Localization of a high-affinity inositol 1,4,5-trisphosphate/inositol 1,4,5,6-tetrakisphosphate binding domain to the pleckstrin homology module of a new 130 kDa protein: Characterization of the determinants of structural specificity
Localization of a high-affinity inositol 1,4,5-trisphosphate/inositol 1,4,5,6-tetrakisphosphate binding domain to the pleckstrin homology module of a new 130 kDa protein: Characterization of the determinants of structural specificity
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DOI：
10.1042/bj3180561
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发表时间：
1996-09-01
影响因子：
4.1
通讯作者：
Hirata, M
中科院分区：
文献类型：
--
作者：
Takeuchi, H;Kanematsu, T;Hirata, M
我们以前已经鉴定出一种新的130 kDa蛋白(P130)，它与Ins(1，4，5)P-3结合，与磷脂酶C-Delta(1)有38%的序列同源性[Kanematsu，Misumi，Watanabe，Ozaki，Koga，Iwanaga，Ikehara和Hirata(1996)Biochem。J.313,319-325]。为了定位INS(1，4，5)P-3的结合区，我们现在已经将编码分子全长的基因或几个截短突变体之一的基因导入COS-1细胞。N-末端残基116-232的缺失完全取消了结合活性，该区域对应于分子的Pleckstrin同源(PH)结构域。当从细菌表达系统中分离到的PH结构域本身(残基95-232)被发现与[H-3]INS(1，4，5)P-3结合时，这一结果得到了证实。我们还发现，Ins(1，4，5，6)P-4与Ins(1，4，5)P-3取代[H-3]Ins(1，4，5)P-3的效果相同，表明这两种多聚磷酸盐与p130的结合具有相似的亲和力。用我们制备的高比放射性的[H-3]Ins(1，4，5，6)P-4进行的直接结合研究证实了这一结论。结合特异性也与各种肌醇磷酸衍生物进行了检测。与迄今为止描述的其他PH结构域的情况一样，我们发现4，5-邻位磷酸对是配基专一性的重要决定因素。然而，p130的PH结构域显示出一些新的特征。例如，3-和/或6-磷酸也可能有助于总的结合；这与其他一些PH结构域形成对比，在这些结构域中，这些磷酸基团通过施加空间约束而降低配体亲和力。其次，游离的单酯1-磷酸大大增加了结合亲和力，这是迄今为止p130的PH结构域所独有的情况。
We have previously identified a novel 130 kDa protein (p130) which binds Ins(1,4,5)P-3 and shares 38 % sequence identity with phospholipase C-delta(1) [Kanematsu, Misumi, Watanabe, Ozaki, Koga, Iwanaga, Ikehara and Hirata (1996) Biochem. J. 313, 319-325]. We have now transfected COS-1 cells with genes encoding the entire length of the molecule or one of several truncated mutants, in order to locate the region for binding of Ins(1,4,5)P-3. Deletion of N-terminal residues 1 16-232, the region which corresponds to the pleckstrin homology (PH) domain of the molecule, completely abolished binding activity. This result was confirmed when the PH domain itself (residues 95-232), isolated from a bacterial expression system, was found to bind [H-3]Ins(1,4,5)P-3. We also found that Ins(1,4,5,6)P-4 was as efficacious as Ins(1,4,5)P-3 in displacing [H-3]Ins(1,4,5)P-3, suggesting that these two polyphosphates bind to p130 with similar affinity. This conclusion was confirmed by direct binding studies using [H-3]Ins(1,4,5,6)P-4 with high specific radioactivity which we prepared ourselves. Binding specificity was also examined with a variety of inositol phosphate derivatives. As is the case with other PH domains characterized to date, we found that the 4,5-vicinal phosphate pair was an essential determinant of ligand specificity. However, the PH domain of p130 exhibited some novel features. For example, the 3- and/or 6-phosphates could also contribute to overall binding; this contrasts with some other PH domains where these phosphate groups decrease ligand affinity by imposing a steric constraint. Secondly, a free monoester 1-phosphate substantially increased binding affinity, which is a situation so far unique to the PH domain of p130.