Coppolino M, Leung-Hagesteijn C, Dedhar S, Wilkins J
Division of Cancer Biology Research, Sunnybrook Health Science Centre, Toronto, Ontario, Canada.
J Biol Chem. 1995 Sep 29;270(39):23132-8. doi: 10.1074/jbc.270.39.23132.
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.
我们之前已经证明，整合素α亚基胞质结构域高度保守的KXGFFKR序列与钙网蛋白之间存在相互作用。由于这个高度保守的序列基序与整合素亲和力状态的调节有关，我们想确定钙网蛋白-整合素相互作用是否也取决于整合素的亲和力状态，以及通过KXGFFKR基序的钙网蛋白对整合素的占据是否参与配体亲和力状态的调节。我们现在证明，抗整合素抗体或佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）诱导的Jurkat细胞上α2β1整合素的激活，通过刺激与I型胶原的黏附来测定，导致与该整合素结合的钙网蛋白量增加。用抗β1或抗α2单克隆抗体激活α2β1会导致更多的钙网蛋白与该整合素共免疫沉淀。用中和性抗整合素抗体使其失活消除了钙网蛋白-整合素相互作用。还发现PMA诱导的α2β1激活与与该整合素结合的钙网蛋白量之间存在相关性。此外，用抗钙网蛋白抗体预处理经链球菌溶血素O通透的Jurkat细胞，会导致对α2β1抗体或PMA诱导的与I型胶原黏附的显著且特异性抑制。这些数据表明，活性、高亲和力形式的α2β1与钙网蛋白结合，并且钙网蛋白与α2胞质结构域的结合可能是稳定该整合素高亲和力状态所必需的。此处呈现的数据还首次证明了整合素与细胞内蛋白之间通过整合素α亚基发生的可诱导相互作用。
