IDENTIFICATION OF PUTATIVE LIGAND-BINDING SITES OF THE INTEGRIN ALPHA-4-BETA-1 (VLA-4, CD49D/CD29)
IDENTIFICATION OF PUTATIVE LIGAND-BINDING SITES OF THE INTEGRIN ALPHA-4-BETA-1 (VLA-4, CD49D/CD29)
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DOI：
10.1042/bj3050945
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发表时间：
1995-02-01
影响因子：
4.1
通讯作者：
TAKADA, Y
中科院分区：
文献类型：
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作者：
KAMATA, T;PUZON, W;TAKADA, Y
整合素α 4 β 1识别纤连蛋白（CS-1序列）和血管细胞粘附分子-1（VCAM-1）。为了定位α 4的配体结合位点，我们定位了功能阻断性抗α 4单克隆抗体（mAb）的表位，包括使用哺乳动物细胞中表达的种间α 4嵌合体识别先前描述的（但尚未物理定位的）功能表位（A，B1，B2和C）的表位。表位B1和B2与配体结合相关，表位A和B2与同型细胞聚集相关。将mAb P4 C2（表位B2）、20 E4和PS/2定位在残基108-182内;将mAb HP 2/1（表位B1）、SG/73和R1-2定位在残基195-268内;将mAb HP 1/3（表位A）和P4 G9定位在残基1-38内;并且将B5 G10（表位C）定位在残基269-548内。数据表明，不包括二价阳离子结合基序的残基108-268与VCAM-1和CS-1结合相关，而α 4的更多N-末端部分（残基1-38和108-182）与同型聚集相关。由于单克隆抗体PS/2和HP 2/1阻断α 4 β 7与粘膜地址素细胞粘附分子-I（MAdCAM-1）的结合，因此MAdCAM-1结合位点接近VCAM-1和CS-1结合位点或与VCAM-1和CS-1结合位点重叠。检测β 1的Asp-130在与VCAM-1和CS-1肽结合中的作用。表达β 1（D130 A）的中国仓鼠卵巢（CHO）细胞（β 1的Asp-130至Ala突变体）和α 4与两种配体的结合比表达野生型β 1和α 4的CHO细胞少得多[β 1的显性负效应（D130 A）]，提示β 1的Asp-130对于与两种配体的结合是关键的，并且两种配体共享共同的结合机制。
Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-38; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1-38 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1(D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effect of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligands share common binding mechanisms.