scholarly journals Negative co-operativity in the EGF receptor

2012 ◽  
Vol 40 (1) ◽  
pp. 15-19 ◽  
Author(s):  
Linda J. Pike
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Egf Receptor ◽  
The Other ◽  
New Approach ◽  
X Ray ◽  
High Affinity ◽  
Binding Data ◽  
Epidermal Growth ◽  
Scatchard Plots

Scatchard analyses of the binding of EGF (epidermal growth factor) to its receptor (EGFR) yield concave up Scatchard plots, indicative of some type of heterogenity in ligand-binding affinity. This was typically interpreted as being due to the presence of two independent binding sites: one of high affinity representing ≤10% of the receptor population, and one of low affinity making up the bulk of the receptors. However, the concept of two independent binding sites is difficult to reconcile with the X-ray structures of the dimerized EGFR that show symmetrical binding of the two ligands. A new approach to the analysis of 125I-EGF-binding data combined with the structure of the singly-occupied Drosophila EGFR have now shown that this heterogeneity is due to the presence of negative co-operativity in the EGFR. Concerns that negative co-operativity precludes ligand-induced dimerization of the EGFR confuse the concepts of linkage and co-operativity. Linkage refers to the effect of ligand on the assembly of dimers, whereas co-operativity refers to the effect of ligand binding to one subunit on ligand binding to the other subunit within a preassembled dimer. Binding of EGF to its receptor is positively linked with dimer assembly, but shows negative co-operativity within the dimer.

scholarly journals Cross talk among tyrosine kinase receptors in PC12 cells: desensitization of mitogenic epidermal growth factor receptors by the neurotrophic factors, nerve growth factor and basic fibroblast growth factor.

1993 ◽  
Vol 4 (7) ◽  
pp. 737-746 ◽  
Author(s):  
I Mothe ◽  
R Ballotti ◽  
S Tartare ◽  
A Kowalski-Chauvel ◽  
E Van Obberghen
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Fibroblast Growth Factor ◽  
Pc12 Cells ◽  
Binding Sites ◽  
Egf Receptor ◽  
Fibroblast Growth ◽  
High Affinity ◽  
Epidermal Growth

We have studied the effects of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) on epidermal growth factor (EGF) binding to PC12 cells. We show that NGF and bFGF rapidly induce a reduction in 125I-EGF binding to PC12 cells in a dose-dependent manner. This decrease amounts to 50% for NGF and 35% for bFGF. Both factors appear to act through a protein kinase C(PKC)-independent pathway, because their effect persists in PKC-downregulated PC12 cells. Scatchard analysis indicates that NGF and bFGF decrease the number of high affinity EGF binding sites. In addition to their effect on EGF binding, NGF and bFGF activate in intact PC12 cells one or several serine/threonine kinases leading to EGF receptor threonine phosphorylation. Using an in vitro phosphorylation system, we show that NGF- or bFGF-activated extracellular regulated kinase 1 (ERK1) is able to phosphorylate a kinase-deficient EGF receptor. Phosphoamino acid analysis indicates that this phosphorylation occurs mainly on threonine residues. Furthermore, two comparable phosphopeptides are observed in the EGF receptor, phosphorylated either in vivo after NGF treatment or in a cell-free system by NGF-activated ERK1. Finally, a good correlation was found between the time courses of ERK1 activation and 125I-EGF binding inhibition after NGF or bFGF treatment. In conclusion, in PC12 cells the NGF- and bFGF-stimulated ERK1 appears to be involved in the induction of the threonine phosphorylation of the EGF receptor and the decrease in the number of high affinity EGF binding sites.

scholarly journals Effect of tunicamycin treatment on ligand binding to the erythropoietin receptor: conversion from two classes of binding sites to a single class

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2503-2510 ◽  
Author(s):  
M Nagao ◽  
S Matsumoto ◽  
S Masuda ◽  
R Sasaki
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Single Phase ◽  
Saturation Curve ◽  
Biochemical Basis ◽  
Bhk Cells ◽  
Tunicamycin Treatment ◽  
High Affinity ◽  
Scatchard Plots ◽  
Asparagine Residue

Scatchard analyses of erythropoietin (EPO) binding to its receptor (EPO- R) have shown that some erythroid cells display a biphasic nature of the ligand-saturation curve, indicating the presence of two classes of binding sites with different affinities. The biochemical basis accounting for this observation is unknown. We found that the culture of a human erythroleukemia cell line with tunicamycin, an inhibitor of N-glycosylation, converted the biphasic Scatchard plot to a single phase with high-affinity sites. Scatchard plots of baby hamster kidney (BHK) cells that had been engineered to express cloned mouse EPO-R were also biphasic and the plots of cells cultured with tunicamycin became a single phase with high-affinity sites. Mouse EPO-R is glycosylated at one asparagine residue in the extracellular region. The mutant EPO-R, in which asparagine residue responsible for N-glycosylation was replaced with glutamine residue, was expressed on BHK cells. Unexpectedly, mutant EPO-R was similar in ligand binding to wild-type EPO-R. BHK cells that expressed mutant EPO-R showed biphasic Scatchard plots that were converted to single-phase plots with only high-affinity sites by tunicamycin treatment. These results indicate that the N- linked sugar of EPO-R is not involved in the manifestation of two classes of binding sites, and that there is a yet unidentified glycoprotein crucial for the ligand-saturation characteristics of EPO-R.

