scholarly journals Physicochemical characterization of the cytoplasmic domain of the epidermal growth factor receptor and evidence for conformational changes associated with its activation by ammonium sulphate

1995 ◽  
Vol 306 (3) ◽  
pp. 667-678 ◽  
Author(s):  
M Gregoriou ◽  
P F Jones ◽  
J F Timms ◽  
J J Yang ◽  
S E Radford ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Conformational Changes ◽  
Egf Receptor ◽  
Specific Activity ◽  
Physicochemical Characterization ◽  
Cytoplasmic Domain ◽  
Epidermal Growth ◽  
Peptide Phosphorylation

The physiochemical properties of the purified cytoplasmic domain of the epidermal growth factor (EGF) receptor, its self-phosphorylation and peptide phosphorylation activities, and its activation by ammonium sulphate have been studied. Highly efficient purification procedures for the isolation of the recombinant cytoplasmic domain (Met644-Ala1186) of the EGF receptor, expressed in the baculovirus/insect cell system, are described. Physicochemical characterization of the protein included investigation of its isoelectric and hydrodynamic properties, stability, oligomeric status, and secondary structure using far-u.v. circular dichroism. The recombinant protein was not recognized by anti-phosphotyrosine antibodies, unless first self-phosphorylated in vitro. Tryptic phosphopeptide maps of self-phosphorylated recombinant cytoplasmic domain and the EGF-stimulated A431-membrane receptor were very similar, suggesting that the recombinant had similar self-phosphorylation capacity and specificity. The preparations were characterized by high specific activity towards peptide tyrosine phosphorylation. Although the cytoplasmic domain was isolated as a homogeneously monomeric protein, storage at 4 degrees C led to slow, spontaneous aggregation with reduction in specific activity. Both high activity and monomeric state were maintained by storage below 0 degree C. The dependence of the initial rate of self-phosphorylation on protein concentration was consistent with cross-phosphorylation but not with the known oligomerization-induced activation of holoreceptor. The peptide phosphorylation activity was stimulated by Mn2+, Mg2+ and (NH4)2SO4 at high concentrations. The substrate specificity of (NH4)2SO4 activation was studied using synthetic peptides. Self-phosphorylation was inhibited by (NH4)2SO4 in the range 0-0.25 M but activated at 1.0-1.5 M, possibly as a result of ionic and hydrophobic protein interactions respectively. Phosphopeptide maps of cytoplasmic domain phosphorylated in the presence of high (NH4)2SO4 showed that the protein was more extensively phosphorylated than in the absence of salt, or than the native receptor. Far-u.v. circular-dichroism spectra of the cytoplasmic domain changed dramatically at 1 M (NH4)2SO4, raising the possibility that (NH4)2SO4 activates the kinase catalytic domain by inducing conformational changes.

scholarly journals An investigation of the role of Glu-842, Glu-844 and His-846 in the function of the cytoplasmic domain of the epidermal growth factor receptor

1995 ◽  
Vol 308 (1) ◽  
pp. 219-229 ◽  
Author(s):  
J F Timms ◽  
M E M Noble ◽  
M Gregoriou
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Active Site ◽  
Egf Receptor ◽  
Cytoplasmic Domain ◽  
Peptide Substrates ◽  
Variable Loop ◽  
Epidermal Growth ◽  
Peptide Phosphorylation

