Making sense out of Ca2+ signals: their role in regulating stomatal movements

Plant cells maintain high Ca²⁺ concentration gradients between the cytosol and the extracellular matrix, as well as intracellular compartments. During evolution, the regulatory mechanisms, maintaining low cytosolic free Ca²⁺ concentrations, most likely provided the backbone for the development of Ca²⁺‐dependent signalling pathways. In this review, the current understanding of molecular mechanisms involved in Ca²⁺ homeostasis of plants cells is evaluated. The question is addressed to which extent the mechanisms, controlling the cytosolic Ca²⁺ concentration, are linked to Ca²⁺‐based signalling. A large number of environmental stimuli can evoke Ca²⁺ signals, but the Ca²⁺‐induced responses are likely to differ depending on the stimulus applied. Two mechanisms are put forward to explain signal specificity of Ca²⁺‐dependent responses. A signal may evoke a specific Ca²⁺ signature that is recognized by downstream signalling components. Alternatively, Ca²⁺ signals are accompanied by Ca²⁺‐independent signalling events that determine the specificity of the response. The existence of such parallel‐acting pathways explains why guard cell responses to abscisic acid (ABA) can occur in the absence, as well as in the presence, of Ca²⁺ signals. Future research may shed new light on the relation between parallel acting Ca²⁺‐dependent and ‐independent events, and may provide insights in their evolutionary origin.     

ER transport signals and trafficking of potassium channels and receptors

Channels and receptors on the cell surface mediate neuronal signaling. It is therefore important to understand how their surface density is controlled. Recent studies on the trafficking of potassium channels and neurotransmitter receptors have revealed unexpected complexity in the regulation of transport from the endoplasmic reticulum to the Golgi apparatus, raising the possibility that the surface composition of channels and receptors may be adjusted by controlling their export from the endoplasmic reticulum.     

Computational modeling of the dynamics of the MAP kinase cascade activated by surface and internalized EGF receptors

We present a computational model that offers an integrated quantitative, dynamic, and topological representation of intracellular signal networks, based on known components of epidermal growth factor (EGF) receptor signal pathways. The model provides insight into signal-response relationships between the binding of EGF to its receptor at the cell surface and the activation of downstream proteins in the signaling cascade. It shows that EGF-induced responses are remarkably stable over a 100-fold range of ligand concentration and that the critical parameter in determining signal efficacy is the initial velocity of receptor activation. The predictions of the model agree well with experimental analysis of the effect of EGF on two downstream responses, phosphorylation of ERK-1/2 and expression of the target gene, c-fos.     

Activation of extracellular-regulated kinases by normal and mutant EGF receptors

Glioblastoma cells express a mutant EGF receptor (EGFRvIII) that has constitutive tyrosine kinase activity and enhances their tumorigenicity. Here we show that EGFRvIII promotes constitutive phosphorylation of extracellular regulated kinases (ERKs) in glioblastoma cells in the absence of EGF. EGFRvIII also promoted constitutive activation of phosphoinositide 3-kinase in these cells, as assessed by phosphorylation of protein kinase B/akt. As expected, phosphorylation of protein kinase B/akt was blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. Less expectedly, we found that this treatment also blocked EGFRvIII-induced phosphorylation of ERKs. In contrast, ERK phosphorylation induced by EGF-activated normal EGF receptor in the same cells was largely unaffected by treatment with phosphoinositide 3-kinase inhibitors. This difference in behavior between the normal receptor and EGFRvIII was not due to differences in the levels of activated EGFRvIII and wild-type EGF receptor, as the two types of receptor were tyrosine phosphorylated to a similar extent under the experimental conditions used. EGFRvIII activation of ERKs was also sensitive to the phospholipase C inhibitor U73122, whereas ERK activation by normal EGF receptor was not. These results show that EGFRvIII and wild-type EGF receptor preferentially use different signaling pathways to induce ERK phosphorylation. The different mechanisms of ERK activation used by normal and mutant EGF receptors may be important in understanding the potent tumorigenic activity of EGFRvIII.     

