Ligand-receptor interactions in the membrane of cultured cells monitored by fluorescence correlation spectroscopy.
We investigated the specific binding of epidermal growth factor (EGF) to its membrane-bound receptors in cultured cells. The specificity of the binding was attested by the consistent displacement of bound rhodamine-labeled EGF (Rh-EGF) 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 Rh-EGF binding, demonstrating no cross-reaction. 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(ass) 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(diss) = 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 ligand-receptor interactions in cell cultures. This is a step forward toward large-scale drug screening in cell cultures.
Pramanik
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A
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Rigler
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R
D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D008024:Ligands; D012235:Rhodamines; D004815:Epidermal Growth Factor; D066246:ErbB Receptors