SEARCH · PubMed Health

Results for “PHOSPHOINOSITIDES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources

PubMed1994

Inhibition of dopamine agonist-induced phosphoinositide hydrolysis by concomitant stimulation of cyclic AMP formation in brain slices.

We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp-cyclic adenosine monophosphothioate (Sp-cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D1 receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp-cyclic adenosine monophosphothioate (Rp-cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 microM Rp-cAMPS, the dose-response curves of the dopamine D1 receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC50 values, however, were not significantly altered by protein kinase A inhibition. Neither Sp-cAMPS nor Rp-cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D1 receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1995

Astroglial phosphoinositide hydrolysis during combined glucose-oxygen deprivation: role of the metabotropic glutamate receptor.

Phosphatidylinositol bisphosphate hydrolysis, leading to the production of myo-inositol trisphosphate and diacylglycerol, may play a significant role in the pathogenesis of hypoxic-ischemic brain injury. We used tritiated myo-inositol phosphate (3H-IP) accumulation as a means to quantitate phosphoinositide hydrolysis in pre-labeled astroglial cultures subjected to combined glucose-oxygen deprivation. Astroglial cultures exposed to combined glucose-oxygen deprivation had significantly greater 3H-IP accumulation compared with cultures exposed to control conditions. To delineate the role of the metabotropic glutamate receptor in astroglial phosphoinositide hydrolysis during combined glucose-oxygen deprivation, we studied the effects of two metabotropic glutamate receptor antagonists, 2-amino-3-phosphonopropionic acid and (+)-methyl-4-carboxyphenylglycine. 2-Amino-3-phosphonopropionic acid attenuated the accumulation of 3H-IP during combined glucose-oxygen deprivation but acted as an agonist under control conditions. (+)-Methyl-4-carboxyphenylglycine had no effect on 3H-IP accumulation during combined glucose-oxygen deprivation or under control conditions. These results suggest that activation of astroglial phosphoinositide hydrolysis during combined glucose-oxygen deprivation may be mediated, at least in part, by the metabotropic glutamate receptor.

Alanine↗

PubMed1996

(1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced increases in cyclic AMP formation in the neonatal rat hippocampus are mediated by a synergistic interaction between phosphoinositide- and inhibitory cyclic AMP-coupled mGluRs.

A pharmacological approach was used to investigate the cellular mechanism and metabotropic glutamate receptor (mGluR) subtypes that mediate stimulation of basal cyclic AMP (cAMP) formation in slices of the neonatal rat hippocampus. (1S,3 R)-1-Aminocyclopentane-1, 3-dicarboxylic acid (1S,3R-ACPD), which is an agonist for phosphoinositide-coupled and inhibitory-coupled cAMP-linked mGluRs in cloned and in situ preparations, produced prominent stimulations of basal cAMP levels (five- to 10-fold). However, the agonists 3,5-dihydroxyphenylglycine (DHPG) and (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), which selectively act on phosphoinositide-coupled and inhibitory cAMP-coupled mGluRs, respectively, only weakly increased cAMP levels. When these two mGluR subtype-selective agonists were added in combination, robust increases in cAMP levels, similar to those observed for 1S,3R-ACPD, were found. Stimulations of cAMP content evoked by 1S,3R-ACPD and combined additions of DHPG plus 2R,4R-APDC occurred at concentrations of these agents that directly couple to other mGluR second messenger responses. However, these stimulatory cAMP responses were prevented by the presence of adenosine deaminase and 8-p-sulfophenyltheophylline (an adenosine receptor antagonist), as well as (+)-alpha-methyl-4-carboxyphenylglycine (an mGluR receptor antagonist). Thus, 1S,3R-ACPD-induced increases in cAMP formation in the neonatal rat hippocampus are mediated by a synergistic interaction between mGluRs coupled to phosphoinositide (group 1) and inhibitory cAMP (group 2), which are indirectly expressed by potentiation of cAMP responses to other agonists (in this case, endogenous adenosine).

Animals↗

PubMedDate not supplied

The germinal vesicle of the mouse oocyte contains elements of the phosphoinositide cycle: what is their role at meiosis resumption?

