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PubMed1991

Phosphoinositide metabolism in adipocytes from hypothyroid rats.

The effect of hypothyroidism on insulin- and epinephrine-stimulated phosphoinositide metabolism was investigated in rat adipocytes. Insulin-mediated phosphoinositide synthesis was enhanced by hypothyroidism (14.5 +/- 1.5% above basal level, control vs. 22.5 +/- 2.0% above basel level, hypothyroid, P less than 0.05). However, insulin did not stimulate hydrolysis of phosphoinositides to inositol phosphates, neither in control nor in hypothyroid rats. The alpha 1-adrenoceptor agonist (e.g. epinephrine) significantly stimulated the incorporation of myo-[3H]inositol into phosphoinositides (P less than 0.01) and hydrolysis of phosphoinositides (P less than 0.01), but this stimulatory action was unaffected by the hypothyroid state. The results suggest that hypothyroidism has differentiated effects on the hormone-regulated phosphoinositide metabolism and that the presumptive G-protein coupled to the alpha 1-adrenoceptor seems to be unaffected by hypothyroidism.

Adipose Tissue↗
PubMed1994

Developmental neurotoxicity of ethanol: further evidence for an involvement of muscarinic receptor-stimulated phosphoinositide hydrolysis.

Various lines of evidence suggest that muscarinic receptor-stimulated phosphoinositide hydrolysis during postnatal development in the rat brain may play a relevant role in glial cell proliferation and neuronal differentiation. We have previously shown that administration of ethanol to developing rats during the brain growth spurt causes microencephaly and selectively decreases muscarinic receptor-stimulated phosphoinositide hydrolysis. In the present study we have investigated the sensitivity of the phosphoinositide system coupled to muscarinic receptors to ethanol inhibition during distinct stages of the brain growth spurt. Different groups of rats were treated for 3 days with ethanol (4 g/kg per day) on postnatal days 2-4 (initial), 6-8 or 10-12 (peak), 13-15 (final stage of the brain growth spurt). The results show that the period of maximal sensitivity to ethanol of muscarinic receptor-stimulated phosphoinositide hydrolysis coincides with the peak of the brain growth spurt and with the period of maximal efficacy of muscarinic receptor agonists to induce inositol phosphates accumulation. Interestingly, only when muscarinic receptor-stimulated phosphoinositide hydrolysis was inhibited, a significant reduction of brain weight was observed. The close parallel between inhibition of this second messenger response and reduction of brain weight suggests that the phosphoinositide system coupled to muscarinic receptors may represent a target for the neurotoxic effects of ethanol during this stage of brain development.

Animals↗
PubMed1996

Receptor-mediated phosphoinositide metabolism in peripheral nerve and cultured Schwann cells.

Peripheral nerve possesses muscarinic cholinergic receptors, predominantly of the M3 subtype, that stimulate phosphoinositide metabolism. Evidence suggests that one site of this response is the myelin sheath. Purified peripheral nerve myelin contains several heterotrimeric GTP-binding proteins. Furthermore, carbachol and guanosine-5'-(3-O-thio)triphosphate-stimulated hydrolysis of exogenous phosphatidylinositol-4,5-bis-phosphate that is blocked by atropine can be reconstituted in a purified peripheral myelin-rich fraction. Nerve phosphoinositide turnover is also stimulated by adenosine analogs and blocked by adenosine receptor antagonists in a pattern consistent with the presence of adenosine A2 receptors in the tissue. Receptor-mediated phosphoinositide metabolism has also been studied in a human tumor-derived Schwann cell line (NF1T) derived from a neurofibromatosis-1 patient. By the same experimental criteria, NF1T cells also appear to contain adenosine A2 receptors which upon activation stimulate phosphoinositide turnover. However, phosphoinositide metabolism in these cells is not increased by either carbachol or ATP. Our findings taken together with other reports suggest that Schwann cells may possess a variety of receptors which regulate phosphoinositide metabolism.

Cells, Cultured↗
PubMed1999

Nerve growth factor-induced PKB/Akt activity is sustained by phosphoinositide 3-kinase dependent and independent signals in sympathetic neurons.

