Reilly Dermot, Larkin Deirdre, Devocelle Marc, Fitzgerald Desmond J, Moran Niamh
Centre for Sythesis and Chemical Biology, Department of Chemistry, Royal College of Surgeons in Ireland, Dublin.
Platelets. 2004 Feb;15(1):43-54. doi: 10.1080/09537100310001640055.
The platelet integrin alphaIIbbeta3 alters conformation in response to platelet activation and ligand binding, although the molecular mechanisms involved are not known. We previously showed that a lipid modified peptide, corresponding to the membrane proximal 989KVGFFKR995 portion of the alphaIIb cytoplasmic tail, independently activates platelet alphaIIbbeta3. Calreticulin (CRT) is a potential integrin regulatory protein based on its interaction with the highly conserved alpha-integrin sequence KxGFFKR. We therefore examined the possible interaction of calreticulin and alphaIIbbeta3 in human platelets. We demonstrate that calreticulin in platelets is localised to the granulomere. In contrast, the known integrin-binding protein talin accumulates at the periphery of spreading platelets and colocalises with alphaIIbbeta3 during the process of adhesion. An interaction between calreticulin and alphaIIbbeta3 could not be demonstrated using co-immunoprecipitation techniques under various platelet activation states, even in the presence of covalent chemical crosslinkers. Thus, calreticulin does not functionally interact with the major integrin in human platelets. In order to identify proteins that interact with the integrin KVGFFKR motif we then used a peptide 'pull-down' assay from platelet lysates with biotinylated peptides and demonstrate that only the alphaIIb and beta3 subunits selectively and individually interact with this sequence. This interaction is divalent cation-dependent, has high-affinity, and occurs both with purified alphaIIbbeta3 complex and with electroeluted alpha and beta subunits. Thus, our data show that the conserved integrin KVGFFKR domain interacts primarily with the alpha and beta cytoplasmic tails and not with CRT in human platelets.
血小板整合素αIIbβ3会因血小板激活和配体结合而改变构象，尽管其中涉及的分子机制尚不清楚。我们之前表明，一种脂质修饰肽，对应于αIIb细胞质尾部膜近端的989KVGFFKR995部分，可独立激活血小板αIIbβ3。基于钙网蛋白（CRT）与高度保守的α整合素序列KxGFFKR的相互作用，它是一种潜在的整合素调节蛋白。因此，我们研究了钙网蛋白与人血小板中αIIbβ3之间可能的相互作用。我们证明血小板中的钙网蛋白定位于颗粒区。相比之下，已知的整合素结合蛋白踝蛋白在铺展血小板的周边积累，并在黏附过程中与αIIbβ3共定位。即使在存在共价化学交联剂的情况下，使用共免疫沉淀技术在各种血小板激活状态下都无法证明钙网蛋白与αIIbβ3之间存在相互作用。因此，钙网蛋白在功能上不与人血小板中的主要整合素相互作用。为了鉴定与整合素KVGFFKR基序相互作用的蛋白质，我们随后使用生物素化肽从血小板裂解物中进行肽“下拉”试验，并证明只有αIIb和β3亚基选择性且分别与该序列相互作用。这种相互作用依赖二价阳离子，具有高亲和力，并且在纯化的αIIbβ3复合物以及电洗脱的α和β亚基中均会发生。因此，我们的数据表明，在人血小板中，保守的整合素KVGFFKR结构域主要与α和β细胞质尾部相互作用，而不与钙网蛋白相互作用。
