期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 284, 期 21, 页码 14524-14536出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M900200200
关键词
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资金
- New Energy and Industrial Technology Development Organization of Japan [17082005, 20370046, 06001294-0]
- Grants-in-Aid for Scientific Research [17082005, 20370046] Funding Source: KAKEN
Integrin alpha 8 beta 1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of alpha 8 beta 1 integrin toward a panel of RGD-containing ligands. Integrin alpha 8 beta 1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 +/- 0.01 nM, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to alpha 8 beta 1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central linker segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to alpha 8 beta 1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrin-ligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of alpha 8 beta 1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was similar to 2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to alpha 8 beta 1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to alpha 8 beta 1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.
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