Mechanisms and consequences of affinity modulation of integrin αvβ3 detected with a novel patch-engineered monovalent ligand
Mechanisms and consequences of affinity modulation of integrin αvβ3 detected with a novel patch-engineered monovalent ligand
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DOI：
10.1074/jbc.274.31.21609
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发表时间：
1999-07-30
影响因子：
4.8
通讯作者：
Shattil, SJ
中科院分区：
文献类型：
--
作者：
Pampori, N;Hato, T;Shattil, SJ
整合素α（v）β（3）介导血管细胞中的多种反应，范围从细胞粘附、迁移和增殖到腺病毒的摄取。然而，α（v）β（3）受受体构象（亲和力）、受体扩散/聚集（亲合力）或受体后事件变化的调节程度尚不清楚。已经使用单价配体模拟抗体PAC 1 Fab建立了相关整联蛋白α（IIb）β（3）的亲和力调节。为了确定α（v）β（3）的亲和力调节作用，通过用来自多价腺病毒五邻体基底的单个α（v）整联蛋白结合结构域替换PAC 1 Fab的重链高变区3来产生新型单价配体模拟抗体（WOW-1）。在受体和细胞结合试验中，WOW-1 Fab和五邻体碱基都选择性地结合活化的α（v）β（3），但不结合α（IIb）β（3）。α（v）β（3）亲和力随细胞类型而变化。未受刺激的B-淋巴母细胞与WORT-1 Fab的结合较差（表观Kd = 2.4 μ M），但用佛波醇12-肉豆蔻酸酯13-乙酸酯急性刺激使受体亲和力增加>30倍（Kd = 80 nM），受体数量无变化。相比之下，黑色素瘤细胞中的α（v）β（3）是组成型活性的，但配体结合可被β（3）胞质尾的过表达抑制。α（v）β（3）亲和力的上调具有功能性后果，因为它增加了细胞粘附和扩散，并促进了腺病毒介导的基因转移。这些研究证实，α（v）β（3）的亲和力发生快速调节变化，从而影响该整合素的生物学功能。
Integrin alpha(v)beta(3) mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which alpha(v)beta(3) is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, alpha(IIb)beta(3), has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of alpha(v)beta(3), a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single alpha(v) integrin binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated alpha(v)beta(3), but not to alpha(IIb)beta(3), in receptor and cell binding assays. alpha(v)beta(3) affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WORT-1 Fab poorly (apparent K-d = 2.4 mu M), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K-d = 80 nM), With no change in receptor number. In contrast, alpha(v)beta(3) in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of beta(3) cytoplasmic tails. Up-regulation of alpha(v)beta(3) affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that alpha(v)beta(3) is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.