title: Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor.
abstract: We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.
authors.affiliation: Department of Biological Sciences and Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37235, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Wilkinson
authors.forename: R A
authors.initials: RA
authors.lastname: Stein
authors.forename: C A
authors.initials: CA
authors.lastname: Guyer
authors.forename: J M
authors.initials: JM
authors.lastname: Beechem
authors.forename: J V
authors.initials: JV
authors.lastname: Staros
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D007377:Interleukin-3 / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D008322:Mammals; D051379:Mice; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013314:Stress, Mechanical; D014162:Transfection
chemical_list: D017077:Culture Media, Conditioned; D007377:Interleukin-3; D007457:Iodine Radioisotopes; D008024:Ligands; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors