Evidence That β3 Integrin-Induced Rac Activation Involves the Calpain-Dependent Formation of Integrin Clusters That Are Distinct from the Focal Complexes and Focal Adhesions That Form as Rac and Rhoa Become Active

Abstract

Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. In the present study, we identified clusters of integrin that formed upstream of Rac activation. These clusters contained a Rac-binding protein(s) and appeared to be involved in Rac activation. The integrin clusters contained calpain and calpain-cleaved β3 integrin, while the focal complexes and focal adhesions that formed once Rac and Rho were activated did not. Moreover, the integrin clusters were dependent on calpain for their formation. In contrast, while Rac- and Rho-GTPases were dependent on calpain for their activation, formation of focal complexes and focal adhesions by constitutively active Rac or Rho, respectively, occurred even when calpain inhibitors were present. Taken together, these data are consistent with a model in which integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin, and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s).

Dynamic Modulation of Cytoskeletal Proteins Linking Integrins to Signaling Complexes in Spreading Cells

“…These are formed by activation of different signaling molecules at different stages of spreading. Moreover, we found that the initial integrin clusters differed from the focal complexes and focal adhesions in that they contained calpain, a protein that we could not detect in focal complexes and adhesions (17). In addition, they contained ␤ 3 -integrin that was present in a calpain-cleaved form whereas only intact ␤ 3 -integrin was detected in the other types of complexes (17).…”

“…In addition, calpain cleaves the cytoplasmic domain of the ␤ 1 -and ␤ 3 -integrins (24,25). We showed that one of the consequences of calpain activation in cultured cells was the formation of clusters of integrin that appear to contain Rac-binding protein(s) and to be required for the activation of Rac (17). Rac induces the formation of submembranous actin filament networks and focal complexes (9,10).…”

“…Although early studies focused on complexes known as focal adhesions that could be readily detected by immunofluorescence in spreading cultured cells, it is becoming apparent that additional types of integrin complexes exist (9,10,17). Recently, we studied the time course of integrin complex formation in spreading cells (17).…”

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

“…First, calpain activity may stimulate integrin activation and clustering that leads to the formation of stress fibers and focal adhesions [140,166,185,186]. This is thought to be achieved in part by calpain cleavage of talin, a cytoskeletal anchor that links integrins to actin filaments.…”

α6β1 integrin induces proteasome-mediated cleavage of erbB2 in breast cancer cells

“…However, erbB2 proteolysis by a6b1 integrin is different from these erbB2 truncations. Integrins are associated with various kinds of proteases, including calpain, urokinase-type plasminogen activator (uPA), MMP and caspase (Brooks et al, 1996;Yebra et al, 1999;Bialkowska et al, 2000;Stupack et al, 2001). However, calpain inhibitors, MMP inhibitors (BB2516 and EDTA) and a caspase inhibitor (z-VAD-fmk) all failed to affect the proteolysis of erbB2 in our experiments (Figure 9 and data not shown).…”