title: 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.
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-delta 1 [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.
authors.affiliation: Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Takeuchi
authors.forename: T
authors.initials: T
authors.lastname: Kanematsu
authors.forename: Y
authors.initials: Y
authors.lastname: Misumi
authors.forename: H B
authors.initials: HB
authors.lastname: Yaakob
authors.forename: H
authors.initials: H
authors.lastname: Yagisawa
authors.forename: Y
authors.initials: Y
authors.lastname: Ikehara
authors.forename: Y
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Z
authors.initials: Z
authors.lastname: Tan
authors.forename: S B
authors.initials: SB
authors.lastname: Shears
authors.forename: M
authors.initials: M
authors.lastname: Hirata
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D001798:Blood Proteins; D007295:Inositol Phosphates; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; C039077:platelet protein P47; C081651:inositol-1,4,5,6-tetrakisphosphate; D015544:Inositol 1,4,5-Trisphosphate