Recombinant p42IP4, a brain-specific 42-kDa high-affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd-Ins(3,4,5)P3.
We have recently cloned the cDNA of p42IP4, a membrane-associated and cytosolic inositol (1,3,4,5)tetrakisphosphate receptor protein [Stricker, R., Hülser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich, F. & Reiser, G. (1997) FEBS Lett. 405, 229-236.] p42IP4 is a protein of 374 amino acids with Mr of 42 kDa. The p42IP4 protein has a zinc finger motif at its N-terminus, followed by two pleckstrin homology domains. To characterize further the biochemical and functional properties of p42IP4, it was expressed as a glutathione-S-transferase fusion protein in Sf9 cells using a recombinant baculovirus vector. The protein was affinity adsorbed on glutathione beads, cleaved from glutathione-S-transferase with the protease factor-Xa and purified on heparin agarose. The recombinant purified protein is active because it shows binding affinities similar to those of the native p42IP4, purified from pig cerebellum or rat brain (Ki for inositol(1,3,4,5)P4 of 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 p42IP4 binds phosphatidylinositol(3,4,5)P3 specifically, as the recombinant protein can associate with lipid membranes (vesicles) containing phosphatidylinositol(3,4,5)P3; this binding occurs in a concentration-dependent manner and is blocked by inositol(1,3,4,5)P4. This specific association and the possibility that endogenous p42IP4 can be converted from a membrane-associated state to a soluble state support the hypothesis that p42IP4 might be redistributed between cellular compartments upon hormonal stimulation.
Hanck
T
T
Institut für Neurobiochemie, Otto-von-Guericke-Universität Magdeburg, Germany.
Stricker
R
R
Krishna
U M
UM
Falck
J R
JR
Chang
Y T
YT
Chung
S K
SK
Reiser
G
G
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D013552:Swine
D007295:Inositol Phosphates; D008563:Membrane Lipids; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; C061107:inositol-1,3,4,5-tetrakisphosphate receptor; C060974:phosphatidylinositol 3,4,5-triphosphate; C081655:inositol-1,3,4,5-tetrakisphosphate