The dual structural roles of the membrane distal region of α integrin cytoplasmic tail in integrin inside-out activation

Abstract

BSTRACTStudies on the mechanism of integrin inside-out activation have been focused on the role of b-integrin cytoplasmic tails, which are relatively conserved and bear binding sites for the intracellular activators including talin and kindlin. Cytoplasmic tails for a-integrins share a conserved GFFKR motif at the membrane-proximal region and this forms a specific interface with the b-integrin membraneproximal region to keep the integrin inactive. The a-integrin membrane-distal regions, after the GFFKR motif, are diverse both in length and sequence and their roles in integrin activation have not been well-defined. In this study, we report that the a-integrin cytoplasmic membrane-distal region contributes to maintaining integrin in the resting state and to integrin inside-out activation.Complete deletion of the a-integrin membrane-distal region diminished talin-and kindlin-mediated integrin ligand binding and conformational change. A proper length and suitable amino acids in a-integrin membrane-distal region was found to be important for integrin inside-out activation. Our data establish an essential role for the a-integrin cytoplasmic membrane-distal region in integrin activation and provide new insights into how talin and kindlin induce the high-affinity integrin conformation that is required for fully functional integrins.

Implications of the differing roles of the β1 and β3 transmembrane and cytoplasmic domains for integrin function

“…This is surprising in light of studies suggesting that the fusion proteins containing the TM-only domain of these integrin subunits can form heterodimers in E. coli (Berger et al, 2010; Schneider and Engelman, 2004). However, these latter studies were conducted in the absence of the β1, α1, and α2 CT, which almost certainly profoundly impact heterodimerization (Briesewitz et al, 1995; Liu et al, 2015). Our results suggest that the α1 and α2 CT may actually inhibit formation of α1β1 and α2β1 TM/CT heterodimers, at least in bicelles.…”

A pivotal role for a conserved bulky residue at the α1-helix of the αI integrin domain in ligand binding

“…HEK293FT cells (ThermoFisher Scientific) were cultured in DMEM plus 10% FBS at 37°C with 5% CO 2 . ICAM-1 binding of HEK293FT cells was as described before (19,21). The cells were co-transfected with ␣ L and ␤ 2 integrin constructs with Lipofectamine 2000 (ThermoFisher Scientific) for at least 24 h. Transfected cells were incubated in HBSGB buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5.5 mM glucose, and 1% BSA) with 15 g/ml each of ICAM-1-Fc and biotin-labeled mouse anti-human IgG1 Fc in the presence of 5 mM EDTA or 1 mM Ca 2ϩ /Mg 2ϩ or 0.2 mM Ca 2ϩ plus 2 mM Mn 2ϩ at 25°C for 30 min.…”

“…Binding of the active conformation-specific anti-␤ 2 mAbs KIM127 and m24 to the HEK293FT transfectants was performed as described before (19,21). In brief, the cells were first incubated with 10 g/ml biotin-labeled KIM127 or m24 in HBSGB buffer containing 1 mM Ca 2ϩ /Mg 2ϩ or 0.2 mM Ca 2ϩ plus 2 mM Mn 2ϩ at 25°C for 30 min and then washed and incubated with 10 g/ml FITC-labeled TS2/4 and Alexa Fluor 647-labeled streptavidin on ice for 30 min.…”

α-integrins dictate distinct modes of type IV collagen recruitment to basement membranes

“…One possible mode of activation is through the diverse cytoplasmic tail region of the α subunits. Studies in mammalian cell lines have shown that the cytoplasmic tails of α of integrin subunits are required for the inside-out activation of integrin receptors and may bestow specificity to integrin activation 38,55 . Supporting this idea, we found that expressing the PAT-2 integrin α cytoplasmic tail fused to the INA-1 extracellular domain in the pharynx activated its ability to recruit laminin, even though INA-1 does not normally function in pharyngeal BM laminin recruitment during larval development.…”