epidermal-growth-factor and inositol-1-3-4-5-tetrakisphosphate

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).

Topics: Adaptor Proteins, Signal Transducing; Biological Transport; Brain; Cell Line; Cell Membrane; Epidermal Growth Factor; Humans; Inositol Phosphates; Mutation; Nerve Tissue Proteins; Phosphatidylinositol Phosphates; Protein Binding; Receptors, Thrombin; Recombinant Fusion Proteins; Thrombin; Type C Phospholipases; Zinc Fingers

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3]. In order to study the effects of PtdIns(3,4,5)P3 on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8% identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P3, inositol 1, 3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], with high affinity (Kd 32. 2+/-5.2 nM) and inositol phosphate specificity [Kd values for Ins(1, 3,4,5,6)P5, InsP6, Ins(1,3,4)P3 and Ins(1,4,5)P3: 283+/-32, >10000, >10000 and >10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P3 in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP-GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P3 and hence constitutes a true PtdIns(3,4,5)P3 receptor.

Topics: ADP-Ribosylation Factors; Animals; Cell Membrane; Cloning, Molecular; Enzyme Activation; Epidermal Growth Factor; GTP-Binding Proteins; Humans; Inositol Phosphates; Mice; Molecular Sequence Data; Nerve Growth Factors; PC12 Cells; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Rats; Receptors, Cytoplasmic and Nuclear; Sequence Analysis, DNA; Structure-Activity Relationship

Several studies have suggested that murine and human keratinocytes respond differently to phorbol 12-myristate 13-acetate (PMA). Using an in vitro assay, we found that in contrast to its effect on murine skin, PMA did not induce ornithine decarboxylase (ODC) activity in human skin biopsies. To explore the signalling induced by PMA and to determine whether an in vitro culture system could be used to predict biological activity of retinoids in human keratinocytes, we studied a simian virus 40 (SV40)-transformed human keratinocyte cell line. Epidermal growth factor (EGF) stimulates ODC activity and increases the steady-state level of ODC mRNA in a dose- and time-dependent manner in these cells [Prystowsky, Clevenger and Zheng (1993) Exp. Dermatol. 2, 125-132]. In this report, 10(-10) M-10(-7) M PMA induced ODC mRNA and enzyme synthesis at 7 h, but did not significantly induce ODC activity and inhibited the EGF induction of ODC activity. To explore the mechanism whereby PMA interfered with EGF signalling, the effect of PMA on EGF binding to its cell-surface receptor was studied; acute treatment with PMA (within 7 h) decreased EGF binding to 41-57% of the baseline level. In contrast, chronic treatment with PMA (24 h) increased EGF binding to 156% of the baseline level and was associated with an increase in quantity of EGF receptor protein. Protein kinase C (PKC) activation correlated with the acute decrease in EGF binding following PMA treatment. In summary, PMA induced ODC mRNA and ODC enzyme synthesis, while steady-state levels of immunoprecipitable ODC enzyme protein and ODC activity were not increased, demonstrating possible increased turnover of ODC enzyme protein. Additionally, PMA inhibited the induction of ODC by EGF through decreased EGF binding, possibly mediated by PKC activation. Finally treatment of the keratinocytes with retinoids including etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cisretinoic acid, and acitretin blocked the PMA induction of ODC mRNA, suggesting this in vitro model could be a valuable screening assay for predicting biological activity in humans.

Topics: Cells, Cultured; Down-Regulation; Epidermal Growth Factor; Humans; Inositol Phosphates; Keratinocytes; Ornithine Decarboxylase; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate

This study investigated the effects of epidermal growth factor (EGF) on cytosolic calcium ([Ca++]i) levels in rat calvarial osteoblasts, the nature of the regulation of this event, and the role these EGF-induced [Ca++]i changes have in osteoblastic cell proliferation. EGF significantly increased [Ca++]i measured in fura-2-loaded, individual cells. This increase was related to extracellular calcium influx. Activation of protein kinase C(PKC) by pretreating the cells with phorbol esters blocked the EGF-induced increase in [Ca++]i. EGF failed to increase inositol trisphosphate levels measured by high performance liquid chromatographic analysis. However, it did increase inositol bisphosphate and inositol tetrakisphosphate production. The EGF-dependent increase in DNA synthesis was partially blocked by the addition of calcium channel blockers. Therefore, it appears that the mechanism of action of EGF-induced osteoblastic cell proliferation is mediated by changes in [Ca++]i primarily due to extracellular calcium influx.

