Dynamic sensitivity of ATP-sensitive K(+) channels to ATP.

ATP and MgADP regulate K(ATP) channel activity and hence potentially couple cellular metabolism to membrane electrical activity in various cell types. Using recombinant K(ATP) channels that lack sensitivity to MgADP, expressed in COSm6 cells, we demonstrate that similar on-cell activity can be observed with widely varying apparent submembrane [ATP] ([ATP](sub)). Metabolic inhibition leads to a biphasic change in the channel activity; activity first increases, presumably in response to a fast decrease in [ATP](sub), and then declines. The secondary decrease in channel activity reflects a marked increase in ATP sensitivity and is correlated with a fall in polyphosphoinositides (PPIs), including phosphatidylinositol 4,5-bisphosphate, probed using equilibrium labeling of cells with [(3)H]myo-inositol. Both ATP sensitivity and PPIs rapidly recover following removal of metabolic inhibition, and in both cases recovery is blocked by wortmannin. These data are consistent with metabolism having a dual effect on K(ATP) channel activity: rapid activation of channels because of relief of ATP inhibition and much slower reduction of channel activity mediated by a fall in PPIs. These two mechanisms constitute a feedback system that will tend to render K(ATP) channel activity transiently responsive to a change in [ATP](sub) over a wide range of steady state concentrations.

Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover.

Caveolae and detergent-insoluble, glycosphingolipid-enriched domains (DIGs) are cholesterol-enriched membrane domains that have been implicated in signal transduction because a variety of signaling proteins as well as phosphatidylinositol bisphosphate (PtdInsP2) are compartmentalized in these domains. We report here that depletion of cellular cholesterol leads to the inhibition of epidermal growth factor- and bradykinin-stimulated PtdIns turnover in A431 cells. This is associated with the loss of compartmentalization of epidermal growth factor receptors, Gq, and PtdInsP2 in the low density membrane domains. Replacement of cellular cholesterol leads to the reorganization of signaling molecules in the low density domains and the reestablishment of hormone-stimulated PtdIns hydrolysis. Oxysterol derivatives show a variable ability to functionally replace the cholesterol in this system. These data are consistent with the hypothesis that localization of signaling proteins and lipids to cholesterol-enriched domains is required for the proper function of hormone-stimulated PtdIns turnover.

Compartmentalization of phosphatidylinositol 4,5-bisphosphate in low-density membrane domains in the absence of caveolin.

In cells that exhibit caveolae, the hormone-sensitive pool of PtdIns 4,5-P2 is localized in a low density, caveolin-enriched membrane fraction (1). Neuro 2a cells do not express caveolin. Nonetheless, the PtdIns 4,5-P2 in these cells is compartmentalized in a low density, detergent-insoluble domain that also contains other signaling-related molecules. Compartmentalization of PtdIns 4,5-P2 was observed regardless of whether Triton X-100-containing or detergent-free methods were used to prepare the membranes. However, the partitioning of receptor tyrosine kinases and GPI-anchored proteins into the low density domains was dependent upon the method of membrane preparation. Treatment of Neuro 2a cells with cyclodextrin delocalized the PtdIns 4,5-P2 and inhibited hormone-stimulated PtdIns turnover. These results suggest that compartmentalization of PtdIns 4,5-P2 does not require caveolin but is necessary for the proper functioning of phosphoinositide-based signaling.

Functional characterization of an epidermal growth factor receptor/RET chimera.