scholarly journals Effect of tunicamycin treatment on ligand binding to the erythropoietin receptor: conversion from two classes of binding sites to a single class

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2503-2510 ◽  
Author(s):  
M Nagao ◽  
S Matsumoto ◽  
S Masuda ◽  
R Sasaki
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Single Phase ◽  
Saturation Curve ◽  
Biochemical Basis ◽  
Bhk Cells ◽  
Tunicamycin Treatment ◽  
High Affinity ◽  
Scatchard Plots ◽  
Asparagine Residue

Abstract Scatchard analyses of erythropoietin (EPO) binding to its receptor (EPO- R) have shown that some erythroid cells display a biphasic nature of the ligand-saturation curve, indicating the presence of two classes of binding sites with different affinities. The biochemical basis accounting for this observation is unknown. We found that the culture of a human erythroleukemia cell line with tunicamycin, an inhibitor of N-glycosylation, converted the biphasic Scatchard plot to a single phase with high-affinity sites. Scatchard plots of baby hamster kidney (BHK) cells that had been engineered to express cloned mouse EPO-R were also biphasic and the plots of cells cultured with tunicamycin became a single phase with high-affinity sites. Mouse EPO-R is glycosylated at one asparagine residue in the extracellular region. The mutant EPO-R, in which asparagine residue responsible for N-glycosylation was replaced with glutamine residue, was expressed on BHK cells. Unexpectedly, mutant EPO-R was similar in ligand binding to wild-type EPO-R. BHK cells that expressed mutant EPO-R showed biphasic Scatchard plots that were converted to single-phase plots with only high-affinity sites by tunicamycin treatment. These results indicate that the N- linked sugar of EPO-R is not involved in the manifestation of two classes of binding sites, and that there is a yet unidentified glycoprotein crucial for the ligand-saturation characteristics of EPO-R.

scholarly journals Molecular Recognition: Perspective and a New Approach

Sensors ◽  
2021 ◽  
Vol 21 (8) ◽  
pp. 2757
Author(s):  
W. Rudolf Seitz ◽  
Casey J. Grenier ◽  
John R. Csoros ◽  
Rongfang Yang ◽  
Tianyu Ren
Keyword(s):  
Molecular Recognition ◽  
Molecularly Imprinted Polymers ◽  
Binding Sites ◽  
Binding Kinetics ◽  
Molecularly Imprinted ◽  
New Approach ◽  
Imprinted Polymers ◽  
High Affinity ◽  
Water Blocking ◽  
High Level

This perspective presents an overview of approaches to the preparation of molecular recognition agents for chemical sensing. These approaches include chemical synthesis, using catalysts from biological systems, partitioning, aptamers, antibodies and molecularly imprinted polymers. The latter three approaches are general in that they can be applied with a large number of analytes, both proteins and smaller molecules like drugs and hormones. Aptamers and antibodies bind analytes rapidly while molecularly imprinted polymers bind much more slowly. Most molecularly imprinted polymers, formed by polymerizing in the presence of a template, contain a high level of covalent crosslinker that causes the polymer to form a separate phase. This results in a material that is rigid with low affinity for analyte and slow binding kinetics. Our approach to templating is to use predominantly or exclusively noncovalent crosslinks. This results in soluble templated polymers that bind analyte rapidly with high affinity. The biggest challenge of this approach is that the chains are tangled when the templated polymer is dissolved in water, blocking access to binding sites.

scholarly journals Adenosine Receptors of Cerebral Microvessels and Choroid Plexus

1986 ◽  
Vol 6 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Rajesh N. Kalaria ◽  
Sami I. Harik
Keyword(s):  
Cerebral Cortex ◽  
Ligand Binding ◽  
Choroid Plexus ◽  
Adenosine Receptors ◽  
Binding Sites ◽  
Specific Binding ◽  
Cerebral Microvessels ◽  
High Affinity ◽  
Receptor Sites ◽  
Ligand Binding Sites

We studied, by ligand binding methods, the two adenosine receptors, A, and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]l-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'- N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity ( Kd ∼ 250 n M) and low-affinity ( Kd ∼ 1–2 μ M) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these microvessels.

