INDUCIBLE INTERACTION OF INTEGRIN ALPHA(2)BETA(1) WITH CALRETICULIN -DEPENDENCE ON THE ACTIVATION STATE OF THE INTEGRIN

We have previously demonstrated an interaction between the highly cons erved KXGFFKR sequence of the integrin alpha-subunit cytoplasmic domai ns and calreticulin. Since this highly conserved sequence motif has be en implicated in the regulation of the integrin affinity state, we wan ted to determine whether the calreticulin-integrin interaction also de pended on the integrin affinity state, and whether calreticulin occupa tion of integrin via the KXGFFKR motif was involved in the regulation of the ligand affinity state; We now demonstrate that anti-integrin an tibody- or phorbol la-myristate 13-acetate (PMA)-induced activation of the alpha(2) beta(1) integrin on Jurkat cells, as determined by stimu lation of adhesion to collagen type I, resulted in an increased amount of calreticulin bound to this integrin. alpha(2) beta(1) activation w ith either anti-beta(1) or anti-alpha(2) monoclonal antibodies resulte d in a greater amount of calreticulin coimmunoprecipitating with this integrin. Inactivation by neutralizing anti-integrin antibodies abroga ted the calreticulin-integrin interaction. A correlation was also foun d between PMA-induced alpha(2) beta(1) activation and the amount of ca lreticulin bound to this integrin. Furthermore, pretreatment of strept olysin O-permeablized Jurkat cells with an anti-calreticulin antibody resulted in a significant and specific inhibition of the adhesion to c ollagen type I that could be induced by antibodies to alpha(2) beta(1) or by PMA. These data suggest that the active, high affinity form of alpha(2) beta(1) binds calreticulin and that calreticulin binding to t he alpha(2) cytoplasmic domain may be required for stabilizing the hig h affinity state of this integrin. The data presented here also demons trate, for the first time, an inducible interaction of an integrin wit h an intracellular protein that occurs via the alpha subunit of the in tegrin.