In T cells, activation of the guanine-nucleotide-binding proteins encoded by the p21ras proto-oncogenes is a common response to triggering of the T-cell antigen receptor, the adhesion molecule CD2 ...and the receptor for the cytokine interleukin 2. This article by Julian Downward and colleagues describes the mechanisms of p21ras regulation and the potential function of p21ras in T cells, and discusses the evidence that multiple intracellular pathways may be involved in the coupling of cell surface receptors to p21ras.
She proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have ...studied the role of She in insulin- and EGF-induced activation of p21
ras
in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21
ras
. Analysis of She immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in She tyrosine phosphorylation and (ii) She antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of She and the presence of Grb2 and mSOS in She immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, She could be detected as well. Furthermore, She immune complexes contained guanine nucleotide exchange activity toward p21
ras
in vitro. From these results, we conclude that after insulin and EGF treatment, She associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21
ras
In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with She was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-mSOS with She may be an important event in insulin-induced, mSOS-mediated activation of p21
ras
.
The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the ...specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161 was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.
A region of the primary amino acid sequence of the epidermal growth factor receptor (EGF) protein‐tyrosine kinase, which is involved in ATP binding, was identified using chemical modification and ...immunological techniques. EGF receptor was 14C‐labelled with the ATP analogue 5′‐p‐fluorosulphonylbenzoyladenosine and from a tryptic digest a single radiolabelled peptide was isolated. The amino acid sequence was determined to be residues 716–724 and hence lysine residue 721 is located within the ATP‐binding site. Antisera were elicited in rabbits to a synthetic peptide identical to residues 716–727 of the EGF receptor and the homologous sequence in v‐erb B transforming protein from avian erythroblastosis virus. The affinity‐purified antibodies precipitated human ECF receptor from A431 cells and placenta, and the v‐erb B protein from erythroblasts. The antibodies inhibited EGF‐stimulated receptor protein‐tyrosine kinase autophosphorylation and phosphorylation of an exogenous peptide substrate containing tyrosine. The antibodies did not immunoprecipitate the transforming proteins pp60v‐src or P120gag‐abl or cAMP‐dependent protein kinase, proteins which have homologous but not identical sequences surrounding the lysine residue within the ATP‐binding site, nor did they react with the platelet‐derived growth factor receptor. The antibodies had no effect on the kinase activity of purified v‐abl protein in solution. The antibodies may therefore be a specific inhibitor of the tyrosine kinase of the EGF receptor.
The human epidermal growth factor receptor has been purified and partial amino acid sequence obtained. A synthetic oligonucleotide was used to select complementary DNA clones from placental and A431 ...clone banks. The nucleotide sequence of a 5.8 kilobase transcript was determined and used to predict the total amino acid sequence of the receptor. We have predicted a model for the receptor which has an external ligand binding domain of 621 amino acids, a transmembrane region of 23 amino acids, and a cytoplasmic domain of 542 amino acids having protein tyrosine kinase activity. The kinase autophosphorylation sites have been mapped onto the primary amino acid sequence. Analysis of protein sequence databases have shown that the erb-B oncogene of avian erythroblastosis virus has acquired part of the avian EGF receptor gene. The hypothesis has been proposed that transformation by this virus is the result of expression of a truncated EGF receptor which lacks the majority of the EGF binding domain and delivers a continuous proliferation signal to transformed cells. We describe here the production of polyclonal and monoclonal antibodies to selected synthetic peptides from the EGF receptor and v-erb B sequences. Antisera to sequences encompassing the three major sites of autophosphorylation and the putative ATP binding site all recognize the native EGF receptor molecule. We have used these reagents to test our model of EGF receptor structure and v-erb B function.
•Plackett-Burman (PB) is a practical design for evaluating uncertain parameters.•Two-Level Full Factorial (2 L FF) is an expensive design for simulation experiments.•Predictions of polynomial models ...built out of PB and 2 L FF designs are comparable.•Forecasts of PB polynomial model is close to Wairakei's actual average.•Wairakei could sustain 353 MWe at least for the next 40 years.
Uncertainty quantification of power (MWe) prediction from a geothermal numerical model is time-consuming. Existing techniques and workflows are still being tested. Our study focuses on using the Experimental Design (ED) and Response Surface Methodology (RSM) framework as an alternative method to facilitate probabilistic MWe estimation. This study involves two types of Experimental Design: the two-level Full Factorial and Plackett Burman, and the investigation is divided into two parts. The first part investigated the difference in the prediction of the Wairakei geothermal field power potential using the polynomial model built from the 64 simulation runs based on the two-level Full Factorial design and the 12 simulation runs based on the Plackett-Burman configuration. The second section compared the MWe predictions using the Plackett-Burman design and the volumetric stored heat method. The simulated MWe potential of Wairakei for 50 years is comparable for both the two-level Full Factorial and Plackett-Burman designs, indicating the latter as a practical design for building a proxy model of a numerical model. Furthermore, the MWe prediction of the polynomial model created based on the ED Framework closely resembles the actual production capacity of Wairakei compared to the volumetric stored heat method. The results based on the proxy modelling further indicate that the 353 MWe production generation could be sustainable at least for another 40 years.
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