NGF stimulation of erk phosphorylation is impaired by a point mutation in the transmembrane domain of trkA receptor.

The nerve growth factor (NGF) trkA receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic tyrosine kinase region by a single transmembrane domain (TMD). To explore the role of TMD in the process of receptor activation, we substituted the hydrophobic amino-acid residue valine 432 with the charged amino-acid glutamic acid (designated V432E mutant) by utilizing in vitro site-directed mutagenesis. NIH 3T3 cells lacking endogenous NGF receptors were stably transfected with a pRc/CMV vector carrying either wild-type (trkA) or mutated (V432E) receptors. Stable transfectants were shown, using 125I-NGF binding and Western-blot analysis, to express the trkA recombinant receptors. Scatchard analysis revealed similar affinity for NGF in wild-type and V432E receptors. Although the level of basal trkA receptor tyrosine phosphorylation was higher in the mutant than in the wild-type, NGF stimulation of WT 11 and V432E transfectants resulted in a rapid increase in receptor tyrosine phosphorylation and of its intracellular adaptor protein SHC. In contrast to WT 11, V432E mutants showed very low levels of NGF-, and moderate levels of FGF-induced erks phosphorylation, respectively. Collectively, these findings suggest that a single substitution (V432E) in the trkA TMD results in a selective impairment of trkA-mediated erks signaling pathway.

Expression of human p140trk receptors in p140trk-deficient, PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis.

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140trk. These biological effects of NGF depend upon the signal-mediating function of p140trk substrates which are likely to differ from cell to cell. To define p140trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140trk cDNA sequence. Two stably transfected clones, one expressing p140trk with higher affinity (PC12EN-trk3; Kd 57.4 pM, Bmax 9.7 pmole/mg) and one expressing p140trk with a lower affinity (PC12EN-trk1; Kd 392.4 pM, Bmax 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140trk receptors show slow NGF-dissociation kinetics, are resistant to trypsin or Triton X-100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140trk receptors. NGF stimulates p140trk tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70S6k, SNT, and phospholipase C gamma, demonstrating that the major NGF-stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [3H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140trk receptor substrates.

Down-regulation of epidermal growth factor receptors by nerve growth factor in PC12 cells is p140(trk)-, Ras-, and Src-dependent.

Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor receptors during the differentiation of PC12 cells. This process is characterized by a progressive decrease in epidermal growth factor (EGF) receptor level measured by 125I-EGF binding, tyrosine phosphorylation, and Western blotting. Treatment of the cells with NGF for 5 days produces a 95% reduction in the amount of [35S]methionine-labeled EGF receptors. This down-regulation does not occur in PC12nnr5 cells, which lack the p140(trk) NGF receptor. However, in PC12nnr5 cells stably transfected with p140(trk), the NGF-induced heterologous down-regulation of EGF receptors is reconstituted in part. NGF-induced heterologous down-regulation, but not EGF-induced homologous down-regulation of EGF receptors, is blocked in Ras- and Src-dominant-negative PC12 cells. Treatment with either pituitary adenylate cyclase-activating peptide (PACAP) or staurosporine stimulates neurite outgrowth in PC12 cell variants, but neither induces down-regulation of EGF receptors. NGF treatment of PC12 cells in suspension induces down-regulation of EGF receptors in the absence of neurite outgrowth. These results strongly suggest a p140(trk)-, Ras- and Src-dependent mechanism of NGF-induced down-regulation of EGF receptors and separate this process from NGF-induced neurite outgrowth in PC12 cells.

Emulsification preparation of calcium alginate beads in the presence of sequesterant.

A biocompatible emulsification method has been developed for microencapsulation of live cells and enzymes within a calcium alginate matrix. Fabrication of alginate beads was achieved by premixing a sequestering agent (sodium polyphosphate) and the calcium source (calcium sulphate) with the hydrogel monomer prior to the introduction to the oil phase. The competition between the sequesterant and sodium alginate in binding the available calcium ions results in a slowing down of the rate of polymerization and thereby lead to a successful calcium alginate bead formation. The mean diameter of the fabricated beads may be easily controlled by employing soy bean lecithin as an emulsifier. The polymerization time in this process may vary between 3 and 35 min depending on the ratio of sequesterant to that of calcium source at constant sodium alginate concentration. This preparation method avoids the use of pH extremes at all times and therefore is particularly suitable for encapsulating pH-sensitive cells and enzymes.

Liposome-encapsulated alginate: controlled hydrogel particle formation and release.

Large unilamellar liposomes of dipalmitoyl phosphatidylcholine have been employed as reaction sites for calcium alginate gelation. Encapsulation of sodium alginate was accomplished by extrusion of phospholipid dispersions through polycarbonate filter of uniform pore size followed by incubation with high concentrations of calcium chloride. The diffusion of calcium into the liposome interior resulted in alginate gelatin within the liposome. Detergent treatment of the liposomes resulted in solubilization of the lipid bilayer with subsequent release of the alginate beads which were measured by laser light scattering and electron microscopy. The release profiles of both the liposomes with entrapped alginate beads and the alginate beads (released by detergent treatment of the liposomes) were determined using cytochrome-c as a marker for release. The release profiles show a rapid release of cytochrome-c over the first 2 for both preparations with slower subsequent release rates for the liposomes with encapsulated alginate beads. The similar early profile may be due to the release of cytochrome-c from bound calcium alginate adsorbed to the outer leaflet of the liposome. This is also supported by calorimetric results which indicate a marked reduction in the enthalpy of the main gel to liquid crystalline phase transition of multilamellar and large unilamellar dispersions of the lipid. This method results in the fabrication of easily controlled, unimodal submicron hydrogel particles which may be used for controlled-release applications.

Kinetics and activity distribution of urease coencapsulated with hemoglobin within polyamide membranes.

A 91.5% mass yield of urease and hemoglobin (Hb), co-encapsulated within polyamide membranes, was determined spectrophotometrically. The specific activity yield of microencapsulation was 84%, twofold higher than values previously reported, as a result of optimization of encapsulation conditions. The kinetic parameters and pH activity profiles of intracapsular urease were determined to be similar to those corresponding to the free enzyme. Similar activities were also observed for intact and microcapsule homogenate, indicating minimal mass transfer and diffusional limitation. The active configuration of the enzyme appears to remain intact upon microencapsulation. The application of a kinetic model for encapsulated urease further indicated that the kinetics were reaction-controlled with minimal mass transfer restrictions.

Activity and distribution of urease following microencapsulation within polyamide membranes.

Urease was microencapsulated by forming a semipermeable polyamide membrane around aqueous microdroplets (266 microns mean diameter) containing the soluble enzyme. The yield of the interfacial polymerization technique, determined spectrophotometrically, was 83% of the original enzyme on a mass basis, resulting in a final intracapsular urease concentration of 62.3 mg ml-1 or 0.1 mM. Similar absorption spectra of broken and intact microcapsules suggested that spectrophotometry may be applied in performing direct studies on the intact microcapsules. The high activity yield of urease microcapsules relative to the mass of entrapped enzyme (92.5%) indicated minimal effects of mass transfer limitation. The mass of active urease incorporated into the nylon membrane represented 6% of the encapsulated enzyme activity. The soluble intracapsular enzyme fraction (94%) was released into solution upon rupture of the membrane. A complete mass and activity balance of the encapsulated enzyme was achieved.