Phosphoinositide 3-kinase and its downstream effector kinase PKB/Akt have been suggested to have crucial roles in suppressing apoptosis in several classes of neurons. However, few studies have conducted a long-term investigation of either kinase activity, many studies relying instead on use of the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. When we added LY294002 or wortmannin to sympathetic neurons, apoptosis in the presence of nerve growth factor (NGF) was very slow compared to that obtained by NGF deprivation. However, expression of a kinase-inactive mutant of PKB/Akt in the presence of NGF induced apoptosis in a significant proportion of the neurons. To understand this discrepancy, we investigated more closely the regulation of PKB/Akt activity by NGF. NGF stimulation induced a rapid increase in PKB/Akt activity which was sustained at approximately 6-fold up to 24 h. Phosphoinositide 3-kinase was also rapidly activated by NGF. However, concentrations of wortmannin which completely blocked phosphoinositide 3-kinase activity in the neurons inhibited no more than 50-70% of cellular PKB/Akt activity. Similarly, approximately 50% of maximal NGF-stimulated PKB/Akt activity remained elevated at concentrations of LY294002 which completely blocked neurite outgrowth, a process known to be phosphoinositide 3-kinase dependent. We suggest that a proportion of the sustained PKB/Akt activity induced by NGF is mediated by phosphoinositide 3-kinase-independent pathways. These results raise a cautionary note as to the usefulness of LY294002 or wortmannin as tools to dissect the role of PKB/Akt in neuronal survival.

Androstadienes↗
PubMed1999

Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells.

Phosphoinositides are a family of inositol-containing phospholipids which are present in all eukaryotic cells. Although in most cells these lipids, with the exception of phosphatidylinositol, constitute only a very minor proportion of total cellular lipids, they have received immense attention by researchers in the past 15-20 years. This is due to the discovery that these lipids, rather than just having structural functions, play key roles in a wide range of important cellular processes. Much less is known about the plant phosphoinositides than about their mammalian counterparts. However, it has been established that a functional phosphoinositide system exists in plant cells and it is becoming increasingly clear that inositol-containing lipids are likely to play many important roles throughout the life of a plant. It is not our intention to give an exhaustive overview of all aspects of the field, but rather we focus on the phosphoinositide kinases responsible for the synthesis of all phosphorylated forms of phosphatidylinositol. Also, we mention some of the aspects of current phosphoinositide research which, in our opinion, are most likely to provide a suitable starting point for further research into the role of phosphoinositides in plants.

Animals↗
PubMed2003

Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry.

Phosphoinositides (phosphorylated derivatives of phosphatidylinositol, PI) are versatile intracellular signaling lipids whose occurrence in low concentrations complicates direct mass measurements. Here we present a sensitive method to detect, identify and quantify phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)) with different fatty acid compositions (phosphoinositide profiles) in total lipid extracts by electrospray ionization mass spectrometry (ESI-MS). Using this method, we detected elevated concentrations of PIP2 in human fibroblasts from patients with Lowe syndrome, a genetic disorder that affects phosphoinositide metabolism. Saccharomyces cerevisiae cells deficient in enzymes involved in PIP metabolism--Sac1p, a phosphoinositide phosphatase, and Vps34p and Pik1p, a PI 3-kinase and PI 4-kinase, respectively--showed not only different PIP concentrations but also differential changes in PIP profiles indicating metabolic and/or subcellular pooling. Mass spectrometric analysis of phosphoinositides offers unique advantages over existing approaches and may represent a powerful diagnostic tool for human diseases that involve defective phosphoinositide metabolism.

Animals↗
PubMed1995

Metabotropic glutamate receptor (mGluR)-mediated potentiation of cyclic AMP responses does not require phosphoinositide hydrolysis: mediation by a group II-like mGluR.

Metabotropic glutamate receptors (mGluRs) in the CNS are coupled to a variety of second messenger systems, the best characterized of which is activation of phosphoinositide hydrolysis. Recently, we found that activation of mGluRs in rat brain slices by the selective mGluR agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) potentiates cyclic AMP (cAMP) responses elicited by activation of other receptors coupled to Gs. It has been suggested that mGluR-mediated potentiation of cAMP responses is secondary to activation of phosphoinositide hydrolysis. However, preliminary evidence suggests that this is not the case. Therefore, we designed a series of experiments to test more fully the hypothesis that mGluR-mediated potentiation of cAMP responses is secondary to phosphoinositide hydrolysis. Inhibitors of both protein kinase C and intracellular calcium mobilization failed to antagonize 1S,3R-ACPD-stimulated potentiation of cAMP responses. Further, coapplication of phorbol esters and 1S,3R-ACPD induced a cAMP response that was greater than additive. Finally, (RS)-3,5-dihydroxyphenylglycine, a selective agonist of mGluRs coupled to phosphoinositide hydrolysis, failed to potentiate cAMP responses, whereas (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine, an mGluR agonist that does not activate mGluRs coupled to phosphoinositide hydrolysis, elicited a robust potentiation of cAMP responses. In total, these data strongly suggest that mGluR-mediated potentiation of cAMP responses is not secondary to activation of phosphoinositide hydrolysis and is likely mediated by a group II mGluR.

