Ionomycin-induced activation of LFA-1 is independent of calpain-mediated cleavage of talin

Integrins are heterodimeric surface receptors that play a critical role in many cellular processes due to their ability to mediate the interactions between cells as well as between cells and the extracellular matrix. Integrin function can be regulated by conformational changes in their extracellular domains that increase their affinity for their ligand. Inside-out signalling is thought to initiate conformational changes through the binding of cytoplasmic proteins such as talin to integrin cytoplasmic domains. Calpains, calcium-dependent proteases, cleave talin upon activation, generating a free talin head domain that exhibits higher affinity for integrins than the intact talin protein. Therefore, the activation of calpains by receptor-induced calcium fluxes may lead to talin cleavage and could be an important step in the activation of integrins via inside-out signalling. In this study, the effects of the calcium ionophore ionomycin on the activation of LFA-1 in murine T cells were tested. Ionomycin treatment induced LFA-1-mediated adhesion in the helper T cell hybridoma line T28, in in vitro generated cytotoxic T cells, and in primary resting splenic T cells. The calpain inhibitors calpeptin and PD 150606 abrogated LFA-1 activation by ionomycin, implicating calpains in this process. However, calpeptin also inhibited LFA-1 activation by PMA, which did not induce calcium influx, suggesting that calpeptin is not entirely specific to calpains. Additionally, both calpain inhibitors induced the rapid apoptosis of cells treated with ionomycin, indicating that their effects on adhesion are non-specific. In contrast, two other calpain inhibitors, namely ALLN and calpain inhibitor III, did not impair LFA-1 activation by ionomycin. Western blotting did not detect cleavage of talin following ionomycin treatment of T cells, or cleavage of a-spectrin, another well-established substrate of calpains. These findings suggest that ionomycin-induced activation of LFA-1 is independent of the calpain-mediated cleavage of talin.Medicine, Faculty ofMedical Genetics, Department ofGraduat