p42(IP4)/centaurin alpha1, a brain-specific PtdIns(3,4,5)P3/Ins(1,3,4,5)P4-binding protein: membrane trafficking induced by epidermal growth factor is inhibited by stimulation of phospholipase C-coupled thrombin receptor.
The brain-specific 42-kDa protein, p42(IP4), contains a N-terminal zinc finger (ZF) motif and a tandem of two pleckstrin homology (PH) domains. p42(IP4) binds in vitro the second messengers phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P3) and inositol(1,3,4,5)tetrakisphosphate (Ins(1,3,4,5)P4). We observed by confocal microscopy in live HEK 293 cells the GFP-p42(IP4), a chimera of human p42(IP4) and green fluorescence protein (GFP). There, we studied the influence of thrombin, which raises Ins(1,3,4,5)P4, on membrane translocation of GFP-p42(IP4), induced by epidermal growth factor (EGF). Thrombin in the presence of LiCl inhibited the EGF-induced membrane recruitment of GFP-p42(IP4). In the absence of LiCl, thrombin weakened the EGF-mediated membrane recruitment of GFP-p42(IP4). Furthermore, the participation of p42(IP4) protein domains on the EGF-mediated membrane translocation was analyzed. We used several p42(IP4) variants, in which one of the domains was deleted. Alternatively, single p42(IP4) domain-GFP fusion proteins were generated. Only the p42(IP4) variant lacking the ZF domain showed a very weak membrane translocation in response to EGF stimulation, but all the other p42(IP4) variants did not translocate. Thus, we conclude that the combination of both PH domains with ZF is required for membrane translocation of p42(IP4).
Sedehizade
Fariba
F
Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität Magdeburg, Magdeburg, Germany.
von Klot
Christoph
C
Hanck
Theodor
T
Reiser
Georg
G
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D018179:Receptors, Thrombin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*; D016335:Zinc Fingers
C100956:ADAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D007295:Inositol Phosphates; D009419:Nerve Tissue Proteins; D018129:Phosphatidylinositol Phosphates; D018179:Receptors, Thrombin; D011993:Recombinant Fusion Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C081655:inositol-1,3,4,5-tetrakisphosphate; D004815:Epidermal Growth Factor; D010738:Type C Phospholipases; D013917:Thrombin