The role of the nuclear phosphoinositide (PI) cycle during meiotic resumption in mouse oocytes was examined. First, using indirect immunofluorescence staining with specific monoclonal antibodies (mAbs) against elements of this cycle, the presence of inositol trisphosphate receptors (IP3Rs) (IP3R-1 or IP3R-3) or phosphoinositide-phospholipase (PLC) isoforms (PLC beta 1 or PLC gamma 1) was monitored in the germinal vesicle (GV). Using confocal laser scanning microscopy, we analysed the effects of the nuclear microinjection of these antibodies on both spontaneous nuclear calcium oscillations and meiosis resumption. Immunostainings showed that IP3R-1 and PLC beta 1 isoforms were both present in the GV, whereas IP3R-3 and PLC gamma 1 isoforms were not. The anti-IP3R-1 mAbs or the anti-PLC beta 1 mAbs microinjected into the GV, induced inhibition of both the nuclear Ca2+ oscillations and the meiotic process, whereas the anti-IP3R-3 mAbs and the anti-PLC gamma 1 mAbs did not. We concluded that a specific nuclear PI cycle is present in the mouse oocyte and meiosis resumption requires a specific nuclear phosphoinositide-dependent Ca2+ signal.

Animals↗

PubMed2002

The phosphatidylinositol signalling system in elongating bovine blastocysts; formation of phosphoinositides, inositol phosphates and stimulation by growth factors.

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo elongating cattle blastocysts was investigated using [3H]myo-inositol. Uptake was examined in 13-, 14- and 16-day-old blastocysts and was largely sodium-dependent throughout (P<0.001), indicating the presence of a sodium-dependent inositol transporter. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP and PtdInsP2, and the inositol phosphates of the phosphatidylinositol signal transduction system was examined at Days 14 and 16; incorporation into the three phosphoinositides and into the inositol phosphate species, InsP1, InsP2, InsP3 (including the second messenger, Ins(1,4,5)P3) and InsP4 was detected in both blastocyst stages. The effects of the peptide growth factor, epidermal growth factor (EGF), and the lipid growth factors, lysophosphatidic acid (LPA) and platelet activating factor (PAF), on the activity of the phosphatidylinositol signalling system in 14- and 16-day-old blastocysts were examined. All growth factors significantly stimulated phosphatidylinositol signalling activity. Epidermal growth factor was stimulatory (P<0.001) only in 16-day-old blastocysts, whereas LPA and PAF were active in both 14- (P<0.005 for LPA and P<0.001 for PAF) and 16-day-old blastocysts (P<0.001 for LPA and PAF). These results indicate that the phosphatidylinositol signalling system is present in cattle blastocysts at the elongation stage and is responsive to stimulation by growth factors.

Animals↗

PubMed1997

Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments.

Phototransduction in retinal rods involves a G protein-coupled signaling cascade that leads to cGMP hydrolysis and the closure of cGMP-gated cation channels that are open in darkness, producing a membrane hyperpolarization as the light response. For many years there have also been reports of the presence of a phosphoinositide pathway in the rod outer segment, though its functions and the molecular identities of its components are still unclear. Using immunocytochemistry with antibodies against various phosphoinositide-specific phospholipase C (PLC) isozymes (beta1-4, gamma1-2, and delta1-2), we have found PLCbeta4-like immunoreactivity in rod outer segments. Similar experiments with antibodies against the alpha-subunits of the G(q) family of G proteins, which are known to activate PLCbeta4, have also demonstrated G(alpha11)-like immunoreactivity in this location. Immunoblots of total proteins from whole retina or partially purified rod outer segments with anti-PLCbeta4 and anti-G(alpha11) antibodies gave, respectively, a single protein band of the expected molecular mass, suggesting specific labelings. The retinal locations of the two proteins were also supported by in situ hybridization experiments on mouse retina with probes specific for the corresponding mouse genes. These two proteins, or immunologically identical isoforms, therefore likely mediate the phosphoinositide signaling pathway in the rod outer segment. At present, G(alpha11) or a G(alpha11)-like protein represents the only G protein besides transducin (which mediates phototransduction) identified so far in the rod outer segment. Although absent in the outer segment layer, other PLC isoforms as well as G(alpha q) (another G(q) family member), are present elsewhere in the retina.

Animals↗

PubMed1995

Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors. A role for pleckstrin's PH domains.

