Oligomerization controls in tissue-specific manner ligand binding of native, affinity-purified p42IP4/centaurin α1 and cytohesins - Proteins with high affinity for the messengers D-inositol 1,3,4,5- tetrakisphosphate/phosphatidylinositol 3,4,5-trisphosphate

Rolf Stricker, Joël Vandekerckhove, Murali U. Krishna, J R Falck, Theo Hanck, Georg Reiser

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Several distinct receptor proteins for the second messengers Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 are already known, such as the brain-specific p42IP4, which we have previously cloned from different species, and cytohesins. However, it is still unclear whether proteins interacting with phosphoinositide and inositolpolyphosphate second messengers are regulated differently in different tissues. Here, we investigated these native proteins for comparison also from rat lung cytosol and purified them by PtdIns(3,4,5)P3 affinity chromatography. Proteins selectively binding Ins(1,3,4,5)P4 with high affinity also showed high affinity and specificity towards PtdIns(3,4,5)P3. In lung cytosol, two prominent protein bands were found in the eluate from a PtdIns(3,4,5)P3 affinity column. We identified these proteins by mass spectrometry as the cytohesin family of Arf guanosine nucleotide exchange factors (cytohesin 1, ARNO, GRP-1) and as Bruton's tyrosine kinase. Western blot analysis indicated that p42IP4 was present in lung only at very low concentrations. Applying the affinity purification scheme established for rat lung cytosol to cytosol from rat brain, however, yielded only p42 IP4. We identified cytohesins in rat brain by Western blotting and PCR, but cytohesins surprisingly did not bind to the PtdIns(3,4,5)P 3-affinity column. Gel filtration experiments of brain cytosol revealed that brain cytohesins are bound to large molecular weight complexes (150 to more than 500 kDa). Thus, we hypothesize that this finding explains why brain cytohesins apparently do not bind the inositolphosphate ligand. In lung cytosol, on the other hand, cytohesins occur as dimers. Gel filtration also showed that p42IP4 in brain cytosol occurs as a monomer. Thus, oligomerization (homomeric or heteromeric) of InsP4/PtdInsP 3 binding proteins can modulate their function in a tissue-dependent manner because it can modify their ability to interact with the ligands.

Original languageEnglish (US)
Pages (from-to)102-115
Number of pages14
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1651
Issue number1-2
DOIs
StatePublished - Sep 23 2003

Keywords

  • Affinity chromatography
  • Brutons tyrosine kinase
  • Centaurin
  • Inositolphosphate
  • Pleckstrin homology domain

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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