Characterization of alpha X I-domain binding to Thy-1

Abstract

The β 2 integrins are found exclusively in leukocytes and they are composed of a common β chain, CD18, and one of four unique α chains, CD11a (α L subunit), CD11b (α M subunit), CD11c (α X subunit), or CD11d (α D subunit). α X-β 2 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 oil expression and regulation, α X-β 2 is less functionally characterized than other β 2 integrins. To understand the biological function of α X-β 2 more, we tested the possibility that α X-β 2 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 α X-β 2, the I-domain, bound Thy-1 in a specific and divalent cation-dependent manner. The dissociation constant (K-D) of α X I-domain binding to Thy-1 was 1.16 μ M and the affinity of the binding was roughly 2-fold higher than that of α M I-domain. Amino acid substitutions on the β D-α 5 of α X I-domain (D249, KE243/244) showed low affinities for Thy-1 while other point mutations on α 3-α 4 and β E-α 6 loops of I-domain did not, suggesting that Thy-1 recognizes the portion of a β D α 5 loop. possibly α 5 helix. Taken together, these results indicate that α X-β 2 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 or T cell homeostasis and proliferation, it is tempting to speculate that α X β 2 may be involved in Thy-1 function.