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

Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hassan Bin Yaakob, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B. Shears, Masato Hirata

研究成果: Contribution to journalArticle査読

60 被引用数 (Scopus)

抄録

We have previously identified a novel 130 kDa protein (p130) which binds Ins(1,4,5)P3 and shares 38% sequence identity with phospholipase C-δ1 [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)P3. Deletion of N-terminal residues 116-232, 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 [3H]Ins(1,4,5)P3. We also found that Ins(1,4,5,6)P4 was as efficacious as Ins(1,4,5)P3 in displacing [3H]Ins(1,4,5)P3, suggesting that these two polyphosphates bind to p130 with similar affinity. This conclusion was confirmed by direct binding studies using [3H]Ins(1,4,5,6)P4 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.

本文言語英語
ページ(範囲)561-568
ページ数8
ジャーナルBiochemical Journal
318
2
DOI
出版ステータス出版済み - 9 1 1996

All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子生物学
  • 細胞生物学

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