Receptor overexpression or inhibition alters cell surface dynamics of EGF-EGFR interaction: new insights from real-time single molecule analysis.
Binding of Epidermal growth factor (EGF) to epidermal growth factor receptor (EGFR) in two types of cancer cells (HeLa; 5 x 10(4) EGFR/cell) and MDA-MB-468; 2 x 10(6) EGFR/cell) was studied using Total Internal Reflectance Fluorescence (TIRF) microscopy at single molecule precision. Mathematical modeling of the binding kinetics revealed that cells respond differently to the same concentration of EGF depending on the expression level of EGFR. Compared to Hela, MDA-MB-468 cells show; (a) higher number of pre-formed dimers, (b) improved EGF-EGFR interaction at lower ligand concentrations, and (c) shorter time-lapse between first and second EGF binding to the dimer. Treatment with a pharmacological inhibitor of EGFR, AG1478, produced strikingly different binding kinetics where the extent of pre-formed EGFR dimers increased substantially. Thus, single molecule approaches produce novel, quantitative information on signaling mechanisms of significant biological importance. Surface kinetics could also serve as surrogate markers to predict biological outcome of signaling pathways.
Yu
Chenxu
C
Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN, USA.
Hale
Jacob
J
Ritchie
Kenneth
K
Prasad
Nagendra K
NK
Irudayaraj
Joseph
J
D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D019281:Dimerization; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011799:Quinazolines; D020032:Tyrphostins / Q000494:pharmacology
D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D020032:Tyrphostins; C101044:RTKI cpd; D004815:Epidermal Growth Factor; D066246:ErbB Receptors