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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September 1996
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Research Article|
September 01 1996
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;
Hiroshi TAKEUCHI
**
*Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-82, Japan
†Department of Maxillofacial Surgery, Faculty of Dentistry, Kyushu University, Fukuoka 812-82, Japan
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Takashi KANEMATSU;
Takashi KANEMATSU
**
*Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-82, Japan
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Yoshio MISUMI;
Yoshio MISUMI
‡Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka 814-80, Japan
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Hassan Bin YAAKOB;
Hassan Bin YAAKOB
††
*Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-82, Japan
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Hitoshi YAGISAWA;
Hitoshi YAGISAWA
§Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo 678-12, Japan
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Yukio IKEHARA;
Yukio IKEHARA
‡Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka 814-80, Japan
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Yutaka WATANABE;
Yutaka WATANABE
‖Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790, Japan
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Zheng TAN;
Zheng TAN
¶Inositol Lipid Section, Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, U.S.A.
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Stephen B. SHEARS;
Stephen B. SHEARS
¶Inositol Lipid Section, Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, U.S.A.
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Masato HIRATA
Masato HIRATA
‡‡
*Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-82, Japan
‡‡To whom all correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
March 14 1996
Revision Received:
April 26 1996
Accepted:
May 07 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 318 (2): 561–568.
Article history
Received:
March 14 1996
Revision Received:
April 26 1996
Accepted:
May 07 1996
Citation
Hiroshi TAKEUCHI, Takashi KANEMATSU, Yoshio MISUMI, Hassan Bin YAAKOB, Hitoshi YAGISAWA, Yukio IKEHARA, Yutaka WATANABE, Zheng TAN, Stephen B. SHEARS, Masato HIRATA; 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. Biochem J 1 September 1996; 318 (2): 561–568. doi: https://doi.org/10.1042/bj3180561
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