Two inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding proteins, having molecular masses of 85 kDa and 130 kDa, were purified from rat brain using an Ins(1,4,5)P3 affinity column; the former protein was found to be the δ1-isozyme of phospholipase C (PLC-δ1) and the latter protein was an unidentified novel protein. Subsequent molecular biology studies isolated the full-length cDNA for the 130 kDa-Ins(1,4,5)P3 binding protein (p130) which encoded 1,096 amino acids. The predicted amino acid sequence of p 130 had 38.2% homology to that of PLC-δ1. The region of p130 responsible for InS(1,4,5)P3 binding was mapped in a pleckstrin homology (PH) domain of the molecule. The PH domain of p130 could also bind to Ins(1,4,5,6)P4 with a similar affinity to Ins(1,4,5)P3. The 85 kDa protein (PLC-δ1) was also analyzed for the binding site by molecular biological, peptide synthetic chemical and immunological studies: the sequence 30-43 of PLC-δ1 was primarily involved in binding, which was mapped in the N-terminal of the PH domain of the molecule. Experiments as to the effect of Ins(1,4,5)P3 on PLC-δ1 activity showed that Ins(1,4,5)P3 at concentrations over 1 μM strongly inhibited PLC activity of PLC-δ1. The PH domains derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and p130, were analyzed for the capability and specificity of binding of inositol phosphates and derivatives of inositol lipids. We concluded from these studies that inositol phosphates and/or inositol lipids might be common ligands for the PH domains, and therefore inositol phosphates/inositol lipids might be involved in more aspects of cellular functions than originally thought, because more than 90 proteins to date are known to include PH domain. Which ligands are physiologically relevant for the PH domain, would depend on binding affinities and their cellular abundance.
|Number of pages||24|
|Journal||ACS Symposium Series|
|Publication status||Published - Dec 1 1999|
All Science Journal Classification (ASJC) codes
- Chemical Engineering(all)