Pleckstrin is a 40-kDa protein present in platelets and leukocytes that contains two PH domains separated by a 150-residue intervening sequence. Pleckstrin is a major substrate for protein kinase C, but its function is unknown. The present studies examine the effects of pleckstrin on second messenger generation. When expressed in cos-1 or HEK-293 cells, pleckstrin inhibited 1) the G alpha-mediated activation of phospholipase C beta initiated by thrombin, M1-muscarinic acetylcholine, and angiotensin II receptors, 2) the stimulation of phospholipase C beta by constitutively active Gq alpha, 3) the G beta gamma-mediated activation of phospholipase C beta caused by alpha 2A-adrenergic receptors, and 4) the tyrosine phosphorylation-mediated activation of phospholipase C gamma caused by Trk A. However, pleckstrin had no effect on either the stimulation or inhibition of adenylyl cyclase. The inhibition of phosphoinositide hydrolysis caused by pleckstrin was similar in magnitude to that caused by activating protein kinase C with phorbol 12-myristate 13-acetate (PMA). When combined, pleckstrin and PMA had an additive effect, inhibiting phosphoinositide hydrolysis by as much as 90%. Structure-function analysis highlighted the role of pleckstrin's N-terminal PH domain in these events. Although deleting the C-terminal PH domain had no effect, deleting the N-terminal PH domain abolished activity (but not expression) and mutating a highly conserved tryptophan residue within the N-terminal PH domain decreased activity by one-third. Notably, however, a pleckstrin variant in which the N-terminal PH domain was replaced with a second copy of the C-terminal PH domain was nearly as active as native pleckstrin. These results show that: 1) pleckstrin can inhibit pathways leading to both phospholipase C beta- and phospholipase C gamma-mediated phosphoinositide hydrolysis, 2) this inhibition affects activation of phospholipase C beta mediated by either G alpha or G beta gamma, but does not affect the regulation of adenylyl cyclase activity by G alpha or G beta gamma, 3) although pleckstrin is a substrate for protein kinase C, the effects of pleckstrin and PMA are at least partially independent, 4) the inhibition caused by pleckstrin appears to be mediated by the PH domain at the N terminus, rather than the C terminus of the molecule, and 5) location of the two PH domains within the molecule clearly contributes to their individual activity.2+1

Animals↗

PubMed1997

Influence of pyruvic acid methyl ester on rat pancreatic islets. Effects on insulin secretion, phosphoinositide hydrolysis, and sensitization of the beta cell.

The methyl ester of pyruvic acid (methyl pyruvate) stimulated a dose-dependent increase in insulin secretion from isolated perifused rat islets. The threshold level for release was about 10 mM, and at 20 mM the addition of MP to perifused islets resulted in a large first phase of secretion followed by an insulin-secretory response that was sustained for at least 40 min. When compared to the effects of 20 mM glucose, peak first-phase release rates in response to 20 mM methyl pyruvate were comparable, but the second phase of release was only about 10-15% of that observed with an equimolar level of the hexose. The stimulatory effects of 20 mM methyl pyruvate on secretion were abolished by the K1+-ATP channel blocker diazoxide (200 microM) and by the calcium channel antagonist nitrendipine (500 nM). The glucokinase inhibitor mannoheptulose (20 mM) had no adverse effect on the secretory response to 20 mM methyl pyruvate, whereas 10 microM forskolin amplified the insulinotropic action of MP. Sodium pyruvate alone or in combination with 10 microM forskolin had no insulinotropic effect. In additional experiments islet phosphoinositide pools were labeled with myo-2-[3H]inositol, and the subsequent accumulation of labeled inositol phosphates was used to monitor the activation of phospholipase C. Methyl pyruvate stimulated a dose-dependent increase in inositol phosphate levels when measured after a 30-min incubation period with a maximal increase of about 300% at 20 mM methyl pyruvate. The increase in phosphoinositide hydrolysis caused by methyl pyruvate (20 mM) was, like insulin secretion, reduced by both diazoxide and nitrendipine but was immune to inhibition by mannoheptulose. Pyruvate (20 mM) had no effect on inositol phosphate accumulation. Prior short-term exposure to methyl pyruvate sensitized islets to subsequent stimulation with 15 mM glucose. Sodium pyruvate did not sensitize islets. These findings support the concept that the mitochondrial metabolism of nutrient molecules is an event sufficient to acutely augment insulin release from the beta cell, to increase phospholipase C-mediated phosphoinositide hydrolysis, and to induce time-dependent potentiation of insulin secretion.

Animals↗

PubMed1998

Synergistic activation of dynamin GTPase by Grb2 and phosphoinositides.