Animals↗
PubMed1998

Selective increases in phosphoinositide signaling activity and G protein levels in postmortem brain from subjects with schizophrenia or alcohol dependence.

Comparisons of the activity of the G protein-mediated phosphoinositide signal transduction system and of G protein levels were made in two regions of frontal cortex from eight schizophrenic, alcohol-dependent, and control subjects. G protein-mediated phosphoinositide hydrolysis was measured by stimulating cortical membranes incubated with [3H]phosphatidylinositol with 0.3-10 microM guanosine 5'-O-(3-thio)triphosphate (GTPgammaS). In frontal cortex areas 8/9, GTPgammaS-induced phosphoinositide hydrolysis was 50% greater in schizophrenic than control or alcohol-dependent subjects, whereas there were no differences among these groups of subjects in the response to GTPgammaS in frontal cortex area 10. Agonists for dopaminergic, cholinergic, purinergic, serotonergic, histaminergic, and glutamatergic receptors coupled to the phosphoinositide signaling system increased [3H]phosphatidylinositol hydrolysis in a GTPgammaS-dependent manner. Responses to most agonists were similar in all three subject groups in both cortical regions, with the largest difference being a 40% greater response to dopaminergic receptor stimulation in frontal cortex 8/9 from schizophrenic subjects. Measurements of the levels of phospholipase C-beta, and of alpha-subunits of Gq, Go, Gi1, Gi2, and Gs, made by immunoblot analyses revealed no differences among the groups of subjects except for increased G alpha(o) in schizophrenic subjects and increased G alpha(o) and G alpha(i1) in alcohol-dependent subjects. These results demonstrate that schizophrenia is associated with increased activity of the phosphoinositide signal transduction system and increased levels of G alpha(o), whereas the phosphoinositide system was unaltered in alcohol dependence, but G alpha(o) and G alpha(i1) were increased.

Adult↗
PubMed2003

Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation.

Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47(phox), a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47(phox) with phosphoinositides. Although the isolated phox homology (PX) domain of p47(phox) can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47(phox) is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47(phox) to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47(phox), because protein kinase C treatment of the wild-type p47(phox) but not of a mutant protein with the S303304328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47(phox) translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47(phox) nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47(phox) enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47(phox) from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.

Amino Acid Substitution↗
PubMed1997

Mycobacterium smegmatis phosphoinositols-glyceroarabinomannans. Structure and localization of alkali-labile and alkali-stable phosphoinositides.

Lipoarabinomannans from fast growing Mycobacterium sp., namely AraLAMs, stimulate the early events of macrophage activation. The immunological activities of all of these AraLAMs drastically decrease with the loss of the mild alkali groups, which were believed to be restricted to the fatty acid residues from the phosphatidyl-myo-inositol anchor. This report reveals the presence and the structure of mild alkali-labile phosphoinositide units linked via the phosphate to the C-5 of the beta-D-Araf in the AraLAMs of Mycobacterium smegmatis, a fast growing mycobacterial species. Their structure was unambiguously established with a strategy based on both one-dimensional 31P and two-dimensional 1H-31P heteronuclear multiple quantum correlation spectroscopy (HMQC) and HMQC-homonuclear Hartmann-Hahn spectroscopy NMR experiments applied to native AraLAMs and to AraLAMs treated in mild alkali conditions. Next to these alkali-labile phosphoinositides estimated at three per molecule, two other mild alkali-stable phosphoinositide units were identified: the expected (myo-inositol-1)-phosphate-(3-glycerol) unit typifying the well known glycosylphosphatidylinositol anchor of the mannan core and, more surprisingly, one (myo-inositol-1)-phosphate-(5-beta-D-Araf) unit having the same structure as the alkali-labile ones. Moreover, these four phosphoinositide units were found capping the arabinan side chains. Thus, their different behavior toward mild alkaline hydrolysis was explained according to their accessibility to the alkali reagent. This novel class of LAMs, namely phosphoinositols-glyceroarabinomannans (PI-GAMs), are characterized by their phosphoinositide units but also by the absence of fatty acid residues. These PI-GAMs were found to elicit the secretion of tumor necrosis factor-alpha, suggesting that phosphoinositides are the major PI-GAM epitope involved in this process.