The RET (recombined in transfection) gene encodes a receptor tyrosine kinase homolog involved in innervation of the gut and renal development. A chimeric epidermal growth factor receptor (EGFR)/RET receptor was constructed which contained the extracellular and transmembrane domains of the EGF receptor fused to the intracellular domain of RET. This construct was expressed in NIH 3T3 cells, and the functional properties of the receptor were characterized and compared with those of the wild type EGF receptor. Whereas the EGF receptor exhibited both high and low affinity binding sites for 125I-EGF, the EGFR/RET chimera exhibited only low affinity binding of 125I-EGF. The chimera was able to internalize EGF more rapidly than the wild type EGF receptor and recycled to the cell surface at twice the rate of the EGF receptor. Pulse-chase experiments indicated that EGF stimulated the degradation of the wild type EGF receptor but had no effect on the rate of degradation of the EGFR/RET receptor. The combination of increased recycling and decreased degradation resulted in the relatively inefficient down-regulation of the EGFR/RET chimera. Incubation of cells expressing the wild type EGF receptor with phorbol 12-myristate 13-acetate led to a reduction in 125I-EGF binding and a loss in EGF-stimulated tyrosine phosphorylation. However, phorbol 12-myristate 13-acetate treatment had only a limited effect on EGF binding and EGF-stimulated tyrosine kinase activity in cells expressing EGFR/RET chimeras. These findings suggest that the ret tyrosine kinase is not regulated by many of the common mechanisms used to terminate signaling via growth factor receptors. Such persistent activation of the Ret tyrosine kinase may be relevant to the physiological function of Ret in cells that normally express this growth factor receptor.

Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains.

Caveolae are small, plasma membrane invaginations that have been implicated in cell signaling. In A431 cells, approximately half of the total cellular phosphatidylinositol 4,5-bisphosphate (PtdIns 4, 5-P2) was found to be localized in low density, Triton-insoluble membrane domains enriched in caveolin. Treatment of cells with either epidermal growth factor or bradykinin for 5 min at 37 degrees C resulted in approximately a 50% decrease in this caveolar PtdIns 4,5-P2 with no change in the levels of plasma membrane PtdIns 4,5-P2. These data suggest that the PtdIns 4,5-P2 present in cells is largely compartmentalized and that the caveolar PtdIns 4,5-P2 is subject to hydrolysis by hormone-stimulated phospholipase C. As growth factor receptors, seven transmembrane domain receptors, heterotrimeric G proteins, and the inositol trisphosphate receptor have all been shown to be enriched in caveolae, these findings suggest that both the generation and response to inositol trisphosphate is highly compartmentalized within the cell.

Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains.

Recent evidence has implicated caveolae/DIGs in various aspects of signal transduction, a process in which polyphosphoinositides play a central role. We therefore undertook a study to determine the distribution of phosphoinositides and the enzymes that utilize them in these detergent-insoluble domains. We report here that the polyphosphoinositide phosphatase, but not several other phosphoinositide-utilizing enzymes, is highly enriched in a low density, Triton-insoluble membrane fraction that contains caveolin. This fraction is also enriched in polyphosphoinositides, containing approximately one-fifth of the total cellular phosphatidylinositol (4,5)P2. Treatment of cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), did not alter the distribution of polyphosphoinositides or the polyphosphoinositide phosphatase. However, PMA treatment did lead to a decrease in the mitogen-activated protein kinase and actin present in these domains. PMA also induced the recruitment of protein kinase C alpha to the caveolae/DIGs fraction. These findings suggest that polyphosphoinositides, the polyphosphoinositide phosphatase and protein kinase C play an important role in the structure or function of detergent-insoluble membrane domains.

The multiple endocrine neoplasia type 2B point mutation alters long-term regulation and enhances the transforming capacity of the epidermal growth factor receptor.

The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand-dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wild-type receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P + 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild-type and M857T EGF receptors. However, significant differences in receptor down-regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation.

Cell-cycle-dependent modulation of EGF-receptor-mediated signaling.

In A431 cells synchronized by treatment with thymidine, the level of EGF-stimulated tyrosine protein kinase activity in cells in S and G2/M phases was reduced approximately 40% relative to that seen in cells in G1. This decrease in receptor tyrosine protein kinase activity did not correlate with a decrease in cell surface EGF receptor expression, indicating that the reduced activity could not be attributed to receptor loss. EGF-stimulated PI 3-kinase activity was also reduced by approximately 60% during S phase as compared to G1 phase. The change was not due to decreased PI 3-kinase expression since Western blot analyses indicated that cellular p85 levels remained constant throughout the cell cycle. These data suggest that the ability of EGF to stimulate biological responses varies during the cell cycle and implicate cell-cycle-dependent processes in the regulation of EGF-receptor-mediated signaling.

Purification and characterization of a polyphosphoinositide phosphatase from rat brain.

