Abstract
Cellular regulation of the ligand binding affinity of integrin adhesion receptors (integrin activation) depends on the integrin beta cytoplasmic domains (tails). The head domain of talin binds to several integrin beta tails and activates integrins. This head domain contains a predicted FERM domain composed of three subdomains (F1, F2, and F3). An integrin-activating talin fragment was predicted to contain the F2 and F3 subdomains. Both isolated subdomains bound specifically to the integrin beta3 tail. However, talin F3 bound the beta3 tail with a 4-fold higher affinity than talin F2. Furthermore, expression of talin F3 (but not F2) in cells led to activation of integrin alpha(IIb)beta3. A molecular model of talin F3 indicated that it resembles a phosphotyrosine-binding (PTB) domain. PTB domains recognize peptide ligands containing beta turns, often formed by NPXY motifs. NPX(Y/F) motifs are highly conserved in integrin beta tails, and mutations that disrupt this motif interfere with both integrin activation and talin binding. Thus, integrin binding to talin resembles the interactions of PTB domains with peptide ligands. These resemblances suggest that the activation of integrins requires the presence of a beta turn at NPX(Y/F) motifs conserved in integrin beta cytoplasmic domains.
Highlights
Integrin adhesion receptors are essential for the development and survival of multicellular animals
To determine whether a portion of the talin head domain was sufficient for these functions, we analyzed a fragment of the talin head containing Glu186–Gln435 that binds to the integrin 1D tail [5]
We report that a predicted PTB-like 96-amino acid subdomain of the talin FERM domain contains a major integrin-binding site and activates integrin ␣IIb3
Summary
Antibodies and cDNAs—Anti-talin (8d4; Sigma), anti-hemagglutinin (12CA5; American Type Culture Collection), anti-Tac (7G7B6; American Type Culture Collection), anti-Syk (4D10; Santa Cruz Biotechnology), anti-Myc (Santa Cruz Biotechnology), and anti-GST (B14; Santa Cruz Biotechnology) monoclonal antibodies and anti-Dok polyclonal. Purification of Recombinant Proteins—Recombinant model proteins of integrin cytoplasmic tails and GST fusion proteins were expressed and purified as previously described [5, 19]. The kinetic data were interpreted in the context of a first-order kinetic model: A ϩ B ϭ AB [23,24,25] For such a model, the association (kon) and dissociation (koff) rate constants are described by Equation 1. Binding of the activation-specific anti-␣IIb3 monoclonal antibody PAC1 to transfected cells FACS Analysis of the Activation State of ␣IIb3—Chinese hamster ovary (CHO) cells expressing integrin ␣IIb3 [3] were transiently transfected with cDNAs encoding Tac-␣5 (1 g) and talin fragments (3 g) using LipofectAMINE Plus (Invitrogen). The quality of the models was assessed with the program WHAT IF [29]
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