Identification of rat brain p42~(IP4), a high-affinity inositol(1, 3, 4, 5)tetrakisphosphate/phosphatidylinositol(3, 4, 5)trisphosphate binding protein

摘要

Inositol(1, 3, 4, 5)tetrakisphosphate (InsP_4) and phosphatidylinositol(3, 4, 5)trisphosphate (PtdInsP_3) are two potential second messengers with a still largely unknown mode of action. We recently cloned the 42 kDa protein p42~(IP4) previously purified from pig cerebellum, which binds InsP_4 (K_d ~ 2 nM) and PtdInsP_3 with comparable affinities (Stricker et al., FEBS Lett. 405 (1997) 229). The protein p42~(IP4) (pig) is highly homologous to centaurin-α, a larger protein of 46 kDa, derived from a rat brain cDNA library clone (Hammonds-Odie et al., J. Biol. Chem. 271 (1996) 18859). Here we investigated whether also p42~(IP4)is expressed in rat brain and how it might be related to centaurin-α. When we carried out RT-PCR using mRNA from brain of rats of different ages we obtained several clones corresponding to p42~(IP4), but not to centaurin-α. The existence of p42~(IP4) in rat brain is supported by the following findings: (1) biochemical analysis of the purified rat brain protein shows inositol phosphate ligand affinities identical to those of the protein from other species; (2) Western blot analysis of rat brain membrane fractions using a peptide-specific antiserum revealed only the 42 kDa protein (p42~(IP4), but did not give evidence for the occurrence of a larger 46 kDa centaurin-α-like protein in rat brain; and (3) the amino acid sequences deduced from p42~(IP4) cDNA are highly homologous in several species and are confirmed by protein fragment microsequences. Thus, p42~(IP4) from rat brain which has two pleckstrin homology domains is a protein largely conserved between different species and most likely has an important function in inositol phosphate or inositol lipid signal transduction.