Characterization of alphaX I-domain binding to Thy-1.
The beta2 integrins are found exclusively in leukocytes and they are composed of a common beta chain, CD18, and one of four unique alpha chains, CD11a (alphaL subunit), CD11b (alphaM subunit), CD11c (alphaX subunit), or CD11d (alphaD subunit). alphaX-beta2 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, alphaX-beta2 is less functionally characterized than other beta2 integrins. To understand the biological function of alphaX-beta2 more, we tested the possibility that alphaX-beta2 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 alphaX-beta2, the I-domain, bound Thy-1 in a specific and divalent cation-dependent manner. The dissociation constant (K(D)) of alphaX I-domain binding to Thy-1 was 1.16muM and the affinity of the binding was roughly 2-fold higher than that of alphaM I-domain. Amino acid substitutions on the betaD-alpha5 of alphaX I-domain (D249, KE243/244) showed low affinities for Thy-1 while other point mutations on alpha3-alpha4 and betaE-alpha6 loops of I-domain did not, suggesting that Thy-1 recognizes the portion of a betaD-alpha5 loop, possibly alpha5 helix. Taken together, these results indicate that alphaX-beta2 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 alphaX-beta2 may be involved in Thy-1 function.
Choi
Jeongsuk
J
Divisions of Science Education and Biology, Kangwon National University, Choonchun, Kangwon, Republic of Korea.
Leyton
Lisette
L
Nham
Sang-Uk
SU
D000818:Animals; D039521:CD11c Antigen / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D016167:Integrin alphaXbeta2 / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018800:Thy-1 Antigens / Q000235:genetics / Q000378:metabolism*
D039521:CD11c Antigen; D016167:Integrin alphaXbeta2; D018800:Thy-1 Antigens