Hydrolysis of GTP by dynamin is essential for budding clathrin-coated vesicles from the plasma membrane. Two distinct domains of dynamin are implicated in the interactions with dynamin GTPase activators. Microtubules and Grb2 bind to the carboxyl-terminal proline/arginine-rich domain (PRD), whereas phosphoinositides bind to the pleckstrin homology (PH) domain. In this study we tested the effect of different phosphoinositides on dynamin GTPase activity and found that the best activator is phosphatidylinositol 4,5-bisphosphate followed by 1-O-(1, 2-di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-D-myo-inositol 3,4,5-triphosphate. Phosphatidylinositol 4-phosphate was a weak activator and phosphatidylinositol 3,4-bisphosphate did not activate GTPase at all. We then addressed the question of whether both domains of dynamin, PRD and PH, can be engaged simultaneously, and determined the effects of dual occupancy on dynamin GTPase activity. We found that Grb2 and phosphatidylinositol 4,5-bisphosphate together increased the dynamin GTPase activity up to 4-fold higher than that obtained by these activators tested separately, and also reduced the dynamin concentration required for half-maximal activities by 3-fold. These results indicate that both stimulators can bind to dynamin simultaneously resulting in superactivation of dynamin GTPase activity. We propose that SH3-containing proteins such as Grb2 bind to the dynamin PRD to target it to clathrin-coated pits and prime it for superactivation by phosphoinositides.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1. Evidence for the pleckstrin homology domain functioning as an allosteric site.

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin beta family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin beta4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P(2) binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P(2) binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf.

ADP-Ribosylation Factors↗

PubMed2000

Characterization of a phosphoinositide-mediated odor transduction pathway reveals plasma membrane localization of an inositol 1,4, 5-trisphosphate receptor in lobster olfactory receptor neurons.

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein alpha subunit of the G(q) family and an inositol 1,4, 5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP(3)) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP(3) receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP(3) receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP(3)R in neurons. The association of the IP(3) receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.

Amino Acid Sequence↗

PubMed2000

Functional characterization of a mammalian Sac1 and mutants exhibiting substrate-specific defects in phosphoinositide phosphatase activity.

The Saccharomyces cerevisiae SAC1 gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex. Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral membrane phosphoinositide phosphatases, the synaptojanins, and these Sac1 domains define novel phosphoinositide phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic phosphoinositide phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in Deltasac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3, 5-bisphosphate phosphatase activities, is essential for the heterologous complementation of Sac1p defects in vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.

Amino Acid Sequence↗

PubMed2001

Characterization of a G protein-activated phosphoinositide 3-kinase in vascular smooth muscle cell nuclei.

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), whereas guanosine 5'-(beta-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPgammaS-induced PtdIns(3,4,5)P(3) synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P(3) accumulation, suggesting that nuclear G(i)/G(0) proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Galpha(0) proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85alpha subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kgamma. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the control of VSMC proliferation and in the pathogenesis of vascular proliferative disorders.

Cell Nucleus↗

PubMed2001

Muscarinic receptors mediate phospholipase C-dependent activation of protein kinase B via Ca2+, ErbB3, and phosphoinositide 3-kinase in 1321N1 astrocytoma cells.

In 1321N1 astrocytoma cells, heterotrimeric G-protein-coupled receptors that activate phosphoinositide-specific phospholipase Cbeta (PLCbeta) isoforms via G(q), induced a prolonged activation of protein kinase B (PKB) after a short delay. For example, the effect of carbachol acting on M3 muscarinic receptors is blocked by wortmannin, suggesting it is mediated via a phosphoinositide 3-kinase (PI 3-kinase). In support of this, carbachol increased PI 3-kinase activity in PI 3-kinase (p85) immunoprecipitates. The pathway linking PLC-coupled receptors to PI 3-kinase was deduced to involve phosphoinositide hydrolysis and Ca2+-dependent ErbB3 transactivation but not protein kinase C on the basis of the following evidence: (i) inhibition of carbachol stimulated PLC by pretreatment with the phorbol ester phorbol 12-myristate 13-acetate concomitantly reduced PKB activity, whereas stimulation of other PLC-coupled receptors also activated PKB; (ii) Ca2+ ionophores and thapsigargin stimulated PKB activity in a wortmannin-sensitive manner, whereas bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked carbachol-stimulated PKB activity; (iii) phorbol 12-myristate 13-acetate alone did not activate PKB, whereas a protein kinase C inhibitor did not prevent the activation of PKB by carbachol; and (iv) carbachol stimulated ErbB3-tyrosine phosphorylation and association with p85, and both these and PKB activity were blocked by tyrphostin AG1478, an epidermal growth factor receptor-tyrosine kinase inhibitor. These experiments define a novel pathway linking G(q)-coupled G-protein-coupled receptors to the activation of PI 3-kinase and PKB.

Astrocytoma↗

PubMed2005

Phosphoinositide binding by the disabled-1 PTB domain is necessary for membrane localization and Reelin signal transduction.

