Recombinant p42^IP4, 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

Abstract

We have recently cloned the cDNA of p42 IP4 , a membrane-associated and cytosolic inositol (1,3,4,5)tetrakisphosphate receptor protein [Stricker, R., Hu Èlser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich, F. & Reiser, G. (1997) FEBS Lett. 405, 229±236.] p42 IP4 is a protein of 374 amino acids 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 further the biochemical and functional properties of p42 IP4 , 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 p42 IP4 , purified from pig cerebellum or rat brain (K i for inositol(1,3,4,5)P 4 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 p42 IP4 binds phosphatidylinositol(3,4,5)P 3 specifically, as the recombinant protein can associate with lipid membranes (vesicles) containing phosphatidylinositol(3,4,5)P 3 ; this binding occurs in a concentration-dependent manner 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 might be redistributed between cellular compartments upon hormonal stimulation.Keywords: baculovirus; inositolP 4 ; membrane interaction; pleckstrin homology domain; phosphatidylinositolP 3 ; receptor; vesicle association.Two second messengers, inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol, are produced from phosphatidylinositol 4,5-bisphosphate [Ptd-Ins(4,5)P 2 ]. The generation of Ptd-Ins(4,5)P 2 and its cleavage by phospholipase C (PLC) are tightly regulated signal transduction pathways [1]. Ins(1,4,5)P 3 mobilizes Ca 2+ from intracellular stores whereas diacylglycerol activates protein kinase C [2]. During the last few years, the putative second messengers Ins(1,3,4,5)P 4 and Ptd-Ins(3,4,5)P 3 , which are generated by phosphorylating the D3 position of the inositol ring, have attracted increasing interest. The functions of Ins(1,3,4,5)P 4 and Ptd-Ins(3,4,5)P 3 are much less clear than that of Ins(1,4,5)P 3 [2]. Ptd-Ins(3,4,5)P 3 is the product of class I phosphoinositide kinases. Phosphoinositide-3-kinase (PI3-kinase) a is regulated by membrane-bound receptor tyrosine kinases [3] whereas PI3-kinase g is activated by heterotrimeric G-proteins [4]. Ins(1,4,5)P 3 can be phosphorylated to Ins(1,3,4,5)P 4 by Ins(1,4,5)P 3 -3-kinase [5].The physiological role of Ins(1,3,4,5)P 4 is still widely disputed [6]. Ptd-Ins(3,4,5)P 3 has been demonstrated to be an important regulator of me...

Tubulin potentiates the interaction of the metalloendopeptidase nardilysin with the neuronal scaffold protein p42IP4/centaurin-α1 (ADAP1)

“…Western blot and Far-Western blotting For Western blots, we used 500 ng recombinant p42 IP4 , purified as we described previously (Hanck et al 1999) and 500 ng bovine serum albumin (Sigma) as control. The protein was adjusted with a loading buffer to a final volume of 20 μl, run on a 10% SDS gel and blotted onto a nitrocellulose membrane (Hybond-C Extra, GE-Healthcare, Freiburg, Germany).…”

“…D-Ins (1,3,4,5)P 4 is the effective ligand for p42 IP4 (Hanck et al 1999). When incubated with p42 IP4 and NRD, D-Ins(1,3,4,5)P 4 induces the dissociation of both proteins.…”

RanBPM, a novel interaction partner of the brain‐specific protein p42IP4/centaurin α‐1

“…The cells were approximately 60-70% confluent at time of transfection. The culture of Sf9 cells (LICR, London, UK), as adapted by Dr T. Hanck, is published elsewhere (Hanck et al 1999).…”

Useful access to enantiomerically pure protected inositols from carbohydrates: the aldohexos-5-uloses route

“…These studies require various regio- and stereoisomers of inositol phosphates [6–7] and have prompted the development of practical and efficient synthetic methods for stereoselectively accessing all natural and unnatural inositols as well as their derivatives.…”