SPECIFIC ASSOCIATION OF CD63 WITH THE VLA-3 AND VLA-6 INTEGRINS
SPECIFIC ASSOCIATION OF CD63 WITH THE VLA-3 AND VLA-6 INTEGRINS
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DOI：
10.1074/jbc.270.30.17784
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发表时间：
1995-07-28
影响因子：
4.8
通讯作者：
HEMLER, ME
中科院分区：
文献类型：
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作者：
BERDITCHEVSKI, F;BAZZONI, G;HEMLER, ME
我们筛选了针对细胞表面蛋白的单克隆抗体，并选择了一种名为 6H1 的抗体，它可以识别假定的整合素相关蛋白。 6H1 单克隆抗体 (mAb) 间接共沉淀多个细胞系 Brij 96 去污剂裂解物中的 α(3) beta(1) 和/或 alpha(6) beta(1)，但不共沉淀 alpha(2) beta(1) 或 alpha(5) beta(1)。使用 6H1 mAb 进行大规模纯化，产生了 45-60 kDa 的单一蛋白质，其氨基末端序列与 CD63 完全匹配。确认 6H1 mAb 识别 CD63 蛋白，6H1 和已知的抗 CD63 mAb 产生了相同的共沉淀结果，并且在含有组织蛋白酶 D 的溶酶体颗粒中具有相同的共定位。此外，我们使用已建立的抗 CD63 mAb 在 α(3) beta(1) 免疫沉淀物中检测该蛋白，并且我们还观察到 VLA-3 和 CD63 在细胞“足迹”中共定位。值得注意的是，α(3) 的细胞质结构域对于 CD63 关联来说既不是必需的，也不是充分的，表明它发生在 α(3) beta(1) 复合物内的其他位置。了解这些特定的 CD63-alpha(3) beta(1) 和 CD63-alpha(6) beta(1) 生化关联应该有助于深入了解 alpha(3) beta(1)、alpha(6) beta(1) 和 CD63 的特殊功能。
We screened monoclonal antibodies to cell surface proteins and selected an antibody, called 6H1, that recognizes a putative integrin-associated protein. The 6H1 monoclonal antibody (mAb) indirectly coprecipitated alpha(3) beta(1) and/or alpha(6) beta(1) but not alpha(2) beta(1), or alpha(5) beta(1) from Brij 96 detergent lysates of multiple cell lines. Large scale purification using the 6H1 mAb yielded a single protein of 45-60 kDa with an amino-terminal sequence that exactly matched CD63. Confirming that the 6H1 mAb recognized the CD63 protein, 6H1 and a known anti-CD63 mAb yielded identical coprecipitation results and identical colocalization into lysosomal granules containing cathepsin D. Furthermore, we used an established anti-CD63 mAb to detect this protein in an alpha(3) beta(1) immunoprecipitate, and also we observed VLA-3 and CD63 colocalization in cellular ''footprints.'' Notably, the cytoplasmic domain of alpha(3) was neither required nor sufficient for CD63 association, suggesting that it occurred elsewhere within the alpha(3) beta(1) complex. Knowledge of these specific CD63-alpha(3) beta(1), and CD63-alpha(6) beta(1) biochemical associations should lead to critical insights into the specialized functions of alpha(3) beta(1), alpha(6) beta(1) and CD63.