We examined whether a phosphotyrosine binding (PTB) domain from the human insulin receptor substrate-1 (hIRS-1) is capable of binding inositol phosphates/phosphoinositides. The binding specificity was compared with that of the pleckstrin homology (PH) domain derived from the same protein because the three dimensional structure was found to be very similar to that of the PH domain, despite the lack of sequence similarity. We also attempted to locate the site of binding of the inositol compounds. The PTB domain bound [3H]Ins(1,4,5)P3, which was displaced most strongly by Ins(1,3,4,5,6)P5 and InsP6, indicating that these inositol polyphosphates show the highest affinity. The PTB domain bound to liposomes containing PtdIns(4,5)P2, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, but not phosphatidylinositol. In contrast, the PH domain showed a preference for Ins(1,4,5)P3, the polar head of PtdIns(4,5)P2. Site-directed mutagenesis studies were performed to map the binding site for inositol phosphates in the PTB domain. Mutation of K169Q, K171Q or K177Q, located in the loop connecting the β1 and β2 strands, which is partially responsible for binding inositol phosphates/phosphoinositides in the PH domains of several other proteins, reduced binding activity, probably because of a reduction in affinity. Mutation of R212Q or R227Q, shown to be involved in the binding of a phosphotyrosine, had little effect on the binding capacity. These results indicate that the PTB domain of hIRS-1 can bind inositol phosphates/phosphoinositides. Therefore signalling through the PTB domain could be regulated by the binding not only of proteins with phosphotyrosine but also of inositol phosphates/phosphoinositides, implying that PTB domains could be involved in a myriad of interconnections between intracellular signalling pathways.
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August 1998
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Research Article|
August 15 1998
PTB domain of insulin receptor substrate-1 binds inositol compounds
Hiroshi TAKEUCHI;
Hiroshi TAKEUCHI
*Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-8582, Japan
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Miho MATSUDA;
Miho MATSUDA
*Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-8582, Japan
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Tada-aki YAMAMOTO;
Tada-aki YAMAMOTO
*Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-8582, Japan
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Takashi KANEMATSU;
Takashi KANEMATSU
*Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-8582, Japan
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Ushio KIKKAWA;
Ushio KIKKAWA
†Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
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Hitoshi YAGISAWA;
Hitoshi YAGISAWA
‡Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo 678-1201, Japan
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Yutaka WATANABE;
Yutaka WATANABE
§Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan
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Masato HIRATA
Masato HIRATA
1
*Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-8582, Japan
1To whom correspondence should be addressed (e-mail hirata1@dent.kyushu-u.ac.jp).
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Publisher: Portland Press Ltd
Received:
April 06 1998
Revision Received:
June 01 1998
Accepted:
June 19 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 334 (1): 211–218.
Article history
Received:
April 06 1998
Revision Received:
June 01 1998
Accepted:
June 19 1998
Citation
Hiroshi TAKEUCHI, Miho MATSUDA, Tada-aki YAMAMOTO, Takashi KANEMATSU, Ushio KIKKAWA, Hitoshi YAGISAWA, Yutaka WATANABE, Masato HIRATA; PTB domain of insulin receptor substrate-1 binds inositol compounds. Biochem J 15 August 1998; 334 (1): 211–218. doi: https://doi.org/10.1042/bj3340211
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