Disabled-1 (Dab1) is an essential adaptor protein that functions in the Reelin signaling pathway and is required for the regulation of neuronal migration during embryonic development. Dab1 interacts with NPXY motifs in the cytoplasmic tails of the lipoprotein receptors ApoER2 and very low density lipoprotein receptor through an amino-terminal phosphotyrosine binding (PTB) domain. Binding of Reelin to these receptors leads to tyrosine phosphorylation of Dab1 and the initiation of a signaling cascade that results in remodeling of the cytoskeleton. Structural and biochemical studies of the Dab1 PTB domain have demonstrated that this domain binds to both the NPXY peptide motif in the lipoprotein receptor tails as well as to the head group of phosphoinositide 4,5-P2 through energetically independent mechanisms. Here we have investigated how phosphoinositide binding by the Dab1 PTB domain influences Reelin signal transduction. Our findings in cultured primary neurons that have been transduced with lentiviral constructs expressing mutant Dab1 forms reveal that phosphoinositide binding by the Dab1 PTB domain is necessary for proper membrane localization of Dab1 and for effective transduction of a Reelin signal.

Amino Acid Sequence↗

PubMed2005

Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain.

Ubiquitination serves as a key sorting signal in the lysosomal degradation of endocytosed receptors through the ability of ubiquitinated membrane proteins to be recognized and sorted by ubiquitin-binding proteins along the endocytic route. The ESCRT-II complex in yeast contains one such protein, Vps36, which harbors a ubiquitin-binding NZF domain and is required for vacuolar sorting of ubiquitinated membrane proteins. Surprisingly, the presumptive mammalian ortholog Eap45 lacks the ubiquitin-binding module of Vps36, and it is thus not clear whether mammalian ESCRT-II functions to bind ubiquitinated cargo. In this paper, we provide evidence that Eap45 contains a novel ubiquitin-binding domain, GLUE (GRAM-like ubiquitin-binding in Eap45), which binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains. The GLUE domain shares similarities in its primary and predicted secondary structures to phosphoinositide-binding GRAM and PH domains. Accordingly, we find that Eap45 binds to a subset of 3-phosphoinositides, suggesting that ubiquitin recognition could be coordinated with phosphoinositide binding. Furthermore, we show that Eap45 colocalizes with ubiquitinated proteins on late endosomes. These results are consistent with a role for Eap45 in endosomal sorting of ubiquitinated cargo.

Amino Acid Sequence↗

PubMed1999

Endothelin-1 stimulates phosphoinositide hydrolysis in the rat pineal gland.

The presence of functional endothelin receptors and their signal transduction mechanism has not been determined so far in the pineal gland. We examined the effect of endothelin-1 (ET-1) on phosphoinositide turnover in whole pineal gland. Endothelin-1 increased monophosphate accumulation in a dose-dependent manner. The phosphoinositide (PI) response elicited by ET-1 was dependent on the presence of extracellular Ca (++) since its chelation resulted in a marked decrease in ET-1-stimulated InsP(1) accumulation. On the contrary, phosphoinositide hydrolysis was not changed by the calcium blocker amlodipine. ET-1 induced PI breakdown was inhibited by neomycin, an inhibitor of phospholipase C. However, mastoparan 7, a G protein activator via Gi/Go s timulation, did not alter ET-1-induced InsP(1) accumulation. Our data indicate that stimulation of PI turnover constitutes one of the signaling pathways of ET in rat pineal gland through the stimulation of a receptor-coupled phospholipase C. And they demonstrate, for the first time, the presence of functional binding sites for endothelin in the pineal gland.

Amlodipine↗

PubMed1996

Normalization of immune response and phosphoinositide fatty acid composition of peripheral blood lymphocytes in an alcoholic patient after alcohol abstinence.

After 10 months of alcohol abstinence a malnourished alcoholic patient improved his nutritional status. The analysis of peripheral blood lymphocyte response to mitogenic stimulation with the antibody anti-CD3 and of the fatty acid composition of the (poly)-phosphoinositide fraction derived from lymphocytes revealed: 1) a similar [3H]-thymidine uptake as in control (non-drinker) subjects; 2) a similar relative molar content of the main fatty acids in the (poly)-phosphoinositides as in control subjects. Alcohol abstinence can normalize both the parameters, which are greatly altered during alcohol abuse. This suggests a link between nutritional status and lymphocyte responsiveness via phosphoinositide fatty acid composition.

Alcoholism↗