Characterization of αX I-domain binding to Thy-1

The β2 integrins are found exclusively in leukocytes and they are composed of a common β chain, CD18, and one of four unique α chains, CD11a (αL subunit), CD11b (αM subunit), CD11c (αX subunit), or CD11d (αD subunit). αX-β2 which binds several ligands including fibrinogen and iC3b is expressed in monocytes/macrophages and dendritic cells playing an important role in the host defense. Despite the unique characteristics on expression and regulation, αX-β2 is less functionally characterized than other β2 integrins. To understand the biological function of αX-β2 more, we tested the possibility that αX-β2 binds Thy-1, a membrane protein involved in cell adhesion and signaling regulation in neurons and T cells. Here we report that a ligand binding moiety of αX-β2, the I-domain, bound Thy-1 in a specific and divalent cation-dependent manner. The dissociation constant (KD) of αX I-domain binding to Thy-1 was 1.16 μM and the affinity of the binding was roughly 2-fold higher than that of αM I-domain. Amino acid substitutions on the βD-α5 of αX I-domain (D249, KE243/244) showed low affinities for Thy-1 while other point mutations on α3-α4 and βE-α6 loops of I-domain did not, suggesting that Thy-1 recognizes the portion of a βD-α5 loop, possibly α5 helix. Taken together, these results indicate that αX-β2 specifically interacts with Thy-1. Additionally, kinetic analysis reveals a moderate affinity interaction in the presence of divalent cations. Given the reported role of Thy-1 in the regulation of T cell homeostasis and proliferation, it is tempting to speculate that αX-β2 may be involved in Thy-1 function.