Abstract
Integrin activation involves global conformational changes as demonstrated by various functional and structural analyses. The integrin beta hybrid domain is proposed to be involved in the propagation of this activation signal. Our previous study showed that the integrin beta(2)-specific monoclonal antibody 7E4 abrogates monoclonal antibody KIM185-activated but not Mg(2+)/EGTA-activated leukocyte function-associated antigen-1 (LFA-1; alpha(L)beta(2))-mediated adhesion to ICAM-1. Here we investigated the allosteric inhibitory property of 7E4. By using human/mouse chimeras and substitution mutations, the epitope of 7E4 was mapped to Val(407), located in the mid-region of the beta(2) hybrid domain. Two sets of constitutively active LFA-1 variants were used to examine the effect of 7E4 on LFA-1/ICAM-1 binding. 7E4 attenuated the binding of variants that have modifications to regions membrane proximal with respect to the beta(2) hybrid domain. In contrast, the inhibitory effect was minimal on variants with alterations in the alpha(L) I- and beta(2) I-like domains preceding the hybrid domain. Furthermore, 7E4 abrogated LFA-1/ICAM-1 adhesion of phorbol 12-myristate 13-acetate-treated MOLT-4 cells. Our data demonstrate that interaction between the hybrid and I-like domain is critical for the regulation of LFA-1-mediated adhesion.
Highlights
Integrins are key proteins involved in cell-cell and cell-matrix interactions, mediating essential biological processes such as embryogenesis, the immune response, and inflammation [1, 2]
We reasoned that the properties exhibited by 7E4 provide a unique opportunity to address how the activation signal is propagated via the hybrid domain of leukocyte function-associated antigen-1 (LFA-1)
Based on electron micrographs of the representative integrins ␣IIb3 and ␣51, the general structure of the integrin molecule had been taken as a two-pronged plug having a globular head and two legs anchoring them into the membrane [33,34,35]
Summary
Reagents and Antibodies—The following antibodies were from various sources: MHM24 (anti-␣L) [17] was obtained from A. Mo(Met1–Asp77) was inserted into the NheI and EcoRV sites of CD18-pcDNA3.1/zeo(ϩ) that had their corresponding human 2 fragments removed. Mo(Met1– Glu298) was inserted into the NheI and EcoRI sites and Mo(Glu298– Asn584) into the EcoRI and SacII sites of CD18-pcDNA3.1/zeo(ϩ) [23] that had their corresponding human 2 fragments removed They were named 2,Hu/MoB, 2,Hu/MoC, and 2,Hu/MoD, respectively. Constructs (3.5 g) were transfected into 293T cells using the calcium phosphate method as described previously [28, 29]. Flow Cytometry—Cells were incubated with primary mAb, at 10 g/ml unless otherwise stated, in RPMI 1640 for 1 h at 4 °C They were washed twice and incubated with fluorescein isothiocyanate-conjugated sheep anti-mouse F(abЈ) secondary antibody (1:400 dilution; Sigma) for 45 min at 4 °C. Positive gates were set to include 5% of the brightest cells stained with the irrelevant antibody KB43
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