Characterization of αX I-domain binding to Thy-1

The beta 2 integrins are found exclusively in leukocytes and they are composed of a common beta chain, CD18, and one of four unique alpha chains, CD11a (alpha L subunit), CD11b (alpha M subunit), CD11c (alpha X subunit), or CD11d (alpha D subunit). alpha X-beta 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, alpha X-beta 2 is less functionally characterized than other beta 2 integrins. To understand the biological function of alpha X-beta 2 more, we tested the possibility that alpha X-beta 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 alpha X-beta 2, the I-domain, bound Thy-1 in a specific and divalent cation-dependent manner. The dissociation constant (K-D) of alpha X I-domain binding to Thy-1 was 1.16 mu M and the affinity of the binding was roughly 2-fold higher than that of alpha M I-domain. Amino acid substitutions on the beta D-alpha 5 of alpha X I-domain (D249, KE243/244) showed low affinities for Thy-1 while other point mutations on alpha 3-alpha 4 and beta E-alpha 6 loops of I-domain did not, suggesting that Thy-1 recognizes the portion of a beta D alpha 5 loop. possibly alpha 5 helix. Taken together, these results indicate that alpha X-beta 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 alpha X beta 2 may be involved in Thy-1 function. (c) 2005 Elsevier Inc. All rights reserved.