ALPHA2 BETA1 INTEGRIN (VLA2) LIGAND INTERACTION

整合素α2β1(VLA-2)是胶原、层粘连蛋白、艾柯病毒的受体 1和整合素α3β1(VLA-3)。α2β1-配体相互作用可以 成为许多疾病的潜在治疗靶点。我们最近 确定α2亚基的I结构域为配体结合部位 通过绘制功能抑制抗α2单抗的表位 利用人/牛α2嵌合体和定点的抗体 诱变。我们还发现了一个含有胶原蛋白的重组I结构域片段 结合功能，提示Alpha2的I结构域可能具有所有 配基结合所必需的成分。此外，我们还确定了 激活依赖的Beta1亚基表位，它是由 通过激活抗β1单抗激活，或刺激细胞(例如，T 细胞)由佛波酯合成。还发现了一个Beta1突变体(D130A)表达 构成激活的表位，表明突变体有一个 高度激活的整合素的构象，而突变失活 配体结合功能。了解α2beta1整合素 基于这些初步数据，粘附性在分子水平上起作用， 我们建议：1)确定I结构域中涉及到的残基 使用定点突变的配体-α2beta1相互作用 多肽和重组α2片段(谷胱甘肽融合蛋白 S-转移酶，命名为GST-α2I)；2)研究α2β1/配体 通过测量重组I结构域片段与 配基和通过可视化胶原/重组I结构域片段 电子显微镜下的相互作用；3)研究β1亚基的作用 利用Asp13O或Asp13O突变体对α2beta1活性的调节 重组Beta1片段。拟议的工作将定义残留物 以及α2亚基的I结构域的识别配体的区域 以及与α2beta1相互作用的胶原残基和区域。 此外，β1亚基在调节α2β1中的作用 功能将被研究。也称合成肽或重组片段 或配体中的识别序列(例如，胶原蛋白) 可能会影响α2β1与配体之间的相互作用，从而 可能是免疫反应、病毒感染、 血栓形成、肿瘤进展或转移。

Integrin alpha2beta1 (VLA-2) is receptor for collagen, laminin, Echovirus 1 and integrin alpha3beta1 (VLA-3). The alpha2beta1-ligand interaction may be a potential therapeutic target for many diseases. We recently identified the I domain of the alpha2 subunit as the ligand binding site by mapping the epitopes of function-inhibiting anti-alpha2 monoclonal antibodies (mAbs) utilizing human/bovine alpha2 chimeras and site-directed mutagenesis. Also we found a recombinant I domain fragment has collagen binding function, suggesting the I domain of alpha2 may have all the necessary components for ligand binding. Also, we identified the activation-dependent epitope of the beta1 subunit, which is induced upon activation by activating anti beta1 mAb, or stimulation of cells (e.g., T cells) by phorbol ester. Also a beta1 mutant (D130A) was found to express constitutively the activated epitope, indicating that the mutant has a conformation of highly activated integrins, while the mutation inactivate the ligand binding function. To understand the alpha2beta1 integrin adhesive function at the molecular level based on these preliminary data, we propose to: 1) identify residues in the I domain that are involved in ligand-alpha2beta1 interaction using site-directed mutagenesis, synthetic peptides and recombinant alpha2 fragments (fusion protein with glutathione S-transferase, designated GST-alpha2I); 2) study the alpha2beta1/ligand interaction by measuring binding of the recombinant I domain fragment to ligand and by visualizing the collagen/recombinant I domain fragment interaction by electron microscopy; 3) study the role of the beta1 subunit in the regulation of alpha2beta1 activity using mutants of Asp13O or recombinant beta1 fragments. The proposed work will define the residues and regions of the I domain of the alpha2 subunit that recognize ligands and the residues and regions of collagen that interact with alpha2beta1. Furthermore, the role of the beta1 subunit in regulation of alpha2beta1 function will be studied. Also synthetic peptides or recombinant fragments of the I domain, or recognition sequences in ligands (e.g., collagen) might affect the interactions between alpha2beta1 and ligands, and thus may represent potential inhibitors for immune response, viral infection, thrombus formation, tumor progression or metastasis.