Activation of several protein kinases is mediated, at least in part, by phosphorylation of conserved Thr or Tyr residues located in a variable loop region, near the active site. In certain kinases, this activation loop also controls access of peptide substrates to the active site. In the corresponding region of the epidermal growth factor (EGF) receptor, a potential phosphorylation site, Tyr-845, does not appear to have a major regulatory role. In order to find out whether this variable loop can modulate the peptide phosphorylation and self-phosphorylation activities of the EGF receptor kinase, we investigated the role of residues around Tyr-845, using site-directed mutagenesis. Multiple sequence alignment showed that residues Glu-842, Glu-844 and His-846 are conserved or nearly conserved in eight members of the EGF receptor family. Mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala were expressed in the baculovirus/insect cell system, purified to near-homogeneity and characterized with respect to their peptide phosphorylation and self-phosphorylation activities. All three mutants were active, and these changes did not affect ATP binding directly. However, all mutations increased the Km(app.) for peptide substrates and MnATP in peptide phosphorylation reactions. The Vmax. for the phosphorylation of peptide RREELQDDYEDD was unaltered, but the Vmax. for self-phosphorylation (with variable [MnATP]) decreased 4-, 2- and 7-fold for mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala respectively, compared with the wild-type. These results suggest that binding of this peptide restored an optimal conformation at the active site that might be impaired by the mutations. A study of the dependence of initial rates of self-phosphorylation on cytoplasmic domain concentration showed that the order of reaction increased with the progress of self-phosphorylation. Both pre-phosphorylation and high concentrations of ammonium sulphate restored maximal or near-maximal levels of self-phosphorylation in the mutants, possibly through compensating conformational changes. A plausible homology model, based on the cyclic AMP-dependent protein kinase catalytic subunit, accommodated the sequence Glu-841-Glu-Lys-Glu as an insertion in the peptide binding loop at the edge of the active site cleft. The model suggests that Glu-844 and His-846 may participate in H-bonding interactions, thus stabilizing the active site region, while Glu-842 does not appear to interact with regions of the catalytic core.

scholarly journals Sulphydryl agents modulate insulin- and epidermal growth factor (EGF)-receptor kinase via reaction with intracellular receptor domains: differential effects on basal versus activated receptors

1993 ◽  
Vol 292 (1) ◽  
pp. 217-223 ◽  
Author(s):  
S Clark ◽  
N Konstantopoulos
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Cytoplasmic Domain ◽  
Receptor Kinase ◽  
Egf Receptors ◽  
Receptor Structure ◽  
Epidermal Growth

Sulphydryl reagents have been shown to produce a variety of effects on insulin-receptor structure and function. However, localization of these effects to specific receptor domains has not been attempted. We have investigated this question with insulin- and epidermal growth factor (EGF)-receptors (both are receptor tyrosine kinases but have different sulphydryl/disulphide structures within the external domain), and the insulin receptor kinase (IRK) protein consisting solely of the insulin-receptor cytoplasmic domain and exhibiting constitutive kinase activity. Results showed a differential response between basal and activated receptors. The physiological reductant GSH stimulated basal receptor autophosphorylation, but was either without effect (EGF) or inhibited (insulin) activated receptors, and occurred without visible reduction of receptor structure. These results contrast with those obtained with dithiothreitol which appears to activate phosphorylation in association with reduction of the extracellular insulin-receptor disulphides, but is without effect on the EGF receptor or the IRK protein. Alkylating agents N-ethylmaleimide (NEM) and iodoacetamide (IAM) had opposing effects on receptor autophosphorylation. However, only in the basal state was IAM able to protect receptors from the inhibitory effect of NEM. Our results suggest that complex sulphydryl interactions can occur within the cytoplasmic domain of insulin- and EGF-receptors to alter receptor kinase activity. The basal and activated state of receptors is not the same with respect to sulphydryl reagent action, possibly due to conformational change in the receptor induced by ligand (insulin, EGF) or constitutive (IRK) activation.

scholarly journals Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility.

1986 ◽  
Vol 103 (2) ◽  
pp. 327-331 ◽  
Author(s):  
E Livneh ◽  
M Benveniste ◽  
R Prywes ◽  
S Felder ◽  
Z Kam ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Diffusion Coefficients ◽  
Egf Receptor ◽  
Lateral Diffusion ◽  
Cytoplasmic Domain ◽  
Protein Kinase Activity ◽  
Lateral Mobility ◽  
Cos Cells ◽  
Epidermal Growth

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.

Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation

1990 ◽  
Vol 10 (6) ◽  
pp. 3048-3055
Author(s):  
S Massoglia ◽  
A Gray ◽  
T J Dull ◽  
S Munemitsu ◽  
H J Kun ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Control Mechanisms ◽  
Egf Receptors ◽  
Intact Cells ◽  
Epidermal Growth

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.

