Membrane-proximal α/β Stalk Interactions Differentially Regulate Integrin Activation

Tetsuji Kamata,Makoto Handa,Yukiko Sato,Yasuo Ikeda,Sadakazu Aiso

doi:10.1074/jbc.m409548200

Abstract

The affinity of integrin-ligand interaction is regulated extracellularly by divalent cations and intracellularly by inside-out signaling. We report here that the extracellular, membrane-proximal alpha/beta stalk interactions not only regulate cation-induced integrin activation but also play critical roles in propagating inside-out signaling. Two closely related integrins, alphaIIbbeta3 and alphaVbeta3, share high structural homology and bind to similar ligands in an RGD-dependent manner. Despite these structural and functional similarities, they exhibit distinct responses to Mn(2+). Although alphaVbeta3 showed robust ligand binding in the presence of Mn(2+), alphaIIbbeta3 showed a limited increase but failed to achieve full activation. Swapping alpha stalk regions between alphaIIb and alphaV revealed that the alpha stalk, but not the ligand-binding head region, was responsible for the difference. A series of alphaIIb/alphaV domain-swapping chimeras were constructed to identify the responsible domain. Surprisingly, the minimum component required to render alphaIIbbeta3 susceptible to Mn(2+) activation was the alphaV calf-2 domain, which does not contain any divalent cation-binding sites. The calf-2 domain makes interface with beta epidermal growth factor 4 and beta tail domain in three-dimensional structure. The effect of calf-2 domain swapping was partially reproduced by mutating the specific amino acid residues in the calf-2/epidermal growth factor 4-beta tail domain interface. When this interface was constrained by an artificially introduced disulfide bridge, the Mn(2+)-induced alphaVbeta3-fibrinogen interaction was significantly impaired. Notably, a similar disulfide bridge completely abrogated fibrinogen binding to alphaIIbbeta3 when alphaIIbbeta3 was activated by cytoplasmic tail truncation to mimic inside-out signaling. Thus, disruption/formation of the membrane-proximal alpha/beta stalk interface may act as an on/off switch that triggers integrin-mediated bidirectional signaling.

Highlights

Integrins are a family of ␣/␤ heterodimeric transmembrane cell surface receptors that mediate cell-extracellular matrix and cell-cell interactions
The results suggest that disruption/formation of the membrane-proximal calf-2/epidermal growth factor (EGF)4-␤ tail domain (␤TD) interface may act as an on/off switch that propagates the conformational signals in integrin-mediated bidirectional signaling
C, Fluorescein isothiocyanate (FITC)-Fbg binding to cells expressing ␣IIb/␣V domainswapping chimeras (TC1C2, TC1, and T) in the presence of 1 mM Ca2ϩ/Mg2ϩ and 1 mM Mn2ϩ and cells pretreated with DTT in the presence of 1 mM Ca2ϩ/Mg2ϩ is shown

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—Anti-␣IIb monoclonal antibody (mAb) PL98DF6 [18] was a generous gift from Drs J. Conformation-dependent anti-␤3 mAbs anti-LIBS1 and anti-LIBS2 [19] were generous gifts from Dr Mark H. Anti-␣IIb␤3 complex-specific ligand-mimetic mAb OP-G2 [20] was a kind gift from Dr Yoshiaki Tomiyama (University of Osaka, Osaka, Japan). The ␣IIb␤3 complexspecific anti-␣IIb mAb P2, anti-␤3 mAb SZ21, and anti-␣V mAb AMF-7 were purchased from Beckman Coulter (Fullerton, CA). Mayor Clinic, AZ) were cloned into mammalian expression vector pBJ-1 (kindly provided by Dr Mark Davis, University of California, San Francisco, CA). The cDNAs for B/V and V/B chimeras were created by replacing the SacI-BamHI fragment between ␣IIb and ␣V. The cDNAs for ␣IIb/␣V domain-swapping chimeras TC1C2, TC1, T, C1, C2, D, C1C2D, TC2D, and TC1D were created using the overlap extension PCR method. The cDNAs for B/V 753–755, B/V 760 –764, B/V 774, B/V 781–783, B/V 787, B/V 899 –900, B/V 902–904,

The abbreviations used are

RESULTS

DISCUSSION