Arf-GAP with dual PH domain-containing protein 2
inositol 1,3,4,5 tetrakisphosphate binding
Identification of gene structure and subcellular localization of human centaurin alpha 2, and p42IP4, a family of two highly homologous, Ins 1,3,4,5-P4-/PtdIns 3,4,5-P3-binding, adapter proteins.
Proteins which recognize the two messengers phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3), a membrane lipid, and inositol 1,3,4,5-tetrakisphosphate (InsP4), a water-soluble ligand, play important roles by integrating external stimuli, which lead to differentiation, cell death or survival. p42IP4, a PtdInsP3/InsP4-binding protein, is predominantly expressed in brain. The recently described centaurin alpha2 of similar molecular mass which is 58% identical and 75% homologous to the human p42IP4 orthologue, is expressed rather ubiquitously in many tissues. Here, elucidating the gene structure for both proteins, we found the human gene for centaurin alpha2 located on chromosome 17, position 17q11.2, near to the NF1 locus, and human p42IP4 on chromosome 7, position 7p22.3. The two isoforms, which both have 11 exons and conserved exon/intron transitions, seem to result from gene duplication. Furthermore, we studied binding of the two second messengers, PtdInsP3 and InsP4, and subcellular localization of the two proteins. Using recombinant baculovirus we expressed centaurin alpha2 and p42IP4 in Sf9 cells and purified the proteins to homogeneity. Recombinant centaurin alpha2 bound both InsP4 and PtdInsP3 equally well in vitro. Furthermore, fusion proteins of centaurin alpha2 and p42IP4, respectively, with the green fluorescent protein (GFP) were expressed in HEK 293 cells to visualize subcellular distribution. In contrast to p42IP4, which was distributed throughout the cell, centaurin alpha2 was concentrated at the plasma membrane already in unstimulated cells. The protein centaurin alpha2 was released from the membrane upon addition of wortmannin, which inhibits PI3-kinase. p42IP4, however, translocated to plasma membrane upon growth factor stimulation. Thus, in spite of the high homology between centaurin alpha2 and p42IP4 and comparable affinities for InsP4 and PtdInsP3, both proteins showed clear differences in subcellular distribution. We suggest a model, which is based on the difference in phosphoinositide binding stoichiometry of the two proteins, to account for the difference in subcellular localization.