Antigens, Bacterial↗
PubMed1998

Specificity and promiscuity in phosphoinositide binding by pleckstrin homology domains.

Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides. We describe use of a convenient "dot-blot" approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound phosphoinositides in the assay, but only two (from phospholipase C-delta1 and Grp1) showed clear specificity for a single species. Using soluble inositol phosphates, we show that the Grp1 PH domain (originally cloned on the basis of its phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binding) binds specifically to D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (the PtdIns(3,4,5)P3 headgroup) with KD = 27.3 nM, but binds D-myo-inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) or D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) over 80-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only when phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inositol phosphate binding by the Grp1 PH domain. By contrast, another PH domain capable of PI 3-K-dependent membrane recruitment (encoded by EST684797) does not distinguish Ins(1,3,4)P3 from Ins(1,3,4,5)P3 (binding both with very high affinity), despite selecting strongly against Ins(1,4,5)P3. The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presented in the literature, our results suggest that many PH domains bind similarly to multiple phosphoinositides (and in some cases phosphatidylserine), and are likely to be regulated in vivo by the most abundant species to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly specific phosphoinositide binding is a characteristic of relatively few cases.

Animals↗
PubMed1999

Regulation of AMP deaminase by phosphoinositides.

AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a K(i) of 110 nM. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/K)X(n)(R/K)XKK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo.

AMP Deaminase↗
PubMed2001

Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

1-Phosphatidylinositol 4-Kinase↗
PubMed2001

The p40phox and p47phox PX domains of NADPH oxidase target cell membranes via direct and indirect recruitment by phosphoinositides.

The Phox homology (PX) domain has recently been reported to bind to phosphoinositides, and some PX domains can localize to endosomes in vivo. Here we show data to support the conclusion that the p40(phox) PX domain binds to phosphatidylinositol 3-phosphate specifically in vitro and localizes to endosomes in intact cells. In addition, its Y59A/L65Q mutant, which has decreased affinity for phosphatidylinositol 3-phosphate in vitro, fails to target EGFP-p40-PX to endosomes. However, unlike published results, we find that the p47(phox) PX domain weakly binds to many phosphoinositides in vitro showing slightly higher affinity for phosphatidylinositol 3,4,5-trisphosphate. Moreover, we show for the first time that upon insulin-like growth factor-1 stimulation of COS cells, the p47(phox) PX domain is localized to the plasma membrane, and this subcellular localization is dependent on PI 3-kinase activity. Unexpectedly, its R42Q mutant that loses in vitro phosphoinositide-binding ability can still target EGFP-p47-PX to the plasma membrane. Our data suggest that the translocation of p47(phox) PX domain to the plasma membrane does involve 3'-phosphoinositide(s) in the process, but the phosphoinositide-binding of p47(phox) PX domain is not sufficient to recruit it to the plasma membrane. Therefore, the p40(phox) and p47(phox) PX domains can target subcellular membranes via direct or indirect recruitment by phosphoinositides, while both are under the control of phosphatidylinositol 3-kinase activity.

Animals↗
PubMed2005

Phosphoinositide binding regulates alpha-actinin dynamics: mechanism for modulating cytoskeletal remodeling.

