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: ジャーナルへの寄稿記事

60 引用 (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.

元の言語英語
ページ(範囲)561-568
ページ数8
ジャーナルBiochemical Journal
318
発行部数2
DOI
出版物ステータス出版済み - 9 1 1996

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Inositol 1,4,5-Trisphosphate
Phosphates
Proteins
Ligands
Polyphosphates
Molecules
Gene encoding
Inositol Phosphates
COS Cells
Radioactivity
Type C Phospholipases
inositol-1,4,5,6-tetrakisphosphate
platelet protein P47
Pleckstrin Homology Domains
Derivatives
Genes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

これを引用

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

:: Biochemical Journal, 巻 318, 番号 2, 01.09.1996, p. 561-568.

研究成果: ジャーナルへの寄稿記事

Takeuchi, Hiroshi ; Kanematsu, Takashi ; Misumi, Yoshio ; Yaakob, Hassan Bin ; Yagisawa, Hitoshi ; Ikehara, Yukio ; Watanabe, Yutaka ; Tan, Zheng ; Shears, Stephen B. ; Hirata, Masato. / 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. :: Biochemical Journal. 1996 ; 巻 318, 番号 2. pp. 561-568.
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abstract = "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.",
author = "Hiroshi Takeuchi and Takashi Kanematsu and Yoshio Misumi and Yaakob, {Hassan Bin} and Hitoshi Yagisawa and Yukio Ikehara and Yutaka Watanabe and Zheng Tan and Shears, {Stephen B.} and Masato Hirata",
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T1 - 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

T2 - Characterization of the determinants of structural specificity

AU - Takeuchi, Hiroshi

AU - Kanematsu, Takashi

AU - Misumi, Yoshio

AU - Yaakob, Hassan Bin

AU - Yagisawa, Hitoshi

AU - Ikehara, Yukio

AU - Watanabe, Yutaka

AU - Tan, Zheng

AU - Shears, Stephen B.

AU - Hirata, Masato

PY - 1996/9/1

Y1 - 1996/9/1

N2 - 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.

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