Translocation between membranes and cytosol of p42^IP4, a specific inositol 1,3,4,5‐tetrakisphosphate/phosphatidylinositol 3,4,5‐trisphosphate‐receptor protein from brain, is induced by inositol 1,3,4,5‐tetrakisphosphate and regulated by a membrane‐associated 5‐phosphatase

Abstract

The highly conserved 42-kDa protein, p42IP4 was identified recently from porcine brain. It has also been identified similarly in bovine, rat and human brain as a protein with two pleckstrin homology domains that binds Ins(1,3,4,5)P 4 and PtdIns(3,4,5)P 3 with high affinity and selectivity. The brain-specific p42 IP4 occurs both as membrane-associated and cytosolic protein. Here, we investigate whether p42 IP4 can be translocated from membranes by ligand interaction. p42 IP4 is released from cerebellar membranes by incubation with Ins(1,3,4,5)P 4 . This dissociation is concentration-dependent (. 100 nm), occurs within a few minutes and and is ligand-specific. p42IP4 specifically associates with PtdIns(3,4,5)P 3 -containing lipid vesicles and can dissociate from these vesicles by addition of Ins(1,3,4,5)P 4 . p42 IP4 is only transiently translocated from the membranes as Ins(1,3,4,5)P 4 can be degraded by a membrane-associated 5-phosphatase to Ins(1,3,4)P 3 . Then, p42IP4 re-binds to the membranes from which it can be re-released by re-addition of Ins(1,3,4,5)P 4 . Thus, Ins(1,3,4,5)P 4 specifically induces the dissociation from membranes of a PtdIns(3,4,5)P 3 binding protein that can reversibly re-associate with the membranes. Quantitative analysis of the inositol phosphates in rat brain tissue revealed a concentration of Ins(1,3,4,5)P 4 comparable to that required for p42 IP4 translocation. Thus, in vivo p42 IP4 might interact with membranes in a ligand-controlled manner and be involved in physiological processes induced by the two second messengers Ins(1,3,4,5)P 4 and PtdIns(3,4,5)P 3 .Keywords: p42 IP4 ; pH domain; centaurin; inositol; 5-phosphatase.Phosphoinositides and inositol polyphosphates are central players in cellular development and intracellular signal transduction. A well characterized pathway in phosphoinositide signalling is the hydrolysis of PtdIns(4,5)P 2 by several distinct receptor-activated phospholipase C isoforms, yielding Ins(1,4,5)P 3 , which releases Ca 2+ from intracellular stores and diacylglycerol which activates protein kinase C [1]. The Ins(1,4,5)P 3 signal is terminated by specific enzymatic dephosphorylation of Ins(1,4,5)P 3 in position 5. Alternatively, Ins(1,4,5)P 3 is phosphorylated by a Ins(1,4,5)P 3 3-kinase to the putative second messenger Ins(1,3,4,5)P 4 . The function of Ins(1,3,4,5)P 4 (reviewed in [2]) is not yet clarified, but has been implicated in regulation of the intracellular Ca 2+ concentration ([3] and references therein). In endothelial cells, an Ins(1,3,4,5)P 4 -sensitive Ca 2+ channel has been demonstrated [4]. In hippocampal pyramidal neurons, Ins(1,3,4,5)P 4 was described to promote Ca 2+ accumulation after ischaemia which resulted in neuronal cell death [5]. We have recently demonstrated that Ins(1,3,4,5)P 4 injected into hippocampal CA1 neurons enhanced long-term potentiation by stimulating Ca 2+ entry either via activating N-type Ca 2+ channels or via enhancing Ins(1,4,5)P 3 -sensitive Ca 2+ release [6]. In order to clarify the physiological role of Ins(...

p42IP4/Centaurin α1, 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

“…Therefore, PI 3-kinase activation and subsequent generation of PtdIns(3,4,5)P 3 are responsible for membrane translocation of p42 IP4 . This is in accordance with previous results showing the in vitro binding of p42 IP4 to lipid vesicles containing PtdIns(3,4,5)P 3 (7).…”

“…Binding of p42 IP4 to Ins(1,3,4,5)P 4 has been demonstrated until now only in vitro (7,14). We and others have shown that the in vitro binding affinity of this protein to [ 3 H]Ins(1,3,4,5)P 4 , was similarly high as the PtdIns(3,4,5)P 3 binding affinity (8,14,33).…”

“…Previously we have shown that in the presence of Ins(1,3,4,5)P 4 the porcine p42 IP4 dissociates from cerebellar membranes and re-binds to the membranes after dephosphorylation of Ins(1,3,4,5)P 4 (7). These data indicate that p42 IP4 could translocate between cytosol and membranes depending on the intracellular levels of its two ligands, the membrane lipid PtdIns(3,4,5)P 3 and the cytosolic Ins(1,3,4,5)P 4 .…”

“…PH domains, which contain approximately 120 amino acids, are found in many proteins, such as protein kinase B (PKB), Bruton's tyrosine kinase (Btk), phosphoinositide-dependent kinase-1 (PDK1) and phospholipase C (PLC)-c1 [reviewed in (3,4)]. PH domains occur in proteins of the cytohesin family [cytohesin-1, Arf nucleotide-binding-site opener (ARNO; cytohesin-2), general receptor for phosphoinositides-1: GRP1 (cytohesin-3)] [reviewed in (5)], in Ras GTPase activating proteins (GAP1 m and GAP1 IP4BP ) (6) and in members of the centaurin family (p42 IP4 /centaurin a1; PIP3BP, and centaurin a2) (7)(8)(9)(10). For these proteins in vitro binding to phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P 3 ) and inositol (1,3,4,5) tetrakisphosphate (Ins(1,3,4,5)P 4 ) has been reported [reviewed in (5,11)].…”

“…In vitro studies on p42 IP4 from pig cerebellum have shown that p42 IP4 dissociates from cerebellar membranes by incubation with Ins(1,3,4,5)P 4 . Interestingly, we could demonstrate that in the presence of membrane-associated 5-phosphatase, which dephosphorylates Ins(1,3,4,5)P 4 to inositol (1,3,4)trisphosphate (Ins(1,3,4)P 3 ), p42 IP4 re-binds to the membranes (7). Therefore, it was of particular interest to investigate the cellular trafficking of p42 IP4 /centaurin a1 in living cells with hormonestimulated PI-3 kinase and PLC activation.…”