Locking in alternate conformations of the integrin alphaLbeta2 I domain with disulfide bonds reveals functional relationships among integrin domains.
We used integrin alphaLbeta2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins. mAbs to the alphaL I domain and beta2 I-like domain inhibit adhesion of wild-type alphaLbeta2 to intercellular adhesion molecule-1. However, with alphaLbeta2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, alphaLbeta2 containing a locked open I domain was completely resistant to inhibition by mAbs to the beta2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type alphaLbeta2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn(2+), as measured with mAb m24, which we map here to the beta2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn(2+)-induced exposure of the KIM127 epitope in the beta2 stalk region. Furthermore, locked open I domains, in alphaLbeta2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg(2+) and Mn(2+). These results suggest that Mn(2+) activates alphaLbeta2 by binding to a site other than the I domain, most likely the I-like domain of beta2.
The Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
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Shimaoka
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Zang
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Takagi
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Springer
D000911:Antibodies, Monoclonal / Q000276:immunology; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D004220:Disulfides; D016169:Lymphocyte Function-Associated Antigen-1; D008345:Manganese