PTB domain of insulin receptor substrate-1 binds inositol compounds.
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 beta1 and beta2 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.
Takeuchi
H
H
Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812-8582, Japan.
Matsuda
M
M
Yamamoto
T
T
Kanematsu
T
T
Kikkawa
U
U
Yagisawa
H
H
Watanabe
Y
Y
Hirata
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains
C526292:IRS1 protein, human; D007295:Inositol Phosphates; D055504:Insulin Receptor Substrate Proteins; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate