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

Theodor Hanck, Rolf Stricker, U. Murali Krishna, John R. Falck, Young Tae Chang, Sung Kee Chung, Georg Reiser

Research output: Contribution to journalArticle

20 Scopus citations

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., Hulser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich, F. and Reiser, G. (1997) FEBS Lett. 405, 229-236.] p42(IP4) is a protein of 374 amine 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)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 p42(IP4) 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 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.

Original languageEnglish (US)
Pages (from-to)577-584
Number of pages8
JournalEuropean Journal of Biochemistry
Volume261
Issue number2
DOIs
StatePublished - Apr 15 1999

Keywords

  • Baculovirus
  • InositolP
  • Membrane interaction
  • PhosphatidylinosilolP
  • Pleckstrin hemology domain
  • Receptor
  • Vesicle association

ASJC Scopus subject areas

  • Biochemistry

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