Dual Function for a Unique Site within the β2I Domain of Integrin αMβ2

Driss Ehirchiou,Yu-Mei Xiong,Shelesa Brew,Li Zhang,Yang Li

doi:10.1074/jbc.m413525200

Driss Ehirchiou, Yu-Mei Xiong + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m413525200

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Abstract

Integrin activation has been postulated to occur in part via conformational changes in the I domain of the beta subunit (the betaI domain), especially near the F-alpha(7) loop, in response to "inside-out" signaling. However, direct evidence for a role of the F-alpha(7) loop in ligand binding and activity modulation is still lacking. Here, we report our finding that the F-alpha(7) loop (residues 344-358) within the beta(2)I domain has dual functions in ligand binding by alpha(M)beta(2). On the one hand, it supports intercellular adhesion molecule 1 (ICAM-1) binding to alpha(M)beta(2) directly as part of a recognition interface formed by five noncontiguous segments (Pro(192)-Glu(197), Asn(213)-Glu(220), Leu(225)-Leu(230), Ser(324)-Thr(329), and Glu(344)-Asp(348)) on the apex of the beta(2)I domain. On the other hand, it controls the open and closed conformation of the alpha(M)beta(2) receptor, thereby indirectly affecting alpha(M)beta(2) binding to other ligands. Switching the five constituent sequences of the ICAM-1-binding site within the beta(2)I domain to their beta(1) counterparts destroyed ICAM-1 binding but had no effect on the gross conformations of the receptor. Of the five ICAM-1 binding-defective mutants, four had normal or even stronger interaction with Fg and C3bi, as reported in our previous study. Synthetic peptides derived from the identified site inhibited alpha(M)beta(2)-ICAM-1 interaction and supported direct binding to ICAM-1. Most importantly, perturbation of the F-alpha(7) loop caused conformational changes within the beta(2)I domain, which was further propagated to other regions of alpha(M)beta(2). Altogether, our data demonstrate that inside-out signaling could modulate ligand binding directly by changing the ligand-binding pocket per se and/or indirectly by inducing multiple conformational changes within the receptor.

Highlights

Integrins are heterodimeric surface receptors that play essential roles in cell-cell and cell-matrix interactions [1, 2]
The results from this study demonstrate that intercellular adhesion molecule 1 (ICAM-1) recognizes a novel region on top of the ␤2I domain, which is well separated from the binding sites for C3bi and Fg
Cell Adhesion to ICAM-1 by ␣M␤2 Is Activation-dependent— Ligand binding by the ␤2 integrins depends on receptor activation [2, 4], especially for ICAM-1 binding by ␣M␤2, which is of low affinity [10]

Summary

EXPERIMENTAL PROCEDURES

Materials—Human kidney 293 cells and the expression vector pCIS2M were gifts from Dr F. Human 293 cells expressing ␣M␤2 mutants that contain the active ␣M subunits with either the wild type or different mutant ␤2 subunits were prepared as described previously. FACS Analysis—A total of 106 cells expressing wild type or mutant ␣M␤2 in Hanks’ balanced salt solution containing 1 mM Mg2ϩ and 1 mM Ca2ϩ were incubated with 1 ␮g of mAb for 30 min at 4 °C, except for mAb 24, which were carried out at 37 °C. A total of 2 ϫ 106 cells in Hanks’ balanced salt solution containing 1 mM Ca2ϩ and 1 mM Mg2ϩ in the presence or absence of 2 mg/ml synthetic peptides or 50 nM NIF (specific for the ␣M subunit) were added to each well and incubated at 37 °C for 20 min. The specificity of peptide binding was further verified using the corresponding scrambled controls

RESULTS

DISCUSSION

Wild type

Another important finding from this work is that sequence