The active association-dissociation of dynamic protein-protein interactions is critical for the ability of the actin cytoskeleton to remodel. To determine the influence of phosphoinositide binding on the dynamic interaction of alpha-actinin with actin filaments and integrin adhesion receptors, fluorescence recovery after photobleaching (FRAP) microscopy was carried out comparing wild-type green fluorescent protein (GFP)-alpha-actinin and a GFP-alpha-actinin mutant with a decreased affinity for phosphoinositides (Fraley, T. S., Tran, T. C., Corgan, A. M., Nash, C. A., Hao, J., Critchley, D. R., and Greenwood, J. A. (2003) J. Biol. Chem. 278, 24039-24045). In fibroblasts, recovery of the mutant alpha-actinin protein was 2.2 times slower than the wild type along actin stress fibers and 1.5 times slower within focal adhesions. FRAP was also measured in U87MG glioblastoma cells, which have higher levels of 3-phosphorylated phosphoinositides. As expected, alpha-actinin turnover for both the stress fiber and focal adhesion populations was faster in U87MG cells compared with fibroblasts with recovery of the mutant protein slower than the wild type along actin stress fibers. To understand the influence of alpha-actinin turnover on the modulation of the actin cytoskeleton, wild-type or mutant alpha-actinin was co-expressed with constitutively active phosphoinositide (PI) 3-kinase. Co-expression with the alpha-actinin mutant inhibited actin reorganization with the appearance of enlarged alpha-actinin containing focal adhesions. These results demonstrate that the binding of phosphoinositides regulates the association-dissociation rate of alpha-actinin with actin filaments and integrin adhesion receptors and that the dynamics of alpha-actinin is important for PI 3-kinase-induced reorganization of the actin cytoskeleton. In conclusion, phosphoinositide regulation of alpha-actinin dynamics modulates the plasticity of the actin cytoskeleton influencing remodeling.

Actinin↗
PubMed2005

Regulation of MDR1 expression and drug resistance by a positive feedback loop involving hyaluronan, phosphoinositide 3-kinase, and ErbB2.

Multidrug resistance is a potent barrier to effective, long term therapy in cancer patients. It is frequently attributed to enhanced expression of multidrug transporters or to the action of receptor kinases, such as ErbB2, and downstream anti-apoptotic signaling pathways, such as the phosphoinositide 3-kinase/Akt pathway. However, very few connections have been made between receptor kinases or anti-apoptotic pathways and multidrug transporter expression or function. Data presented herein show that constitutive interaction of the pericellular polysaccharide, hyaluronan, with its receptor, CD44, regulates assembly and activation of an ErbB2-containing signaling complex, which in turn stimulates phosphoinositide 3-kinase activity in multidrug-resistant MCF-7/Adr human breast carcinoma cells. Phosphoinositide 3-kinase activates Akt and downstream anti-apoptotic events, which contribute to drug resistance. However, hyaluronan and phosphoinositide 3-kinase stimulate expression of the multidrug transporter, MDR1 (P-glycoprotein), in an interdependent, but Akt-independent, manner. Furthermore, constitutively active phosphoinositide 3-kinase, but not Akt, stimulates hyaluronan production. These Akt-independent effects are dominant over the effects of Akt on doxorubicin resistance in MCF-7/Adr cells. Thus hyaluronan, phosphoinositide 3-kinase, and ErbB2 form a positive feedback loop that strongly amplifies MDR1 expression and regulates drug resistance in these cells. This pathway may also be important in progression of other malignant characteristics. These results illustrate the potential importance of hyaluronan as a therapeutic target in multidrug-resistant carcinomas.

Breast Neoplasms↗
PubMed1998

Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts.

Both phosphoinositides and small GTP-binding proteins of the Rho family have been postulated to regulate actin assembly in cells. We have reconstituted actin assembly in response to these signals in Xenopus extracts and examined the relationship of these pathways. We have found that GTPgammaS stimulates actin assembly in the presence of endogenous membrane vesicles in low speed extracts. These membrane vesicles are required, but can be replaced by lipid vesicles prepared from purified phospholipids containing phosphoinositides. Vesicles containing phosphatidylinositol (4,5) bisphosphate or phosphatidylinositol (3,4,5) trisphosphate can induce actin assembly even in the absence of GTPgammaS. RhoGDI, a guanine-nucleotide dissociation inhibitor for the Rho family, inhibits phosphoinositide-induced actin assembly, suggesting the involvement of the Rho family small G proteins. Using various dominant mutants of these G proteins, we demonstrate the requirement of Cdc42 for phosphoinositide-induced actin assembly. Our results suggest that phosphoinositides may act to facilitate GTP exchange on Cdc42, as well as to anchor Cdc42 and actin nucleation activities. Hence, both phosphoinositides and Cdc42 are required to induce actin assembly in this cell-free system.

Actins↗
PubMed1999

Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.

Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane.

Animals↗