Ligand-Receptor Interactions in the Membrane of Cultured Cells Monitored by Fluorescence Correlation Spectroscopy
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Aladdin Pramanik
Abstract
We investigated the specific binding of epidermal growth factor (EGF) to its membranebound receptors in cultured cells. The specificity of the binding was attested by the consistent displacement of bound rhodaminelabeled EGF (RhEGF) following addition of 1000-fold molar excess of unlabeled EGF. The binding specificity of EGF was further confirmed when vascular EGF was unable to displace RhEGF binding, demonstrating no crossreaction. Evidence for the specific interactions was verified by an equilibrium saturation binding experiment. EGF binding to the cell membranes is saturated at nanomolar concentration. The Scatchard plots show a binding process with K of 1.5 x 10[9]M[-1]. The dissociation kinetics follow a single exponential function characteristic for a relatively slow dissociation process with k = 2.9 x 10[-4] s[-1]. The appearance of two binding complexes through the distribution of diffusion times may suggest that these are representatives of two different forms or subtypes of EGF receptors. This study is of pharmaceutical significance as it provides evidence that fluorescence correlation spectroscopy can be used as a rapid technique for studying ligandreceptor interactions in cell cultures. This is a step forward toward largescale drug screening in cell cultures.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
Articles in the same Issue
- Highlight: Fluorescence Correlation Spectroscopy
- Heuristic Statistical Analysis of Fluorescence Fluctuation Data with Bright Spikes: Application to Ligand Binding to the Human Serotonin Receptor Expressed in Escherichia coli Cells
- Fluorescence Correlation Spectroscopy as a Tool to Investigate Single Molecule Probe Dynamics in Thin Polymer Films
- Ligand-Receptor Interactions in the Membrane of Cultured Cells Monitored by Fluorescence Correlation Spectroscopy
- Fluorescence Fluctuation Analysis for the Study of Interactions between Oligonucleotides and Polycationic Polymers
- Determination of the Net Exchange Rate of Tubulin Dimer in Steady-State Microtubules by Fluorescence Correlation Spectroscopy
- UV-Fluorescence Correlation Spectroscopy of 2-Aminopurine
- Isolation of Two cDNAs Encoding Functional Human Cytoplasmic Cysteinyl-tRNA Synthetase
- Involvement of Intracellular Ca2+in the Regulation of Bovine Leukemia Virus Expression
- Nucleotide-Binding Sites in the Functional Unit of Sarcoplasmic Reticulum Ca2+-ATPase as Studied by Photoaffinity Spin-Labeled 2-N3-SL-ATP
- Interaction between Lipopolysaccharide (LPS), LPS-Binding Protein (LBP), and Planar Membranes
- Differential Binding of Urokinase and Peptide Antagonists to the Urokinase Receptor: Evidence from Characterization of the Receptor in Four Primate Species
- Effects of Cysteine to Serine Substitutions in the Two Inter-Chain Disulfide Bonds of Insulin
- Tricorn Protease in Bacteria: Characterization of the Enzyme from Streptomyces coelicolor
- Structures of Tryparedoxins Revealing Interaction with Trypanothione
- Quantitative Two-Color Fluorescence Cross-Correlation Spectroscopy in the Analysis of Polymerase Chain Reaction
- Fluorescence Correlation Spectroscopy as a New Method for the Investigation of Aptamer/Target Interactions
- Localization of Lysobisphosphatidic Acid-Rich Membrane Domains in Late Endosomes
- Fluorescence Correlation Spectroscopy for the Characterisation of Drug Delivery Systems
- Accessing Molecular Dynamics in Cells by Fluorescence Correlation Spectroscopy
- Tailor-Made Dyes for Fluorescence Correlation Spectroscopy (FCS)
- Synergistic Inhibition of the Glucocorticoid Receptor by Radicicol and Benzoquinone Ansamycins
Articles in the same Issue
- Highlight: Fluorescence Correlation Spectroscopy
- Heuristic Statistical Analysis of Fluorescence Fluctuation Data with Bright Spikes: Application to Ligand Binding to the Human Serotonin Receptor Expressed in Escherichia coli Cells
- Fluorescence Correlation Spectroscopy as a Tool to Investigate Single Molecule Probe Dynamics in Thin Polymer Films
- Ligand-Receptor Interactions in the Membrane of Cultured Cells Monitored by Fluorescence Correlation Spectroscopy
- Fluorescence Fluctuation Analysis for the Study of Interactions between Oligonucleotides and Polycationic Polymers
- Determination of the Net Exchange Rate of Tubulin Dimer in Steady-State Microtubules by Fluorescence Correlation Spectroscopy
- UV-Fluorescence Correlation Spectroscopy of 2-Aminopurine
- Isolation of Two cDNAs Encoding Functional Human Cytoplasmic Cysteinyl-tRNA Synthetase
- Involvement of Intracellular Ca2+in the Regulation of Bovine Leukemia Virus Expression
- Nucleotide-Binding Sites in the Functional Unit of Sarcoplasmic Reticulum Ca2+-ATPase as Studied by Photoaffinity Spin-Labeled 2-N3-SL-ATP
- Interaction between Lipopolysaccharide (LPS), LPS-Binding Protein (LBP), and Planar Membranes
- Differential Binding of Urokinase and Peptide Antagonists to the Urokinase Receptor: Evidence from Characterization of the Receptor in Four Primate Species
- Effects of Cysteine to Serine Substitutions in the Two Inter-Chain Disulfide Bonds of Insulin
- Tricorn Protease in Bacteria: Characterization of the Enzyme from Streptomyces coelicolor
- Structures of Tryparedoxins Revealing Interaction with Trypanothione
- Quantitative Two-Color Fluorescence Cross-Correlation Spectroscopy in the Analysis of Polymerase Chain Reaction
- Fluorescence Correlation Spectroscopy as a New Method for the Investigation of Aptamer/Target Interactions
- Localization of Lysobisphosphatidic Acid-Rich Membrane Domains in Late Endosomes
- Fluorescence Correlation Spectroscopy for the Characterisation of Drug Delivery Systems
- Accessing Molecular Dynamics in Cells by Fluorescence Correlation Spectroscopy
- Tailor-Made Dyes for Fluorescence Correlation Spectroscopy (FCS)
- Synergistic Inhibition of the Glucocorticoid Receptor by Radicicol and Benzoquinone Ansamycins