Characterization of a Factor VII Molecule Carrying a Mutation in the Second Epidermal Growth Factor-like Domain

1998 ◽  
Vol 79 (06) ◽  
pp. 1136-1143 ◽  
Author(s):  
Anita Kavlie ◽  
Lars Örning ◽  
Anne Grindflek ◽  
Helge Stormorken ◽  
Hans Prydz
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tissue Factor ◽  
Specific Activity ◽  
Factor Vii ◽  
Wild Type ◽  
Fold Reduction ◽  
Epidermal Growth ◽  
Compound Heterozygotes

SummaryA missense mutation at codon 100 in the second epidermal growth factor-like domain, resulting in Gln100→Arg, was detected in 19 out of 21 available severely factor VII (FVII) deficient patients in Norway. Seventeen patients were homozygous, and the two remaining were compound heterozygotes. In the homozygous patients, FVII antigen was measured to 10-28%, and activity to 0.6-6.5% of that in normal pooled plasma. Recombinant FVII containing the mutation was expressed transiently in CHO cells to a mean antigen level of 57% of the wild type FVII protein, and with a specific activity of 6% of wild type. The mutant protein had a 14-fold reduction in affinity for tissue factor (TF), whereas binding of FX seemed unaffected. In line with the experimental data, molecular modelling of the mutation based on the coordinates of the tissue factor/FVIIa complex showed that substituting arginine for glutamine disrupts the interface between the catalytic and second epidermal growth factor-like domains.

scholarly journals Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation.

1990 ◽  
Vol 10 (6) ◽  
pp. 3048-3055 ◽  
Author(s):  
S Massoglia ◽  
A Gray ◽  
T J Dull ◽  
S Munemitsu ◽  
H J Kun ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Control Mechanisms ◽  
Egf Receptors ◽  
Intact Cells ◽  
Epidermal Growth

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.

scholarly journals Cellular Characterization of Epidermal Growth Factor-expanded Free-floating Neurospheres

2003 ◽  
Vol 51 (1) ◽  
pp. 89-103 ◽  
Author(s):  
Maria V.T. Lobo ◽  
F. Javier M. Alonso ◽  
Carolina Redondo ◽  
Miguel A. López-Toledano ◽  
Enrique Caso ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Cell Types ◽  
Specific Subset ◽  
Cell Processes ◽  
Epidermal Growth ◽  
Dying Cells ◽  
Electron Lucent

Neural stem cells proliferate in liquid culture as cell clusters (neurospheres). This study was undertaken to characterize the epidermal growth factor (EGF)-expanded free-floating neurospheres derived from rat fetal striatum. We examined the ultrastructural and antigenic characteristics of these spheres. They consisted of two cell types, electron-dense and electron-lucent cells. Lucent cells were immunopositive to actin, vimentin, and nestin, whereas dense cells were immunopositive to actin, weakly positive to vimentin, and nestin-negative. Neurospheres contained healthy, apoptotic, and necrotic cells. Healthy cells were attached to each other by adherens junctions. They showed many pseudopodia and occasionally a single cilium. Sphere cells showed phagocytic capability because healthy cells phagocytosed the cell debris derived from dead cells in a particular process that involves the engulfment of dying cells by cell processes from healthy cells. Sphere cells showed a cytoplasmic and a nuclear pool of fibroblast growth factor (FGF) receptors. They expressed E- and N-cadherin, α- and β-catenin, EGF receptor, and a specific subset of FGF receptors. Because sphere cells expressed this factor in the absence of exogenous FGF-2, we propose that they are able to synthesize FGF-2.

scholarly journals Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) Is a Step Separable from Ligand-induced EGFR Signaling

2002 ◽  
Vol 13 (7) ◽  
pp. 2547-2557 ◽  
Author(s):  
Xiaochun Yu ◽  
Kailash D. Sharma ◽  
Tsuyoshi Takahashi ◽  
Ryo Iwamoto ◽  
Eisuke Mekada
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Cytoplasmic Domain ◽  
Erythropoietin Receptor ◽  
Mutant Form ◽  
Chimeric Receptor ◽  
Heparin Binding ◽  
Egfr Ligands ◽  
Epidermal Growth

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between835Ala and918Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events.

Sign in / Sign up

Export Citation Format

Share Document