The Talin Rod IBS2 α-Helix Interacts with the β3 Integrin Cytoplasmic Tail Membrane-proximal Helix by Establishing Charge Complementary Salt Bridges

Sophie Rodius,Olivier Chaloin,Michèle Moes,Elisabeth Schaffner-Reckinger,Isabelle Landrieu,Guy Lippens,Minghui Lin,Ji Zhang,Nelly Kieffer

doi:10.1074/jbc.m709704200

Abstract

Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a second, IBS2, that maps to helix 50 of the talin rod domain and is essential for linking integrin beta subunits to the cytoskeleton ( Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J. Biol. Chem. 282, 17280-17288 ). Through the combined approach of mutational analysis of the beta3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies, as well as site-specific antibody inhibition experiments, we provide evidence that the integrin beta3-talin rod interaction relies on a helix-helix association between alpha-helix 50 of the talin rod domain and the membrane-proximal alpha-helix of the beta3 integrin cytoplasmic tail. Moreover, charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin beta3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the beta3 subunit cytoplasmic helix as the integrin alphaIIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the beta3 subunit following integrin activation.

Highlights

Integrins are ␣␤ heterodimeric receptors that mediate attachment of cells to the extracellular matrix (ECM) and play important roles in cell adhesion, migration, proliferation, and survival [2, 3]
A Polyclonal Antibody Raised against Helices 50 and 51 of the Human Talin Rod Domain Inhibits the Talin integrin binding site 2 (IBS2)-Integrin ␤3 Cytoplasmic Tail Interaction—We have previously identified the 23-residue amphipathic ␣-helix 50 of talin rod as the minimal functional IBS2 structure able to interact with the integrin ␤3 subunit cytoplasmic tail [1, 21]
When tested in indirect immunofluorescence assays using Chinese hamster ovary (CHO) ␣IIb␤3 cells adherent on fibrinogen (Fig. 1D), the anti-IBS2 Ab reacted with endogenous hamster talin and preferentially labeled lamellipodia, where its staining overlapped with anti-talin rod or anti-␤3 integrin labeling

Summary

Introduction

Integrins are ␣␤ heterodimeric receptors that mediate attachment of cells to the extracellular matrix (ECM) and play important roles in cell adhesion, migration, proliferation, and survival [2, 3]. We have previously identified ␣-helix 50 of the talin rod domain as the minimal functional structure of the integrin binding site 2 (IBS2), able to interact with the ␤3 integrin cytoplasmic tail [1, 21].

Results

Conclusion