A novel membrane-bound polyphosphoinositide phosphatase has been purified 7700-fold from rat brain. A combination of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated that the enzyme is a monomer with a molecular weight of 85,000-90,000. Biochemical analysis of the polyphosphoinositide phosphatase demonstrated that the enzyme utilizes phosphatidylinositol(4)phosphate (PtdIns(4)P), phosphatidylinositol(3)phosphate (PtdIns(3)P), and phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P2) as substrates. In the case of PtdIns(4,5)P2, the substrate is doubly dephosphorylated to yield PtdIns. The apparent Km values for PtdIns(4)P and PtdIns(4,5)P2 are 45 and 5 microM, respectively. Inositol(1,4)bisphosphate and inositol(1,4,5)trisphosphate neither serve as direct substrates for the polyphosphoinositide phosphatase nor inhibit its activity even at concentrations as high as 100 microM. Thus, the substrate specificity of the polyphosphoinositide phosphatase is distinct from that of previously identified phosphatases that utilize both inositol phospholipids and soluble inositol phosphates as substrates. The ability of the polyphosphoinositide phosphatase to hydrolyze phosphate from the 3-, 4-, or 5-position of the inositol ring suggests that this enzyme may play a key role in maintaining homeostasis among all forms of polyphosphoinositides.

Mutation of proline-1003 to glycine in the epidermal growth factor (EGF) receptor enhances responsiveness to EGF.

We have shown previously that the epidermal growth factor (EGF) receptor is phosphorylated at Ser-1002 and that this phosphorylation is associated with desensitization of the EGF receptor. Ser-1002 is followed immediately by Pro-1003, a residue that may promote the adoption of a specific conformation at this site or severe as a recognition element for the interaction of the EGF receptor with other proteins. To examine these possibilities, we have mutated Pro-1003 of the EGF receptor to a Gly residue and have analyzed the effect of this mutation on EGF-stimulated signaling. Cells expressing the P1003G EGF receptors exhibited higher EGF-stimulated autophosphorylation and synthetic peptide phosphorylation compared to cells expressing wild-type EGF receptors. In addition, the ability of EGF to stimulate PI 3-kinase activity and mitogen-activated protein kinase activity was enhanced in cells expressing the P1003G EGF receptor. Cells expressing P1003G receptors also demonstrated an increased ability to form colonies in soft agar in response to EGF. These results indicate that mutation of Pro-1003 leads to a potentiation of the biological effects of EGF. The findings are consistent with the hypothesis that Pro-1003 plays a role in a form of regulation that normally suppresses EGF receptor function.

Serine 1002 is a site of in vivo and in vitro phosphorylation of the epidermal growth factor receptor.

We have shown previously that treatment of A431 cells with epidermal growth factor (EGF) induces desensitization of the EGF receptor. We now show that this desensitization is associated with an increase in the phosphorylation of the receptor on Ser-1002. Using a synthetic peptide corresponding to the sequence surrounding Ser-1002, p34cdc2 was identified as a kinase capable of phosphorylating this serine residue. Purified Xenopus p34cdc2 was found to phosphorylate the synthetic peptide on the serine residue corresponding to Ser-1002. This kinase also phosphorylated purified EGF receptor in vitro on Ser-1002. Phosphorylation of the EGF receptor by p34cdc2 was associated with a decrease in its tyrosine protein kinase activity. These data indicate that the EGF receptor may be a target for phosphorylation by a cyclin-dependent kinase in vivo and imply that receptor function may be regulated in a cell cycle-dependent fashion.

Phosphatidylinositol 4-kinases and the role of polyphosphoinositides in cellular regulation.

