Membrane association of a new inositol 1,4,5-trisphosphate binding protein, p130 is not dependent on the pleckstrin homology domain.
The 130-kDa protein was isolated as a novel inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding protein from rat brain and was molecularly cloned to be found similar to phospholipase C-delta 1 (Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. and Hirata, M., 1992. Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol, J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. and Hirata, M., 1996. A new inositol 1,4,5-trisphosphate binding protein similar to phospholipase C-delta 1, Biochem. J. 313, 319-325). The 130-kDa protein and its deleted protein expressed in COS-1 cells were seen in both the membrane and the cytosol fractions. Truncation of 232 residues from the N-terminus, the protein molecule lacking the pleckstrin homology (PH) domain was also localized in the membrane fraction as much as seen with a full-length protein and other deleted proteins, thereby indicating that the PH domain is not primarily involved in the membrane localization. The addition of Mg2+ to homogenates of COS-1 cells caused the translocation of expressed proteins from the cytosol to the membrane fraction, yet further addition of AlF4- which induced the activation of GTP binding proteins did not cause a further translocation. The protein translocated to the membrane by the addition of Mg2+ was hardly extracted with Triton X-100. The inclusion of Ins(1,4,5)P3 or phosphatidylinositol 4,5-bisphosphate in cell homogenates caused the very small reduction in the amounts of membrane-associated proteins expressed by some constructs. These results indicate that (i) the PH domain is not primarily involved in the membrane localization of the 130-kDa protein, (ii) the activation of GTP binding protein does not appear to cause the translocation of the 130-kDa protein, and (iii) intrinsic phosphatidylinositol 4,5-bisphosphate present in the membrane appears to be involved in the membrane association of the 130-kDa protein to a very small extent, probably through the binding site in the PH domain.
Takeuchi
H
H
Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
Kanematsu
T
T
Misumi
Y
Y
Hirata
M
M
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D019204:GTP-Binding Proteins