Getting a sense for signals: regulation of the plant iron deficiency response

Understanding the Fe deficiency response in plants is necessary for improving both plant health and the human diet, which relies on Fe from plant sources. In this review we focus on the regulation of the two major strategies for iron acquisition in plants, exemplified by the model plants Arabidopsis and rice. Critical to our knowledge of Fe homeostasis in plants is determining how Fe is sensed and how this signal is transmitted and integrated into a response. We will explore the evidence for an Fe sensor in plants and summarize the recent findings on hormones and signaling molecules which contribute to the Fe deficiency response. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Preparation and properties of monoclonal and polyclonal antibodies to mouse epidermal growth factor (EGF) receptors: evidence for cryptic EGF receptors in embryonal carcinoma cells

Monoclonal antibodies to mouse epidermal growth factor (EGF) receptor were prepared by the immunization of rats with receptor glycoprotein purified from mouse liver by affinity chromatography on immobilized EGF. Purified mouse EGF receptor retained EGF-inducible autophosphorylating activity and was antigenic in rats and rabbits. The monoclonal antibodies cross react very poorly with human EGF receptor, while polyclonal rabbit antibodies immunoprecipitate human, rat and mouse EGF receptor equally well. The rabbit antibody blocks EGF binding to mouse fibroblast cells and, at 20-fold higher concentrations, stimulates uptake of tritiated thymidine into DNA. This indicates that antibodies bind at or close to the EGF-binding site and can mimic the effects of the growth factor. None of the monoclonals bind at the EGF site of the receptor. Immunoprecipitation, immunoblotting, 125I-EGF cross linking, 125I-surface labelling, immunohistochemistry and autophosphorylation techniques were used to delineate the basis for the induction of EGF receptors when OC15 embryonal carcinoma (EC) cells differentiate into endodermal derivatives (END). EGF-stimulated autophosphorylation of a 170 X 10(3) Mr protein in solubilized OC15 EC cells is readily detectable, although intact EC cells do not bind or respond to EGF by all other tests. The results suggest that cryptic EGF receptors are present in EC stem cells, a finding with implications in development.     

Protamine enhances epidermal growth factor (EGF)-stimulated mitogenesis by increasing cell surface EGF receptor number. Implications for existence of cryptic EGF receptors

Treatment of Swiss mouse 3T3 cells and human epidermoid carcinoma A431 cells with protamine at 37 degrees C increased the 125I-epidermal growth factor (EGF) binding activity at 4 degrees C. The effect of protamine on the increase of 125I-EGF binding activity appeared to be time, temperature, and dose dependent. This up-modulation of 125I-EGF binding by protamine correlated with protamine enhancement of EGF-stimulated mitogenesis, with respect to the magnitude of the effect and the dose response curves. Scatchard plot analyses indicated that protamine induced an increase in numbers of both high and low affinity EGF receptors without affecting their affinities. Protamine also increased functionally active EGF receptors in plasma membranes and solubilized membranes. This was evidenced by Scatchard plot analyses and by a protamine-induced increase of 125I-EGF-EGF receptor complex and an increase in EGF-stimulated phosphorylation of the EGF receptor. Combined with column chromatography of the solubilized EGF receptor on protamine-agarose gel, these results suggest that protamine may increase the EGF receptor number by directly activating cryptic EGF receptors in the plasma membrane.     

Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding

"Transactivation" of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, alpha(2A)-adrenergic receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB-EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, alpha(2A)AR expressing "donor" cells are co-cultured with "acceptor" cells lacking the alpha(2A)AR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the alpha(2)AR selective agonist UK14304 rapidly increases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB-EGF, indicating that it results from the release of HB-EGF from the alpha(2A)AR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after alpha(2A)AR stimulation requires Gbetagamma subunits upstream and dynamin-dependent endocytosis downstream of HB-EGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.     

Touch sense: Functional organization and molecular determinants of mechanosensitive receptors

Cutaneous mechanoreceptors are localized in the various layers of the skin where they detect a wide range of mechanical stimuli, including light brush, stretch, vibration and noxious pressure. This variety of stimuli is matched by a diverse array of specialized mechanoreceptors that respond to cutaneous deformation in a specific way and relay these stimuli to higher brain structures. Studies across mechanoreceptors and genetically tractable sensory nerve endings are beginning to uncover touch sensation mechanisms. Work in this field has provided researchers with a more thorough understanding of the circuit organization underlying the perception of touch. Novel ion channels have emerged as candidates for transduction molecules and properties of mechanically gated currents improved our understanding of the mechanisms of adaptation to tactile stimuli. This review highlights the progress made in characterizing functional properties of mechanoreceptors in hairy and glabrous skin and ion channels that detect mechanical inputs and shape mechanoreceptor adaptation.     

