Inhibition of Ca(2+) signalling by p130, a phospholipase-C-related catalytically inactive protein: critical role of the p130 pleckstrin homology domain.
Inhibition of Ca(2+) signalling by p130, a phospholipase-C-related catalytically inactive protein: critical role of the p130 pleckstrin homology domain.
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p130（一种磷脂酶-C 相关催化失活蛋白）对 Ca(2 ) 信号传导的抑制：p130 pleckstrin 同源结构域的关键作用。
作者：
H. Takeuchi;M. Oike;Hugh Paterson;Victoria Allen;T. Kanematsu;Yushi Ito;Christophe Erneux;M. Katan;Masato Hirata
p130最初被鉴定为类似于磷脂酶C-δ但缺乏任何磷脂酶活性的Ins（1，4，5）P（3）结合蛋白。在本研究中，我们进一步分析了p130与肌醇化合物在体外的相互作用。为了确定哪些潜在的配体与p130在细胞中相互作用，我们分析了这种蛋白的细胞定位，从细胞裂解物中分离蛋白-配体复合物，并通过使用透化和瞬时或稳定转染的COS-1细胞（COS-1（p130））研究了p130对Ins（1，4，5）P（3）介导的Ca（2+）信号传导的影响。在体外，p130与Ins（1，4，5）P（3）的结合亲和力高于与磷酸肌醇的结合亲和力。当通过免疫沉淀从COS-1（p130）细胞中分离该蛋白时，发现它与Ins（1，4，5）P（3）相关。定位研究表明全长p130存在于活细胞的细胞质中，而不是在质膜上。在基于细胞的测定中，p130对Ca（2+）信号传导具有抑制作用。当用缓激肽、表皮生长因子或ATP刺激载有fura-2的COS-1（p130）细胞时，发现在对照细胞中观察到的激动剂诱导的游离Ca（2+）浓度增加在COS-1（p130）中被抑制。这种抑制并不伴随着Ins（1，4，5）P（3）的产生减少;完整的p130 pleckstrin同源结构域，已知是体外配体结合位点，是细胞中这种效应所必需的。这些结果表明，Ins（1，4，5）P（3）可能是细胞中主要的p130配体，并且这种结合具有抑制Ins（1，4，5）P（3）介导的Ca（2+）信号传导的潜力。
p130 was originally identified as an Ins(1,4,5)P(3)-binding protein similar to phospholipase C-delta but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P(3)-mediated Ca(2+) signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P(3) with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P(3). Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca(2+) signalling. When fura-2-loaded COS-1(p130) cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca(2+) concentration, observed in control cells, was inhibited in COS-1(p130). This inhibition was not accompanied by the decreased production of Ins(1,4,5)P(3); the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P(3) could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P(3)-mediated Ca(2+) signalling.
DOI：
10.1073/pnas.92.23.10472
发表时间：
1995-11-07
影响因子：
11.1
作者：
LEMMON, MA;FERGUSON, KM;SCHLESSINGER, J
通讯作者：
SCHLESSINGER, J