The N-terminal Domains of Talin Cooperate with the Phosphotyrosine Binding-like Domain to Activate β1 and β3 Integrins

Mohamed Bouaouina,Yatish Lad,David A Calderwood

doi:10.1074/jbc.m709527200

Abstract

The activation of integrin adhesion receptors from low to high affinity in response to intracellular cues controls cell adhesion and signaling. Binding of the cytoskeletal protein talin to the beta3 integrin cytoplasmic tail is required for beta3 activation, and the integrin-binding PTB-like F3 domain of talin is sufficient to activate beta3 integrins. Here we report that, whereas the conserved talin-integrin interaction is also required for beta1 activation, and talin F3 binds beta1 and beta3 integrins with comparable affinity, expression of the talin F3 domain is not sufficient to activate beta1 integrins. beta1 integrin activation could, however, be detected following expression of larger talin fragments that included the N-terminal and F1 domains, and mutagenesis indicates that these domains cooperate with talin F3 to mediate beta1 activation. This effect is not due to increased affinity for the integrin beta tail and we hypothesize that the N-terminal domains function by targeting or orienting talin in such a way as to optimize the interaction with the integrin tail. Analysis of beta3 integrin activation indicates that inclusion of the N-terminal and F1 domains also enhances F3-mediated beta3 activation. Our results therefore reveal a role for the N-terminal and F1 domains of talin during integrin activation and highlight differences in talin-mediated activation of beta1 and beta3 integrins.

Highlights

A large body of evidence points to regulation of integrin activation through interactions of the ␤ subunit tail [3, 14], ␣ tail-binding proteins have a role [15, 16]
We have compared the ability of talin fragments to activate ␤1 and ␤3 integrins, and find that ␤1 integrins require larger fragments of talin to produce detectable activation
This observation allowed us to identify a role for the N-terminal 205 amino acids of talin in the activation of both ␤1 and ␤3 integrins

Summary

EXPERIMENTAL PROCEDURES

Antibodies and DNAs—Monoclonal antibodies, anti-talin 8d4 (Sigma), activating anti-␤1 integrin 9EG7 (BD Biosciences), ligand-mimetic anti-␣IIb␤3 PAC1 (BD Biosciences), anti-hamster ␣5␤1 PB1 (Developmental Studies Hybridoma Bank), anti-HA tag (Covance), and polyclonal anti-GST (Chemicon) were purchased. CDNAs encoding integrins ␣IIb, ␣IIb␣5, ␤3␤1A, and GST-, GFP- or HA-tagged mouse talin F2, F3, or F2F3 (residues 206 –305, 309 – 405, and 206 – 405, respectively, of NP_006280.2), ␣IIb and ␤1A integrin tail model proteins, and GST-fibronectin type III repeats 9 –11 (FN9 –11) have been described previously (18, 37, 39 – 41). CHO or HT1080 cells were transfected with the indicated cDNAs using Lipofectamine (Invitrogen) and 24 h later cells were suspended and incubated with biotinylated recombinant GST-FN9 –11 in the presence or absence of integrin activators or inhibitors. The activation state of wild-type or chimeric ␣IIb␤3 integrins was assessed by measuring the binding of the ligand mimetic anti-␣IIb␤3 monoclonal antibody PAC1 in threecolor flow cytometric assays as described previously [17, 19, 21, 39]. PAC1 binding to live, integrin expressing (D57 positive) cells was measured and an activation index calculated as above

RESULTS

Talin fragments did not induce

DISCUSSION