The Integrin Binding Site 2 (IBS2) in the Talin Rod Domain Is Essential for Linking Integrin β Subunits to the Cytoskeleton

Abstract

Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984 -2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258 -22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in ␣ helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this ␣ helix, which disrupted the ␣-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to ␣IIb␤3 integrin in focal adhesions and to inhibit in vitro this association as shown by an ␣IIb␤3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1 ؊/؊ cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.Talin1 is a multifunctional ϳ270-kDa (2541 amino acids, aa) 3 cytoskeletal protein that functions both as a key structural component of focal adhesions linking integrins to the cytoskeleton and also as a major regulator of integrin-dependent bidirectional signal transduction (2). In vivo, talin is found in equilibrium between a functionally inactive cytoplasmic form and a membrane-bound active form (3, 4). Talin activation is a complex process that is still poorly understood. In some cells, integrin outside-in signaling leads to association of the large isoform of type 1 phosphatidylinositol phosphate kinase with talin. This activates the phosphatidylinositol phosphate kinase, and together they translocate to the plasma membrane (5). Local phosphatidylinositol 4,5-biphosphate production by the phosphatidylinositol phosphate kinase is then thought to activate talin (6), possibly by relieving an autoinhibitory head-tail association unmasking the integrin binding site in the talin head (IBS1). In platelets, talin becomes activated through a protein kinase C ␣-dependent signaling pathway that stimulates activation and translocation of the small GTPase Rap1 to the plasma membrane where it interacts with its effector Riam, leading to the recruitment of talin and the unmasking of its integrin binding site in the head domain (7). Active talin, which can form anti-parallel homodimers (3), finally binds to and activates integrins allowing the exposure of a high affinity binding site for integrin extracellular ligands ...

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

“…Interestingly, two of the three Glu residues identified here (Glu-726 and Glu-733) are located on the same face of the ␣-helix as residue , involved in the electrostatic interface with residue Arg-995 of the ␣IIb membrane-proximal ␣-helix, suggesting that the glutamic acids Glu-726 and Glu-733 are not accessible in the resting receptor because of the close contact with the ␣IIb subunit cytoplasmic tail. These results correlate with our previous pull- down data, showing that native integrin ␣IIb␤3 purified from platelets had to be activated with Mn 2ϩ and the ligand mimetic mAb PAC-1 to allow its interaction with talin IBS2 (1). These data are also in agreement with the model of Tanentzapf and Brown (37), suggesting that the talin rod domain may only interact with high affinity, ligand-bound integrins.…”

“…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). Here, we have further characterized the integrin ␤3-talin rod interaction.…”

“…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). To further characterize this IBS2-␤3 interaction, we have generated an anti-IBS2 polyclonal antibody (Ab) raised in rabbits against a recombinant 7-kDa GST-tagged talin rod fragment, corresponding to the ␣-helices 50 and 51 (residues 2071-2135).…”

“…3B) opposite to the hydrophobic face corresponding to the potential vinculin binding site (27), suggesting that they might function as direct contact sites in the IBS2-␤3 integrin interaction. In addition, we have previously shown by sitedirected mutagenesis that residue Lys-2099, located at the C-terminal end of helix 50, is not involved in this interaction (1).…”

Structure and mechanics of integrin-based cell adhesion

“…Formation and transformation of focal contacts-While F3 binding to the integrin proximal β3-tail is sufficient to switch the ectodomain into the ligand-competent state, it does not mediate integrin linkage to the cytoskeleton at focal complexes [62]; a second interaction between the rod domain of talin, formed of multiple amphipathic helical bundles, and ligandoccupied integrin β-tails appears necessary [63]. The structural basis of this integrin-talin rod interaction is unknown; multivalent ligand and force applied on the early matrix-integrin-talin complexes by Rho-activated actomyosin motors may expose a binding-site in the β3-tail for the talin rod [52,64].…”