Calpain cleavage of integrin β cytoplasmic domains

Abstract

We showed previously that the calcium-dependent protease, calpain, cleaves the cytoplasmic domain of the integrin β3 subunit. To investigate whether susceptibility to calpain is a common feature of all integrin β subunits, and to map calpain cleavage sites in different integrin β tails, we treated recombinant cytoplasmic domains of integrin β1A, β1D, β2, β3 and β7 subunits with purified calpain in vitro. We found that the cytoplasmic domains of all these integrin chains were cleaved by calpain. HPLC followed by mass spectrometry was used to identify calpain cleavage sites. These sites were clustered in the C-terminal half of the integrin β cytoplasmic domains in regions flanking the two NXXY motifs, suggesting the possibility that the structural framework provided by these motifs is recognized by calpain. We used the knowledge of these cleavage sites to develop cleavage site-specific antibodies and to demonstrate cleavage of the β1A cytoplasmic domain in intact platelets stimulated with calcium ionophore or thrombin. Thus susceptibility to calpain cleavage is common to integrin β subunits, can be induced in intact cells, and appears to favor regions surrounding two conserved NXXY motifs.