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

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)561-568
Number of pages8
JournalBiochemical Journal
Volume318
Issue number2
DOIs
Publication statusPublished - Sep 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

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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.

In: Biochemical Journal, Vol. 318, No. 2, 01.09.1996, p. 561-568.

Research output: Contribution to journalArticle

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. In: Biochemical Journal. 1996 ; Vol. 318, No. 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.",
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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

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