Teixidó J, Parker C M, Kassner P D, Hemler M E
Division of Tumor Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
J Biol Chem. 1992 Jan 25;267(3):1786-91.
The cell surface heterodimer VLA-4 (alpha 4 beta 1), a member of the integrin family of adhesion receptors, is involved in both cell-extracellular matrix and cell-cell adhesion. Unlike any other integrin alpha subunit, the intact (150 kDa) alpha 4 subunit of VLA-4 can sometimes be cleaved into two noncovalently associated fragments (80 and 70 kDa). Using biosynthetic and mixing experiments, we found that human alpha 4 cleavage is a regulated, compartmentalized event, occurring soon after maturation of the beta 1-associated alpha 4 subunit. Cleavage of alpha 4, which is increased following T cell activation, has been suggested to correlate with altered VLA-4 functions. To address directly the functional importance of alpha 4 cleavage, we have studied VLA-4-mediated adhesion functions in cells expressing intact alpha 4 in comparison with cells expressing cleaved alpha 4. For this purpose, we first sequenced the N terminus of the endogenously produced 70-kDa alpha 4 fragment and identified the alpha 4 cleavage site between Lys557-Arg558 and Ser559. To abolish cleavage, we converted Arg558 to Leu or Lys557 to Gln by site-directed mutagenesis of the alpha 4 cDNA and then transfected both mutant and wild type alpha 4 cDNAs into VLA-4-negative K562 cells. Whereas transfection with wild type alpha 4 cDNA yielded predominantly cleaved alpha 4 subunit, the Leu558-alpha 4 yielded only intact alpha 4 subunit, and Gln557-alpha 4 yielded mostly intact alpha 4 subunit. Transfectants with the intact or the cleaved alpha 4 were equally capable of engaging in VLA-4-dependent adhesion to vascular cell adhesion molecule-1 and to the Hep II fragment of fibronectin (40 kDa) and aggregated equally well in response to anti-alpha 4 antibodies. Thus, cleavage of the alpha 4 subunit in these transfectants did not alter any of the known VLA-4-mediated adhesion functions.
细胞表面异二聚体VLA-4（α4β1）是黏附受体整合素家族的成员，参与细胞与细胞外基质以及细胞与细胞之间的黏附。与其他任何整合素α亚基不同，VLA-4完整的（150 kDa）α4亚基有时可被切割成两个非共价结合的片段（80 kDa和70 kDa）。通过生物合成和混合实验，我们发现人α4的切割是一个受调控的、分隔化的事件，发生在与β1相关的α4亚基成熟后不久。α4的切割在T细胞活化后增加，有人认为这与VLA-4功能改变有关。为了直接探讨α4切割的功能重要性，我们研究了表达完整α4的细胞与表达切割后α4的细胞中VLA-4介导的黏附功能。为此，我们首先对内源性产生的70 kDaα4片段的N端进行测序，并确定了α4在Lys557-Arg558和Ser559之间的切割位点。为了消除切割，我们通过对α4 cDNA进行定点诱变，将Arg558转换为Leu或将Lys557转换为Gln，然后将突变型和野生型α4 cDNA转染到VLA-4阴性的K562细胞中。用野生型α4 cDNA转染主要产生切割后的α4亚基，而Leu558-α4只产生完整的α4亚基，Gln557-α4大多产生完整的α4亚基。表达完整或切割后α4的转染细胞同样能够参与VLA-4依赖的与血管细胞黏附分子-1以及纤连蛋白的Hep II片段（40 kDa）的黏附，并且对抗α4抗体的反应聚集程度相同。因此，这些转染细胞中α4亚基的切割并未改变任何已知的VLA-4介导的黏附功能。
