α2β1 integrin is not recognized by rhodocytin but is the specific, high affinity target of rhodocetin, an RGD-independent disintegrin and potent inhibitor of cell adhesion to collagen

被引:105
作者
Eble, JA
Beermann, B
Hinz, HJ
Schmidt-Hederich, A
机构
[1] Univ Munster, Inst Physiol Chem & Pathobiochem, D-48149 Munster, Germany
[2] Univ Munster, Inst Phys Chem, D-48149 Munster, Germany
关键词
D O I
10.1074/jbc.M009338200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have recombinantly expressed a soluble form of human alpha (2)beta (1) integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble alpha (2)beta (1) integrin binds to its collagen ligands the same way as the wildtype alpha (2)beta (1) integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrin-activating antibody. However, it does not bind to rhodocytin, a postulated agonist of alpha (2)beta (1) integrin from the snake venom of Calloselasma rhodostoma, which elicits platelet aggregation, Tacking advantage of the recombinantly expressed, soluble alpha (2)beta (1) integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble alpha (2)beta (1) integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble alpha (2)beta (1) integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to alpha (2)beta (1) integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with alpha (2)beta (1) integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to alpha (2)beta (1) integrin, which binds to collagenous molecules much more avidly than to noncollagenous Ligands, such as laminin. Blocking alpha (2)beta (1) integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, alpha (2)beta (1) integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, alpha (2)beta (1) integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit alpha (2)beta (1) integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.
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页码:12274 / 12284
页数:11
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