Recombinant p42(IP4), a brain-specific 42-kDa high-affinity Ins(1,3,4,5)P-4 receptor protein, specifically interacts with lipid membranes containing Ptd-Ins(3,4,5)P-3

We have recently cloned the cDNA of p42(IP4), a membrane-associated and cyt osolic inositol (1,3,4,5)tetrakisphosphate receptor protein [Stricker, R., Hulser, E., Fischer, J., Jarchau, T., Waiter, U., Lottspeich, F. & Reiser, G. (1997) FEES Lett. 405, 229-236.] p42(IP4) is a protein of 374 amino acid s with M-r of 42 kDa. The p42(IP4) protein has a zinc finger motif at its N -terminus, followed by two pleckstrin homology domains. To characterize fur ther the biochemical and functional properties of p42(IP4), it was expresse d as a glutathione-S-transferase fusion protein in Sf9 cells using a recomb inant baculovirus vector. The protein was affinity adsorbed on glutathione beads, cleaved from glutathione-S-transferase with the protease factor-Xa a nd purified on heparin agarose. The recombinant purified protein is active because it shows binding affinities similar to those of the native p42(IP4) , purified from pig cerebellum or rat brain (K-i for inositol(1,3,4,5)P-4 o f 4.1 nM and 2.2 nM, respectively). Moreover the ligand specificity of the recombinant protein for various inositol polyphosphates is similar to that of the native protein purified from brain. Importantly, we show here that p 42(IP4) binds phosphatidylinositol(3,4,5)P-3 specifically, as the recombina nt protein can associate with lipid membranes (vesicles) containing phospha tidylinositol(3,4,5)P-3; this binding occurs in a concentration-dependent m anner and is blocked by inositol(1,3,4,5)P-4. This specific association and the possibility that endogenous p42(IP4) can be converted from a membrane- associated state to a soluble state support the hypothesis that p42(IP4) mi ght be redistributed between cellular compartments upon hormonal stimulatio n.