Interaction of epidermal growth factor with receptors on human and porcine thyroid membranes

1984 ◽  
Vol 102 (1) ◽  
pp. 57-61 ◽  
Author(s):  
H. Humphries ◽  
S. MacNeil ◽  
D. S. Munro ◽  
S. Tomlinson
Keyword(s):  
Enzyme Activity ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Sites ◽  
Affinity Constant ◽  
Maximal Inhibition ◽  
High Affinity ◽  
Epidermal Growth ◽  
And Function ◽  
Bovine Tsh

ABSTRACT Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1·4 × 109 l/mol); the density of binding sites was low compared with the TSH receptor. At 37 °C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0·5 nmol/l) using 0·5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity. J. Endocr. (1984) 102, 57–61

Generation of an agonistic binding site for blockers of the M3 muscarinic acetylcholine receptor

2008 ◽  
Vol 412 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Doreen Thor ◽  
Angela Schulz ◽  
Thomas Hermsdorf ◽  
Torsten Schöneberg
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Binding Sites ◽  
Expression System ◽  
Muscarinic Acetylcholine Receptor ◽  
Intracellular Loop ◽  
Inverse Agonists ◽  
Yeast Expression System ◽  
High Affinity

GPCRs (G-protein-coupled receptors) exist in a spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and inverse agonists respectively. Because ligand binding of agonists and inverse agonists often occurs in a competitive manner, one can assume an overlap between both binding sites. Only a few studies report mutations in GPCRs that convert receptor blockers into agonists by unknown mechanisms. Taking advantage of a genetically modified yeast strain, we screened libraries of mutant M3Rs {M3 mAChRs [muscarinic ACh (acetylcholine) receptors)]} and identified 13 mutants which could be activated by atropine (EC50 0.3–10 μM), an inverse agonist on wild-type M3R. Many of the mutations sensitizing M3R to atropine activation were located at the junction of intracellular loop 3 and helix 6, a region known to be involved in G-protein coupling. In addition to atropine, the pharmacological switch was found for other M3R blockers such as scopolamine, pirenzepine and oxybutynine. However, atropine functions as an agonist on the mutant M3R only when expressed in yeast, but not in mammalian COS-7 cells, although high-affinity ligand binding was comparable in both expression systems. Interestingly, we found that atropine still blocks carbachol-induced activation of the M3R mutants in the yeast expression system by binding at the high-affinity-binding site (Ki ∼10 nM). Our results indicate that blocker-to-agonist converting mutations enable atropine to function as both agonist and antagonist by interaction with two functionally distinct binding sites.

Transmetalation reactions with nitrogen-containing "pincer"-class ligands on platinum(II) centers

2001 ◽  
Vol 79 (5-6) ◽  
pp. 709-718 ◽  
Author(s):  
Martin Albrecht ◽  
Stuart L James ◽  
Nora Veldman ◽  
Anthony L Spek ◽  
Gerard van Koten
Keyword(s):  
Ligand Binding ◽  
Platinum Complexes ◽  
Binding Mode ◽  
Unit Cell ◽  
The Other ◽  
Pincer Ligand ◽  
X Ray ◽  
Spectroscopic Analyses ◽  
Structure Determinations ◽  
Independent Molecules

The transmetalation reaction of the aryllithium compound [Li(NCN)]2 (NCN is the monoanionic "pincer" ligand [C6H3(CH2NMe2)2-2,6]-) with the cyclometalated arylplatinum complex [PtCl(NCN)] afforded the bisaryl platinum(II) complex [Pt(NCN)2] (3) containing one η3-N,C,N-terdentate and the other η1-C-monodentate-bonded pincer ligand. Spectroscopic analyses on 3 suggest that η3 to η1 interconversion of the ligand binding mode (or vice versa) is inhibited. Two independent X-ray structure determinations on crystals of 3 revealed the existence of a rare polymorph containing one and three crystallographically independent molecules, respectively, in the unit cell. A similar transmetalation reaction with lithium and platinum complexes containing heteroleptic NCNRR' ligands (NCNRR' is [C6H3(CH2NRR')-2,6]- with R = R' = Me or R = Me, R' = Et) pointed to the formation of a heterodinuclear cationic bisaryl platinum lithium species as an intermediate of a preequilibrium to the final transmetalation products, involving, rapid transcyclometalation (TCM) reactions. These TCM reactions comprise the exchange of the monoanionic NCNRR' ligands between the platinum(II) and lithium centers. A consequence of the latter properties is that the strong Pt—N bonds in [PtX(NCN)] complexes are considerably weakened by the presence of Li+ cations.Key words: transmetalation, transcyclometalation (TCM), platinum, bisaryl complex, polymorphism.

Sign in / Sign up

Export Citation Format

Share Document