Topics: Analysis of Variance; Animals; Calcium; Calcium Channel Blockers; Cell Division; Cells, Cultured; Chromatography, High Pressure Liquid; Cytosol; DNA; Enzyme Activation; Epidermal Growth Factor; Fura-2; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Osteoblasts; Protein Kinase C; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate

In PC12 cells, preincubated with [3H]inositol, nerve growth factor (NGF) stimulated an approximately 100% increase in the levels of [3H]inositol 1,3,4-trisphosphate ([3H]-Ins(1,3,4)P3], [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3], and [3H]inositol 1,3,4,5-tetrakisphosphate ([3H]Ins(1,3,4,5)P4] as early as 5-15 s after addition of NGF. This NGF-mediated response was apparent only when the cells had been cultured in the absence of fetal bovine serum (FBS). PC12 cells cultured in FBS-containing medium did not display NGF-mediated increases in [3H]Ins(1,3,4)P3, [3H]Ins(1,4,5)P3, and [3H]Ins(1,3,4,5)P4 levels. Using cells cultured in the absence of FBS, epidermal growth factor (EGF) and fibroblast growth factor also stimulated production of [3H]Ins(1,3,4)P3, [3H]Ins(1,4,5)P3, and [3H]Ins(1,3,4,5)P4. Lavendustin A, a tyrosine kinase inhibitor, inhibited both the EGF- and NGF-stimulated increases in the levels of these tritiated inositol phosphates. These results suggest that NGF stimulates the production of Ins(1,3,4)P3, Ins(1,4,5)P3, and Ins(1,3,4,5)P4 and that this response is dependent on tyrosine kinase activity. Furthermore, although the production of Ins(1,3,4)P3, Ins(1,4,5)P3, and Ins(1,3,4,5)P4 may be a common response to factors stimulating neuronal differentiation, it is not sufficient for stimulation of neuronal differentiation.

Topics: Animals; Epidermal Growth Factor; Fibroblast Growth Factors; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Nerve Growth Factors; PC12 Cells

Injection of D-myo-inositol-1,4,5-trisphosphate (IP3) was found to induce a transient increase of intracellular Ca2+ concentration in cancerous mammary cells (MMT060562) and in normal mammary cells treated with epidermal growth factor. Responses to injection of either D-myo-inositol-1,4-bisphosphate (IP2) or D-myo-inositol-1,3,4,5-tetrakisphosphate (IP4) were small or absent. Furthermore, normal mammary cells cultivated with low-protein serum replacement alone or in the presence of differentiation-inducing hormones (insulin + cortisol + prolactin) were less sensitive to IP3. Thapsigargin induced a transient increase of Ca2+ due to the release of Ca2+ from an intracellular pool. There was no difference in the peak heights of the thapsigargin-induced Ca2+ increase when mammary cells were cultivated in the presence or absence of epidermal growth factor or insulin + cortisol + prolactin. These findings suggest that the releasable intracellular Ca2+ pool remained unchanged whereas sensitivity to IP3 increases during the proliferation stage. Mechanical stimulus of a mammary cell induces an increase of intracellular Ca2+ in the stimulated cell. A certain stimulating factor is released from the mechanically stimulated cell into the extracellular space, and it induces an increase of Ca2+ in surrounding cells. In contrast, the IP3-induced Ca2+ increase in both cancerous and epidermal growth factor-treated normal mammary cells did not spread to adjacent cells. Therefore, increase of Ca2+ is not sufficient to account for the release of stimulating substances from mammary cells in the mechanically-induced spreading response.

Topics: Animals; Caffeine; Calcium; Cell Division; Cells, Cultured; Drug Resistance; Epidermal Growth Factor; Epithelial Cells; Epithelium; Female; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Inbred ICR; Pregnancy; Ryanodine; Terpenes; Thapsigargin; Tumor Cells, Cultured

A short-term treatment with phorbol 12,13-dibutyrate (PDBu) was found to inhibit totally the epidermal growth factor (EGF)-stimulated phosphoinositide hydrolysis in A431 cells, whereas long-term pretreatment with PDBu, which is known to down regulate protein kinase C, induced a greater accumulation of the EGF-triggered inositol phosphate accumulation, particularly of Ins(1,3,4,5)P4. The increased Ins(1,4,5)P3/Ins(1,3,4,5)P4 formation in the PDBu long-term pretreated cells was coincident with the increased Ca2+ influx stimulated by EGF in the same cells. Since long-term pretreatment with PDBu was found to enhance the EGF signals, an explanation for the synergism between EGF and phorbol esters in the induction of DNA synthesis is provided.

Topics: Chlorides; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Epidermal Growth Factor; Humans; Hydrolysis; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Lithium; Lithium Chloride; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphatidylinositols; Protein Kinase C; Tumor Cells, Cultured

A human hepatocellular carcinoma-derived cell line, PLC/PRF/5, was examined for its ability to respond to epidermal growth factor (EGF) exposure with increased phosphatidylinositol 4,5-bisphosphate hydrolysis. Upon addition of EGF (25 ng/ml), a rapid (10-15 s) but transient increase in Ins(1,4,5)P3 levels and large, prolonged (2 min) increases in Ins(1,3,4,5)P4 and Ins(1,3,4)P3 levels were detected. Increases in cytosolic Ca2+ were observed after a 10 to 20 s lag, reaching peak value at 1 min, and remaining elevated for 10 min. The initial burst of cytosolic Ca2+ occurred in the absence of extracellular Ca2+ and probably reflects mobilization of intracellular Ca2+ stores. In cells pretreated with EGTA, the sustained component of the Ca2+ response was not observed. Comparison of the inositol phosphate and Ca2+ responses of PLC/PRF/5 cells to responses reported in other cell types indicates that this cell line is a good model for EGF action in liver.

Topics: Calcium; Carcinoma, Hepatocellular; Chromatography, High Pressure Liquid; Cytosol; Egtazic Acid; Epidermal Growth Factor; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Liver Neoplasms; Sugar Phosphates; Tumor Cells, Cultured