On the Role of Spectrin and Spectrin SH3 Domain in Integrin-induced Signaling.

Spectrin is a cytoskeletal protein that is considered to be a structural component of submembranous skeletons. However, spectrin contains motifs such as the src homology 3 domain (SH3) that suggests it may also be involved in signal transduction. In platelets, spectrin is a component of a membrane skeleton that interacts with β1 and β3 integrins. In the present study, we have used bovine aortic endothelial (BAE) cells and Chinese Hamster Ovary (CHO) cells to determine whether spectrin is involved in integrin-induced signaling and to determine whether the SH3 domain is involved. Previously, we found that signaling across β1-and β-containing integrins activates calpain which leads to the formation of integrin clusters. The clusters contain active calpain , calpain cleaved β3 integrin and putative Rac-binding protein(s). Rac appears to be activated in these clusters and then Rac-induced focal complexes and, later, RhoA-induced focal adhesions form. In the present study, immunofluorescence experiments showed that spectrin colocalized with β3 integrin in the calpain-induced integrin clusters but it was absent from focal complexes and focal adhesions. To test the possibility that spectrin SH3 domain might regulate integrin-induced signaling, we overexpressed the SH3 domain. This domain accumulated in the integrin clusters and the subsequent formation of focal complexes and focal adhesions was inhibited. Overexpression of constitutively active Rac in the presence of SH3 domain restored cell spreading, suggesting that the SH3 domain regulates a signaling step upstream of Rac activation. The direct measurement of Rac activation showed inhibition of Rac GTP-loading in the presence of SH3 domain. A two-hybrid system approach identified a novel protein (Da51) that interacted with the SH3 domain of spectrin. This protein contains a proline-rich domain that was shown in in vitro binding assays to mediate the interaction of Da51 with the spectrin SH3 domain. When overexpressed as a GFP-fusion protein in BAE cells, Da51 coimmuno-precipitated with spectrin indicating an in vivo interaction. As with overexpression of the spectrin SH3 domain, overexpression of Da51 in BAE cells, inhibited integrin-induced cell spreading. Taken together our data suggest i) that calpain-induced cleavage of spectrin and recruitment of spectrin to transient integrin clusters is an initial event following integrin-ligand interactions and ii) recruitment of signaling molecule(s) such as Da51 to the SH3 domain of spectrin in the integrin clusters participates in activation of signaling steps leading to Rac activation.