TY - JOUR
T1 - 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
AU - Stricker, Rolf
AU - Vandekerckhove, Joël
AU - Krishna, Murali U.
AU - Falck, J R
AU - Hanck, Theo
AU - Reiser, Georg
N1 - Funding Information:
This work was supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 426, and Fonds der Chemischen Industrie (G.R.), NIH grant (number GM31278 to J.R.F.). The technical help of K. Christoph is gratefully acknowledged. Furthermore, we thank Prof. G. Vogel, University Wuppertal, Germany, for generously providing some inositol phosphate analogs; Dr. W. Kolanus, University Bonn, Germany, for providing the antibody MAb 2D7; and Dr. N. Brose, Göttingen, Germany for the rat cytohesin vectors.
PY - 2003/9/23
Y1 - 2003/9/23
N2 - 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.
AB - 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.
KW - Affinity chromatography
KW - Brutons tyrosine kinase
KW - Centaurin
KW - Inositolphosphate
KW - Pleckstrin homology domain
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U2 - 10.1016/S1570-9639(03)00241-3
DO - 10.1016/S1570-9639(03)00241-3
M3 - Article
C2 - 14499594
AN - SCOPUS:1242341393
SN - 1570-9639
VL - 1651
SP - 102
EP - 115
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 1-2
ER -