Journal of Molecular Biology
Characterization of an Actin-binding Site within the Talin FERM Domain
Introduction
Talin is a large (270 kDa, 2541 amino acid residues) cytoskeletal protein that is localized primarily in cell-extracellular matrix adherens-type junctions. Thus, it is found in the focal adhesions of adherent cells in culture,1 the myotendinous junction,2 neuromuscular junctions3 and costameres of skeletal muscle cells, the intercalated discs and costameres of cardiac muscle cells,4 and the membrane-associated dense plaques of smooth muscle cells.5 However, it is also found in membrane ruffles of migrating cells,1 the junctions between T-cells and antigen presenting cells6 and human platelets.7 At these locations, talin is believed to play a key role in linking the cytoplasmic domains of the integrin family of cell adhesion molecules to the actin cytoskeleton.8, 9 Evidence for such a role has come from a variety of sources. When antibodies against talin were microinjected into fibroblasts, migration of the cells was inhibited, and focal adhesions and actin stress fibers were disrupted.10, 11 Ablation of talin in neuronal growth cones using chromophore-assisted laser inactivation resulted in neurite retraction,12 and down-regulation of talin in HeLa cells using antisense RNA technology caused decreased cell spreading and reduced the size of focal adhesions.13 A similar phenotype was observed in mouse embryonic stem cells carrying mutations in both talin alleles,14 and studies with talin (−/−) fibroblasts show that it is required to establish the initial weak link between integrins and actomyosin,15 and the subsequent assembly of focal complexes.16 Gene knockout studies in Caenorhabditis elegans,17 Drosophila18 and mouse19 confirm that talin is essential for integrin-mediated developmental processes in vivo.
The biochemical properties of talin are also consistent with a role in linking integrins to F-actin. The N-terminal head of talin contains a FERM (band 4.1-ezrin-radixin-moesin) domain, which both binds to and activates integrins,20 probably by disrupting the interaction between the α and β-subunit cytodomains.21 The talin head also contains binding sites for (i) layilin, a C-type lectin that colocalizes with talin in membrane ruffles,22 (ii) the type 1γ neuronal isoform of phosphatidylinositol 4-phosphate 5-kinase (PIPkin),23, 24 which is important in focal adhesion assembly, and (iii) FAK,25 which is implicated in stimulating focal adhesion turnover and cell migration.26 The large C-terminal rod domain of talin contains a second integrin-binding site,27 three vinculin-binding sites,28 and is able to bind F-actin.29, 30, 31, 32 The talin molecule is comprised of two subunits that are likely arranged in an antiparallel manner,33, 34 which probably explains why it can cross-link actin into networks and bundles.31, 35, 36, 37, 38 The ability of talin to crosslink F-actin is highly dependent on pH, ionic strength and temperature, and talin exhibits optimal actin binding activity in vitro at pH 6.4, low ionic strength and 37 °C.31
The best characterized actin-binding site in talin ABS3 (residues 2345–2541) is located close to the C-terminal region of the protein,39 and is highly conserved across species. It contains a so-called I/LWEQ module that is also found in several other actin-binding proteins such as Sla2p and Hip-1.40, 41 A recombinant polypeptide containing this module was shown to inhibit binding of intact talin to F-actin, suggesting that it is a major determinant of actin binding in vitro.41 Preliminary evidence for two other actin-binding sites in talin has been presented,39 one of which (ABS1) is located within the N-terminal head domain. Here, we show that (i) the purified talin head isolated from talin after calpain cleavage does indeed cosediment with F-actin, (ii) the actin-binding site is within the talin FERM domain, (iii) enhanced green fluorescent protein (EGFP) fusion proteins containing talin FERM subdomains colocalize with actin-rich structures in COS cells, (iv) the talin FERM domain F2F3 polypeptide binds to subdomain 4 of F-actin, and (v) PIPkin, a FERM domain ligand, cosediments with F-actin in the presence, but not the absence of the FERM domain. Together, these data provide strong evidence for the presence of an actin-binding site in the talin head domain.
Section snippets
Purified talin head binds to F-actin
The Ca2+-dependent protease type II (m-calpain) readily cleaves talin between residues 433 and 434,42 generating an ∼47 kDa N-terminal head and an ∼190 kDa C-terminal rod domain. Previous reports have indicated that the ∼190 kDa rod domain, but not the ∼47 kDa head, binds to F-actin,30, 32, 36 but these studies were conducted under conditions that were suboptimal for talin binding.31 Because recombinant talin head polypeptides bind to F-actin,39 we re-examined the ability of the purified N-terminal
Discussion
Here, we present six independent lines of evidence that the talin head contains an actin-binding site referred to as ABS1: (i) the talin head purified from a calpain-digest of smooth muscle talin cosedimented with F-actin; (ii) recombinant talin head and polypeptides containing the F2 and F3 FERM subdomains also cosedimented with F-actin; (iii) F-actin protected the recombinant talin head from proteolytic cleavage by trypsin; (iv) PIPkin, which binds to the talin F3 FERM subdomain, cosedimented
Protein expression constructs
DNA fragments encoding various regions of the talin head and the C-terminal ABS3 of talin were amplified by PCR from chicken talin cDNAs39 using oligonucleotides containing 5′ BamHI and 3′ EcoRI restriction enzyme cleavage sites and the products cloned into the pGEX4T1 vector (Amersham Biosciences, Piscataway, NJ). Recombinant plasmids were expressed in Escherichia coli (BL21-Codon+-RIL, Stratagene, La Jolla, CA) and the GST fusion proteins purified using glutathione-agarose (Amersham
Acknowledgements
We thank Dr Elisabeth Huff-Lonergan for providing the highly purified m-calpain and Dr G. Dipaolo (Yale) for the GST-PIPkin28 construct. This research was supported in part by a grant from the United States Department of Agriculture, NRICGP Award 2003-35206-12823. This is Journal Paper No. J-19650 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Projects 6616, 3900 and 2127, and supported by Hatch Act and State of Iowa funds. The electron microscopy and image analysis
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Present address: R. M. Bellin, Department of Biology, College of the Holy Cross, Worcester, MA 01610, USA.