Calpain Cleavage Promotes Talin Binding to the β3Integrin Cytoplasmic Domain

Abstract

Talin links integrin ␤ cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin ␤ cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the ␤ 3 integrin cytoplasmic domain. Here we report that the N-terminal ϳ47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the ␤ 3 tail. The affinity difference is mainly due to a difference in k on . Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K a and a 100-fold increase in apparent k on . The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the ␤ 3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band fourpoint-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.Integrins play important roles in the development and functioning of all multicellular animals (1). Integrins are non-covalent heterodimers of type I transmembrane protein subunits termed ␣ and ␤. Each subunit has a large (Ͼ700 residue) N-terminal extracellular domain. A single membrane-spanning domain links this extracellular domain to a generally (with the exception of ␤4) short (13-70 residues) cytoplasmic domain (2). Integrins bind to insoluble ligands (e.g. collagen fibrils) and link them to the intracellular cytoskeleton. In addition to forming these physical linkages, integrins regulate cell growth, survival, and differentiation (1). These signaling activities and cytoskeletal linkages depend on the integrin cytoplasmic domains (3). Conversely, integrin-mediated adhesion is rapidly and precisely regulated by changes in integrin affinity for ligand (activation) (4, 5) and by affinity-independent mechanisms such as changes in integrin clustering (6). Integrin linkages with the cytoskeleton can affect both integrin activation (7, 8) and affinity-independent (7, 9) regulation of integrinmediated cell adhesion. Thus, integrin-cytoskeletal linkages play a pivotal role in their regulation and in their signaling properties.Talin is an actin-binding protein that links integrins to the actin cytoskeleton (10). Talin co-localizes with clustered integrins in all known species that possess these receptors. Furthermore, genetic and cell biological analyses show that talin is required for integrin clustering into focal adhesions (11). Talinintegrin interactions can also regulate integrin activation (12). Talin consists of an N-terminal ϳ47-kDa globular head domain (talin-H) 1 and a ϳ190-kDa C-terminal rod (talin-R) domain (13...

Myoblast migration is prevented by a calpain‐dependent accumulation of MARCKS

“…However, the major role of these enzymes may be to alter structural integrity of focal contacts, thus mediating the detachment of the cell from the extracellular matrix allowing migration (Huttenlocher et al, 1997;Palecek et al, 1998;Perrin and Huttenlocher, 2002). Several studies have identified, at least in vitro, different key calpain targets, such as talin, paxillin, vinculin, alpha-actinin or integrin (beta-1, 3 and 4), localized in the focal complexes that are involved in adhesion and cell migration (Pfaff et al, 1999;Huttenlocher et al, 1997;Carragher et al, 1999;Yan et al, 2001;Dourdin et al, 2001;Liu and Schnellmann, 2003). Here, we reported a significant accumulation of the myris- Protein extracts from transfected myoblasts were used to perform an immunoblot using antibodies directed against MARCKS (A) and myoblast migration was analyzed by wound assay as described in experimental procedures (B).…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…This is thought to be achieved in part by calpain cleavage of talin, a cytoskeletal anchor that links integrins to actin filaments. Calpain cleavage of talin exposes the N-terminal FERM domain that is then free to bind directly to integrin β subunits [187][188][189] and forge linkages with actin filaments.…”

Regulating cell migration: calpains make the cut

“…However, these clusters do not seem to be precursors of typical adhesion complexes; so their significance is not known. Calpain-mediated proteolysis of talin 1 might be involved in assembling adhesion complexes, since proteolysis of talin 1 by calpains promotes its binding to integrin ␤-tails, which is known to be crucial for inside-out activation of integrins (Calderwood, 2004;Calderwood et al, 2002;Calderwood et al, 1999;Yan et al, 2001). Proteolysis of the actin-binding protein myristoylated alanine-rich protein kinase C substrate (MARCKS) might also play a role in the formation of adhesion complexes, since inhibition of calpains in myoblasts leads to defects in new adhesion formation and migration coincident with an accumulation of MARCKS (Dedieu et al, 2004).…”