Characterization of the binding properties and retrograde axonal transport of a monoclonal antibody directed against the rat nerve growth factor receptor

We have demonstrated in vitro and in vivo the specific binding of a monoclonal antibody to the rat nerve growth factor (NGF) receptor. Previous work had shown that this antibody, designated 192-IgG, does not compete with NGF for binding to the NGF receptor of PC12 cells, but instead interacts with the receptor to increase NGF binding to PC12 cells (Chandler, C. E., L. M. Parsons, M. Hosang, and E. M. Shooter, 1984, J. Biol. Chem., 259:6882-6889). In the present study, a solid-phase separation assay verified the specific formation of a ternary complex of 192-IgG, the NGF receptor, and NGF: 125I-labeled 192-IgG precipitated from solution only when incubated with both solubilized NGF receptor and NGF covalently linked to a solid phase (Sepharose 4B). Filtration assays using plasma membrane preparations of various tissues showed strict correlation of 125I-192-IgG and 125I-labeled NGF binding; only membranes obtained from superior cervical ganglion bound significant amounts of the monoclonal antibody and NGF. Injection of 125I-192-IgG into the rat anterior eye chamber led to accumulation of intact antibody molecules in the ipsilateral superior cervical ganglion, indicating retrograde axonal transport of 125I-192-IgG from the neuronal termini, located at the iris, to the cell bodies situated in the ganglion. The time course and saturation characteristics of 125I-192-IgG retrograde transport were very similar to those previously reported for 125I-NGF transport, indicating that 192-IgG can be internalized and transported by the same mechanisms as is NGF. Consistent with results of the in vitro binding assays, 192-IgG and NGF failed to compete for retrograde transport and were actually co-transported. Retrograde axonal transport of 192-IgG appears to be species specific, since 125I-192-IgG was transported in the rat, but not in mice, gerbils, hamsters, or guinea pigs. These results establish monoclonal antibody 192-IgG as a specific probe for the rat NGF receptor in vitro and in vivo.     

Obesity-inducing amygdala lesions: examination of anterograde degeneration and retrograde transport

Small lesions centered in the posterodorsal region of the medial amygdala resulted in excessive weight gains in female rats. Unilateral lesions were nearly as effective as bilateral lesions in the first 48 h after surgery (+21 to +32 g). Assessment of lesion damage was done by both qualitative evaluation and by a quantitative grid-point counting method. The critical sites for weight gain were the intra-amygdaloid bed nucleus of the stria terminalis and the posterodorsal medial amygdaloid nucleus. Incidental damage to the overlying globus pallidus was negatively related to weight gain. The cupric silver method for demonstrating axonal degeneration was applied to brains with obesity-inducing lesions. A dense pattern of degenerating terminals was found in the lateral septum, amygdala, ventral striatum, and ventromedial hypothalamus. Degeneration in the paraventricular nucleus of the hypothalamus was scarce or absent. Small retrograde tracer injections made in either the intra-amygdaloid bed nucleus of the stria terminalis or in the posterodorsal medial amygdaloid nucleus labeled cells in the amygdala, lateral septum, and hypothalamus, reciprocating the anterograde projections from the amygdala to these areas. The data suggest that subdivisions of the posterodorsal amygdala participate in the regulation of feeding in a manner that is similar to the better-known role of this part of the brain in mediating reproductive behavior. Although topographical differences may exist within the amygdaloid and hypothalamic subdivisions regulating these two sexually dimorphic behaviors, the relays engaged by feeding-related connections and those related to reproduction are remarkably parallel.     

Host innate immune receptors and beyond: making sense of microbial infections

The complexity of the immune system mirrors its manifold mechanisms of host-microbe interactions. A relatively simplified view was posited after the identification of host innate immune receptors that their distinct mechanisms of sensing "microbial signatures" create unique molecular switches to trigger the immune system. Recently, more sophisticated and cooperative strategies for these receptors have been revealed during receptor-ligand interactions, trafficking, and intra- and intercellular signaling, in order to deal with a diverse range of microbes. Continued mapping of the complex networks of host-microbe interactions may improve our understanding of self/non-self discrimination in immunity and its intervention.     

VegT induces endoderm by a self-limiting mechanism and by changing the competence of cells to respond to TGF-beta signals

The maternal determinant VegT is required for both endoderm and mesoderm formation by the Xenopus embryo. An important downstream mediator of VegT action is Xsox17, which has been proposed to be induced in cell-autonomous, then signal-dependent phases. We show that Xsox17 is a direct VegT target, but that direct induction of Xsox17 by VegT is rapidly inhibited. This inhibition is relieved by TGF- beta signalling, to which the future endoderm cell is sensitised by VegT, resulting in the observed dependence on cell contact for maintained Xsox17 expression. We propose that this change in regulation is a consequence of a VegT-induced repressor, inhibiting direct induction of early endoderm markers by VegT, and contributing to the formation of the boundary of the endodermal domain.     

