| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥2,200,000 (Direct Cost: ¥2,200,000)
|
|---|
| Research Abstract |
The 130-kDa protein was isolated as a novel inositol 1,4,5-trisphosphate (D-Ins (1,4,5) P_3) binding protein from rat brain and was recently found to be similar to phospholipase C-delta_1. Here we expressed proteins in COS-1 cells by transfection with genes encoding an entire-length of the molecule and its deletion mutants to locate the binding domain for D-Ins (1,4,5) P_3. The cytosol fraction of COS-1 cells transfected with a gene encoding a full-length of the molecule exhibited an D- [^3H] Ins (1,4,5) P_3 binding activity. Truncation of 232 residues from the N-terminus completely abolished the binding activity of D- [^3H] Ins (1,4,5) P_3, while truncation of N-terminal 115 residues kept the binding activity with a similar affinity as a full-length molecule. Deletion of several hundred residues from the C-terminus did not affect the binding. These data indicate that the binding domain of D-Ins (1,4,5) P_3 resides in the region of residues from 116 to 232, which corresponds to the pleckstrin homology omain (PH domain) of the molecule. The PH domain (residues 95-232) isolated from a bacterial expression system was capable to bind-D [^3H] Ins (1,4,5) P_3. Binding specificity was examined with a construct deleted at C-terminals and isolated PH domain, using inositol phosphate positional isomers and D-Ins (1,4,5) P_3 analogues to explore the structural requirement of D-Ins (1,4,5) P_3 for recognition by the PH domain of the 130-kDa protein. The 4,5-bisphosphates on myo-inositol appeared to be required for the recognition and an additional 1-phosphate increased the affinity. Hydroxyl group at 3-position seemed to provide hydrogen bonding interaction.
|
