The b-tail domain (βTD) regulates physiologic ligand binding to integrin CD11β/CD18. Blood

Harvard University, Cambridge, Massachusetts, United States
Blood (Impact Factor: 10.45). 05/2007; 109(8):3513-20. DOI: 10.1182/blood-2005-11-056689
Source: PubMed


Crystallographic and electron microscopy studies revealed genuflexed (bent) integrins in both unliganded (inactive) and physiologic ligandbound (active) states, suggesting that local conformational changes are sufficient for activation. Herein we have explored the role of local changes in the contact region between the membrane-proximal beta-tail domain (betaTD) and the ligand-binding betaA domain of the bent conformation in regulating interaction of integrin CD11b/CD18 (alphaMbeta2) with its physiologic ligand iC3b. We replaced the betaTD CD loop residues D658GMD of the CD18 (beta2) subunit with the equivalent D672SSG of the beta3 subunit, with AGAA or with NGTD, expressed the respective heterodimeric receptors either transiently in epithelial HEK293T cells or stably in leukocytes (K562), and measured their ability to bind iC3b and to conformation-sensitive mAbs. In the presence of the physiologic divalent cations Ca(2+) plus Mg(2+) (at 1 mM each), the modified integrins showed increased (in HEK293) or constitutive (in K562) binding to iC3b compared with wild-type receptors. K562 expressing the betaTD-modified integrins bound in Ca(2+)Mg(2+) to the betaA-directed high-affinity reporter mAb 24 but not to mAb KIM127, a reporter of the genu-straightened state. These data identify a role for the membrane proximal betaTD as an allosteric modulator of integrin activation.

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Available from: Annette Gylling (Lindroos)
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    • "However, protonation of metalcoordinating Asp and Glu residues at the low pH at which  v  3 crystallizes is probably responsible for variable absence of bound metal (Dong et al., 2012), and unliganded, closed  IIb  3 , which crystallizes at higher pH, has all metal sites bound (Zhu et al., 2008, 2010). Arguments for regulation by a deadbolt and then an interface between the  and  knees have been raised (Gupta et al., 2007; Xiong et al., 2009) and contradicted (Zhu et al., 2007a; Smagghe et al., 2010; Xie et al., 2010; Dong et al., 2012). In contrast, mutational studies that shorten the I 7 helix (Yang et al., 2004) or introduce disulfide bonds (Luo et al., 2004c; Kamata et al., 2010), N-glycan wedges (Luo et al., 2003, 2004b), or point mutations (Mould et al., 2003a; Barton et al., 2004; Luo et al., 2009) consistently show that the closed and open headpiece conformations of intact  3 integrins on cell surfaces have low and high affinity for ligand, respectively. "
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    ABSTRACT: Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the αIIbβ3 integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca(2+), α1 helix, α1' helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward αIIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening.
    Full-text · Article · Jun 2013 · The Journal of Cell Biology
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    • "Stable transfection of wild-type (WT) CD11b/CD18 or the same integrin containing the activating I316G mutation in CD11bA [16] was carried out in K562 cells (from American Type Culture Collection, Manassas, VA) using published protocols as previously described [20]. Transformed cells were maintained in IMDM supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin and 0.5 mg/ml G418. "
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    ABSTRACT: One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS). We used electron microscopy to determine the 3D structure of the αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMβ2) in its inactive state. A well defined density for αA was observed within a bent ectodomain conformation, while the structure of the ectodomain in complex with the Fab fragment of mAb107, which binds at the MIDAS face of CD11b and stabilizes the inactive state, further revealed that αA is restricted to a relatively small range of orientations relative to the Propeller domain. Using Fab 107 as probe in fluorescent lifetime imaging microscopy (FLIM) revealed that αA is positioned relatively far from the membrane surface in the inactive state, and a systematic orientation search revealed that the MIDAS face would be accessible to extracellular ligand in the inactive state of the full-length cellular integrin. These studies are the first to define the 3D EM structure of an αA-containing integrin ectodomain and to position the ligand-binding face of αA domain in relation to the plasma membrane, providing new insights into current models of integrin activation.
    Full-text · Article · Feb 2013 · PLoS ONE
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    • "K562 cells stably transfected with plasmid encoding wild-type integrin CD11b/CD18 (K562 WT cells) have been described previously [37] [38] and were maintained in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS and G418 (0.5 mg/mL). The murine macrophage cell line (RAW 264.7 cells) was obtained from ATCC and the cells were maintained in DMEM supplemented with 10% heat-inactivated FBS according to the manufacturer's instructions. "
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    ABSTRACT: BACKGROUND: CD11b/CD18 is a key adhesion receptor that mediates leukocyte adhesion, migration and immune functions. We recently identified novel compounds, leukadherins, that allosterically enhance CD11b/CD18-dependent cell adhesion and reduce inflammation in vivo, suggesting integrin activation to be a novel mechanism of action for the development of anti-inflammatory therapeutics. Since a number of well-characterized anti-CD11b/CD18 activating antibodies are currently available, we wondered if such biological agonists could also become therapeutic leads following this mechanism of action. METHODS: We compared the two types of agonists using in vitro cell adhesion and wound-healing assays and using animal model systems. We also studied effects of the two types of agonists on outside-in signaling in treated cells. RESULTS: Both types of agonists similarly enhanced integrin-mediated cell adhesion and decreased cell migration. However, unlike leukadherins, the activating antibodies produced significant CD11b/CD18 macro clustering and induced phosphorylation of key proteins involved in outside-in signaling. Studies using conformation reporter antibodies showed that leukadherins did not induce global conformational changes in CD11b/CD18 explaining the reason behind their lack of ligand-mimetic outside-in signaling. In vivo, leukadherins reduced vascular injury in a dose-dependent fashion, but, surprisingly, the anti-CD11b activating antibody ED7 was ineffective. CONCLUSIONS: Our results suggest that small molecule allosteric agonists of CD11b/CD18 have clear advantages over the biologic activating antibodies and provide a mechanistic basis for the difference. General Significance CD11b/CD18 activation represents a novel strategy for reducing inflammatory injury. Our study establishes small molecule leukadherins as preferred agonists over activating antibodies for future development as novel anti-inflammatory therapeutics.
    Full-text · Article · Feb 2013 · Biochimica et Biophysica Acta
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