A Ca2+ transport system associated with the plasma membrane of Dictyostelium discoideum is activated by different chemoattractant receptors

Amebae of Dictyostelium exhibit a transient uptake of extracellular Ca2+ approximately 5 s after activation of surface folate or cAMP receptors (Bumann, J., B. Wurster, and D. Malchow. 1984. J. Cell Biol. 98:173-178). To further characterize these Ca2+ entry systems, we analyzed 45Ca2+ uptake by resting and activated amebae. Like the surface chemoreceptors, folate- and cAMP-induced Ca2+ uptake responses were developmentally regulated; the former response was evident in vegetative but not aggregation-competent cells, whereas the latter response displayed the opposite pattern of expression. In contrast, other characteristics of these Ca2(+)-uptake pathways were remarkably similar. Both systems (a) exhibited comparable kinetic properties, (b) displayed a high specificity for Ca2+, and (c) were inhibited effectively by Ruthenium Red, sodium azide, and carbonylcyanide m-chlorophenyl-hydrazone. These results, together with the finding that vegetative cells transformed with a plasmid expressing the surface cAMP receptor exhibit a cAMP-induced Ca2+ uptake, suggest that different chemoreceptors activate a single Ca2+ entry pathway. Additional pharmacological and ion competition studies indicated that receptor-mediated Ca2+ entry probably does not involve a Na+/Ca2+ exchanger or voltage-activated channels. Chemoattractant binding appears to generate intracellular signals that induce activation and adaption of the Ca2(+)-uptake response. Analysis of putative signaling mutants suggests that Ca2+ entry is not regulated by the guanine nucleotide-binding (G) protein subunits G alpha 1 or G alpha 2, or by G protein-mediated changes in intracellular cAMP or guanosine 3,'5'-cyclic monophosphate (cGMP).     

Products of endocytosis and autophagy are retrieved from axons by regulated retrograde organelle transport

Cellular homeostasis in neurons requires that the synthesis and anterograde axonal transport of protein and membrane be balanced by their degradation and retrograde transport. To address the nature and regulation of retrograde transport in cultured sympathetic neurons, I analyzed the behavior, composition, and ultrastructure of a class of large, phase-dense organelles whose movement has been shown to be influenced by axonal growth (Hollenbeck, P. J., and D. Bray. 1987. J. Cell Biol. 105:2827-2835). In actively elongating axons these organelles underwent both anterograde and retrograde movements, giving rise to inefficient net retrograde transport. This could be shifted to more efficient, higher volume retrograde transport by halting axonal outgrowth, or conversely shifted to less efficient retrograde transport with a larger anterograde component by increasing the intracellular cyclic AMP concentration. When neurons were loaded with Texas red- dextran by trituration, autophagy cleared the label from an even distribution throughout the neuronal cytosol to a punctate, presumably lysosomal, distribution in the cell body within 72 h. During this process, 100% of the phase-dense organelles were fluorescent, showing that they contained material sequestered from the cytosol and indicating that they conveyed this material to the cell body. When 29 examples of this class of organelle were identified by light microscopy and then relocated using correlative electron microscopy, they had a relatively constant ultrastructure consisting of a bilamellar or multilamellar boundary membrane and cytoplasmic contents, characteristic of autophagic vacuoles. When neurons took up Lucifer yellow, FITC-dextran, or Texas red-ovalbumin from the medium via endocytosis at the growth cone, 100% of the phase-dense organelles became fluorescent, demonstrating that they also contain products of endocytosis. Furthermore, pulse-chase experiments with fluorescent endocytic tracers confirmed that these organelles are formed in the most distal region of the axon and undergo net retrograde transport. Quantitative ratiometric imaging with endocytosed 8-hydroxypyrene-1,3,6- trisulfonic acid showed that the mean pH of their lumena was 7.05. These results indicate that the endocytic and autophagic pathways merge in the distal axon, resulting in a class of predegradative organelles that undergo regulated transport back to the cell body.     

An analytical approach to the measurement of equilibrium binding constants: application to EGF binding to EGF receptors in intact cells measured by flow cytometry

In ligand binding studies, ligand depletion often limits the accuracy of the results obtained. This problem is approached by employing the simple observation that as the concentration of receptor in the assay is reduced, ligand depletion is also reduced. Measuring apparent K(D)'s of a ligand at multiple concentrations of receptor with extrapolation to infinitely low receptor concentration takes ligand depletion into account and, depending on the binding model employed, yields a K(D) within the defined limits of accuracy. We apply this analysis to the binding of epidermal growth factor (EGF) to the EGF receptor expressed in intact 32D cells, using a homogeneous fluorescein-labeled preparation of EGF and measuring binding by flow cytometry. Binding isotherms were carried out at varying cell densities with each isotherm fit to the generally applied model with two independent binding sites. Examination of the variation in the K(D)'s versus cell density yields a high-affinity site that accounts for 18% of the sites and a lower affinity site that accounts for the remainder. However, further examination of these data suggests that while consistent with each individual isotherm, the simple model of two independent binding sites that is generally applied to EGF binding to the EGF receptor is inconsistent with the changes in the apparent K(D)'s seen across varying cell densities.