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 conserved KXGFFKR sequence of the integrin alpha-subunit cytoplasmic domains and calreticulin. Since this highly conserved sequence motif has been implicated in the regulation of the integrin affinity state, we wanted to determine whether the calreticulin-integrin interaction also depended on the integrin affinity state, and whether calreticulin occupation of integrin via the KXGFFKR motif was involved in the regulation of the ligand affinity state. We now demonstrate that anti-integrin antibody- or phorbol 12-myristate 13-acetate (PMA)-induced activation of the alpha 2 beta 1 integrin on Jurkat cells, as determined by stimulation of adhesion to collagen type I, resulted in an increased amount of calreticulin bound to this integrin. alpha 2 beta 1 activation with either anti-beta 1 or anti-alpha 2 monoclonal antibodies resulted in a greater amount of calreticulin coimmunoprecipitating with this integrin. Inactivation by neutralizing anti-integrin antibodies abrogated the calreticulin-integrin interaction. A correlation was also found between PMA-induced alpha 2 beta 1 activation and the amount of calreticulin bound to this integrin. Furthermore, pretreatment of streptolysin O-permeablized Jurkat cells with an anti-calreticulin antibody resulted in a significant and specific inhibition of the adhesion to collagen 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 the alpha 2 cytoplasmic domain may be required for stabilizing the high affinity state of this integrin. The data presented here also demonstrate, for the first time, an inducible interaction of an integrin with an intracellular protein that occurs via the alpha subunit of the integrin.
Coppolino
M
M
Division of Cancer Biology Research, Sunnybrook Health Science Centre, Toronto, Ontario, Canada.
Leung-Hagesteijn
C
C
Dedhar
S
S
Wilkins
J
J
D000595:Amino Acid Sequence; D000906:Antibodies / Q000494:pharmacology; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D037282:Calreticulin; D002448:Cell Adhesion; D002455:Cell Division; D002460:Cell Line; D003094:Collagen; D017124:Conserved Sequence; D006801:Humans; D016023:Integrins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D038981:Receptors, Collagen; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D016023:Integrins; D037102:Lectins; D038981:Receptors, Collagen; D012261:Ribonucleoproteins; D003094:Collagen; D013755:Tetradecanoylphorbol Acetate