Phosphoinositides play a central role in the transduction of signals for a variety of hormone and growth factor receptors. Multiple derivatives of phosphatidylinositol are present within the cell including phosphatidylinositol 4,5-bisphosphate, the phosphorylated derivative that is hydrolyzed by phospholipase C to produce the two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. The synthesis, degradation, and subsequent resynthesis of the phosphoinositides form a metabolic cycle known as the phosphoinositide cycle. The phosphoinositide cycle begins with the phosphorylation of phosphatidylinositol to form phosphatidylinositol 4-phosphate, a reaction catalyzed by phosphatidylinositol 4-kinase. Phosphatidylinositol kinase activity has been reported to be present in a variety of cellular membranes, and multiple isozymes of phosphatidylinositol 4-kinase are present within the cell, suggesting that the product of this reaction may have more than one biological function. The activity of phosphatidylinositol 4-kinase is regulated by growth factors, further underscoring the importance of this enzyme in cellular regulation. Recent data suggest that in addition to serving as substrates for phospholipase C, the polyphosphoinositides may themselves function as intracellular mediators of hormone action. For example, polyphosphoinositides have marked effects on the activity of certain actin binding proteins that may allow these lipids to participate in the regulation of actin polymerization. This review focuses on the properties of the phosphatidylinositol 4-kinases and the potential role of polyphosphoinositides in the regulation of cellular processes.

Growth factors promote inositol uptake in BC3H1 cells.

BC3H1 cells induced to differentiate by serum withdrawal were found to incorporate substantially less [3H]inositol into their phosphoinositides than cells induced to differentiate by growth in the presence of high serum. This decrease was found to be due to a decline in the rate of [3H]inositol uptake by the serum-starved cells. Addition of purified growth factors such as TGF-beta, EGF and FGF to these cells promoted inositol uptake and lead to an increase in the incorporation of [3H]inositol into phosphoinositides. Stimulation of inositol uptake by TGF-beta required at least a 24 hr exposure to the growth factor. These data indicate that growth factors regulate phosphoinositide metabolism at many different levels including at the level of inositol uptake.

Lack of correlation between changes in polyphosphoinositide levels and actin/gelsolin complexes in A431 cells treated with epidermal growth factor.

The polyphosphoinositides, PIP and PIP2, have been proposed to regulate actin polymerization in vivo because they dissociate actin/gelsolin complexes in vitro. We tested this hypothesis by comparing the ability of EGF and bradykinin to affect PI metabolism and the actin cytoskeleton in A431 cells. EGF, but not bradykinin, was found to induce ruffling and dissociation of actin/gelsolin complexes in these cells. However, both EGF and bradykinin stimulated the accumulation of inositol phosphates in [3H]inositol-labeled cells indicating that stimulation of PI turnover is not sufficient for the induction of changes in actin/gelsolin complex levels. EGF stimulated a twofold increase in the level of PIP in A431 cells. Other phosphoinositide levels were not markedly altered. Treatment of the cells with cholera toxin abrogated the EGF-induced rise in PIP levels without altering the dissociation of actin from gelsolin. These data indicate that increases in PIP and/or PIP2 are not necessary for dissociation of actin/gelsolin complexes. Overall, these experiments suggest that in A431 cells, the effects of EGF on the actin cytoskeleton are unlikely to be mediated through changes in PIP or PIP2 levels.

Desensitization of the EGF receptor alters its ability to undergo EGF-induced dimerization.

Treatment of A431 cells with EGF has been shown to induce the formation of EGF receptor dimers. Sucrose density gradient centrifugation as well as surface radio-iodination followed by crosslinking were used to study further the properties of EGF receptor monomers and dimers as well as the regulation of dimer formation. We have shown previously that treatment of A431 cells with high doses of EGF at 37 degrees C leads to the desensitization of the EGF receptor without a significant loss of cell surface 125I-EGF binding [Kuppuswamy and Pike (1989) J. biol. Chem. 264, 3357-3363; Cunningham et al. (1989) J. biol. Chem. 264, 15351-15356]. Desensitization of the EGF receptor led to a decrease in the ability of receptor monomers to be induced to form dimers by EGF both in vivo and in vitro. These data suggest that the sensitivity of a cell to EGF may be modulated by altering the capacity of the EGF receptor to form oligomers.

Stimulation of purified phosphatidylinositol 4-kinase by cobra venom cardiotoxin.

Cobra venom cardiotoxin was found to stimulate the phosphatidylinositol kinase activity present in A431 cell membranes and in detergent extracts of these membranes. Incubation of highly purified phosphatidylinositol 4-kinase from this source with cardiotoxin resulted in a 2- to 3-fold stimulation of phosphatidylinositol kinase activity. The activation of the purified phosphatidylinositol 4-kinase by cardiotoxin was time- and dose-dependent and appeared to be associated with a decrease in the Km apparent of the enzyme for phosphatidylinositol with no change in the Vmax apparent of the enzyme. The data suggest that the phosphatidylinositol 4-kinase is activated by direct interaction of the enzyme with cobra venom cardiotoxin.

