Yokosaki Yasuyuki, Tanaka Kumi, Higashikawa Fumiko, Yamashita Keisuke, Eboshida Akira
Molecular Division, Department of Public Health, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima, 734-8551, Japan.
Matrix Biol. 2005 Sep;24(6):418-27. doi: 10.1016/j.matbio.2005.05.005.
The extracellular matrix protein, osteopontin, is a ligand for several members of the integrin family, including alpha5beta1, alphavbeta3, alphavbeta5 and alpha9beta1. Osteopontin is a substrate for a number of extracellular proteases, including thrombin and the metalloproteases MMP-3 and MMP-7, which cleave osteopontin at sites close to or within the mapped integrin binding sites. Using affinity chromatography and cell adhesion assays, we now identify the integrin alphavbeta6 as an additional osteopontin receptor. Utilizing a series of recombinant forms of osteopontin, we compared the structural requirements for alphavbeta6 binding with those for the 4 other osteopontin-binding integrins. Like alpha5beta1, alphavbeta3 and alphavbeta5 (but not alpha9beta1), alphavbeta6 binds to the RGD site in osteopontin, since RGD peptide or mutation of this site to RAA completely inhibits alphavbeta6-mediated cell adhesion. For both alpha9beta1 and alpha5beta1, the N-terminal fragment generated by thrombin cleavage is a much better ligand than full length osteopontin, whereas thrombin-cleavage does not appear to be required for optimal adhesion to alphavbeta3, alphavbeta5 or alphavbeta6. A recombinant fragment predicted to be generated by MMP cleavage no longer supported alpha5beta1 or alpha9beta1-mediated adhesion, but adhesion mediated by alphavbeta5 or alphavbeta6 was unaffected. Finally, adhesion of alphavbeta5 or alphavbeta6 was inhibited by mutation of two aspartic acid residues upstream of the RGD site, whereas adhesion mediated by alphavbeta3, alpha5beta1 or alpha9beta1 was unaffected by these mutations. These results suggest that the hierarchy of integrin interactions with osteopontin can undergo complex regulation at least in part through the action of extracellular proteases.
细胞外基质蛋白骨桥蛋白是整合素家族多个成员的配体，包括α5β1、αvβ3、αvβ5和α9β1。骨桥蛋白是多种细胞外蛋白酶的作用底物，包括凝血酶以及金属蛋白酶MMP - 3和MMP - 7，这些蛋白酶在靠近或位于已定位的整合素结合位点处切割骨桥蛋白。通过亲和层析和细胞黏附试验，我们现在确定整合素αvβ6是另一种骨桥蛋白受体。利用一系列重组形式的骨桥蛋白，我们比较了αvβ6结合与其他4种骨桥蛋白结合整合素的结构要求。与α5β1、αvβ3和αvβ5（但不包括α9β1）一样，αvβ6与骨桥蛋白中的RGD位点结合，因为RGD肽或该位点突变为RAA会完全抑制αvβ6介导的细胞黏附。对于α9β1和α5β1而言，凝血酶切割产生的N端片段是比全长骨桥蛋白更好的配体，而对于αvβ3、αvβ5或αvβ6的最佳黏附似乎不需要凝血酶切割。预测由MMP切割产生的重组片段不再支持α5β1或α9β1介导的黏附，但αvβ5或αvβ6介导的黏附不受影响。最后，RGD位点上游两个天冬氨酸残基的突变抑制了αvβ5或αvβ6的黏附，而αvβ3、α5β1或α9β1介导的黏附不受这些突变影响。这些结果表明，整合素与骨桥蛋白相互作用的层级至少部分可通过细胞外蛋白酶的作用进行复杂调控。
