微克/毫升整合素激活单克隆抗体TS2/16共同激活后测量结合时，KD值为18 pM。KD值的大范围几乎完全由整合素 - BIO1211复合物的解离速率差异决定，解离速率范围从0.17×10(-4) s-1到>140×10(-4) s-1。在相同条件下，结合速率常数仅略有变化，均落在0.9至2.7×10(6) M-1 s-1的狭窄范围内。与单独在金属离子或TS2/16饱和浓度下观察到的情况相比，通过二价阳离子和TS2/16共同激活后观察到的亲和力进一步增加，这表明这些因素导致激活的机制是不同的，并确定了α4β1上一种以前未被传统检测方法检测到的未被识别的高亲和力状态。当研究血管细胞粘附分子-1和CS1与α4β1的结合特性时，观察到了类似的亲和力变化，表明用BIO1211检测到的不同亲和力状态是整合素的固有特性。