Purification and characterization of a cytosolic transglutaminase from a cultured human tumour-cell line.

Transglutaminases are a family of Ca2(+)-dependent enzymes that catalyse the formation of isopeptide bonds between the side chains of glutamine and lysine residues. The enzymes have been hypothesized to be involved in a wide range of cellular processes, including growth and differentiation and stabilization of the cytoskeleton. The human epidermal carcinoma-cell line, A431 cells, have relatively high amounts of a cytosolic transglutaminase activity that varies upon treatment of the cells with epidermal growth factor. We demonstrate here that this cytosolic activity has the biochemical and immunological properties of a tissue transglutaminase. We also report the purification of this enzyme to apparent homogeneity by a protocol which involves a novel affinity-elution step. Polyclonal antibodies to the transglutaminase were raised and used to identify the enzyme by Western blotting. The availability of purified transglutaminase and antitransglutaminase antibodies will permit further study of the role of this enzyme in the growth of this hormone-responsive human tumour-cell line.

Treatment of A431 cells with epidermal growth factor (EGF) induces desensitization of EGF-stimulated phosphatidylinositol turnover.

Epidermal growth factor (EGF) stimulates the turnover of phosphoinositides in A431 cells. In cells that were pretreated with EGF for 30 min at 37 degrees C and then washed to remove surface-bound hormone, a 70-100% decrease in the EGF-stimulated production of inositol monophosphate, inositol bisphosphate, and inositol triphosphate was noted when the cells were exposed to the agonist a second time. Since only a 15% decrease in receptor number was observed in these pretreated cells, the loss of responsiveness to EGF for the production of inositol phosphates could not be attributed to a down-regulation of the EGF receptors. These data suggest that pretreatment of A431 cells with high concentrations of EGF leads to a desensitization of the EGF receptor. This desensitization of the receptor by EGF is apparent within 10-15 min of the addition of EGF and is maximal by 30 min. The desensitization appears to be homologous in nature since pretreatment of cells with EGF did not diminish their responsiveness to bradykinin; and conversely, pretreatment with bradykinin did not diminish the subsequent responsiveness of the cells to EGF. Desensitization to EGF was observed in cells in which protein kinase C had been down-regulated by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate, implying that EGF receptor desensitization is independent of protein kinase C. The desensitizing effects of EGF on growth factor-induced phosphatidylinositol turnover could be prevented by pretreatment of the cells with the calmodulin antagonist trifluoperazine, suggesting that calmodulin may be involved in the regulation of EGF receptor sensitivity.

Ligand-induced desensitization of 125I-epidermal growth factor internalization.

The internalization of 125I-epidermal growth factor (EGF) by A431 cells was investigated. Control cells were able to internalize over 80% of receptor-bound 125I-EGF. By contrast, cells treated with EGF before incubation with 125I-EGF internalized only 50% of the surface-bound radioligand. The ligand-induced decrease in 125I-EGF internalization showed a dose response to EGF with half-maximal effect occurring at 3 nM. The alteration in the extent of 125I-EGF internalization did not require extended treatment with high concentrations of the hormone. When the internalization of picomolar versus nanomolar concentrations of EGF were compared, the lower concentrations of 125I-EGF were more completely internalized than the higher concentrations of radioligand. These data are consistent with the hypothesis that occupation of the EGF receptor by hormone rapidly leads to the activation of cellular processes which effectively desensitize the system to further ligand-induced internalization. The decrease in the extent of ligand internalization occurred in cells in which the protein kinase C (Ca2+/phospholipid-dependent enzyme) activity had been down-regulated by prolonged treatment with 12-O-tetradecanoyl-phorbol-13-acetate implying that the desensitization process is independent of protein kinase C. However, the effects of EGF on the extent of hormone internalization could be mimicked by the addition of A23187 and could be prevented by pretreatment of the cells with calmodulin antagonists suggesting the possibility that Ca2+-calmodulin is involved in the regulation of EGF receptor internalization in A431 cells.