title: Quantitative analysis of the EGF receptor autocrine system reveals cryptic regulation of cell response by ligand capture.
abstract: Autocrine signaling is important in normal tissue physiology as well as pathological conditions. It is difficult to analyze these systems, however, because they are both self-contained and recursive. To understand how parameters such as ligand production and receptor expression influence autocrine activity, we investigated a human epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) loop engineered into mouse B82 fibroblasts. We varied the level of ligand production using the tet-off expression system and used metalloprotease inhibitors to modulate ligand release. Receptor expression was varied using antagonistic blocking antibodies. We compared autocrine ligand release with receptor activation using a microphysiometer-based assay and analyzed our data using a quantitative model of ligand release and receptor dynamics. We found that the activity of our autocrine system could be described in terms of a simple ratio between the rate of ligand production (V(LT)) and the rate of receptor production (V(R)). At a V(LT)/V(R) ratio of <0.3, essentially no ligand was found in the extracellular medium, but a significant number of cell receptors (30-40%) were occupied. As the V(LT)/V(R) ratio increased from 0.3 towards unity, receptor occupancy increased and significant amounts of ligand appeared in the medium. Above a V(LT)/V(R) ratio of 1.0, receptor occupancy approached saturation and most of the released ligand was lost into the medium. Analysis of human mammary epithelial cells showed that a V(LT)/V(R) ratio of < 5 x 10(-4)was sufficient to evoke >20% of a maximal proliferative response. This demonstrates that natural autocrine systems can be active even when no ligand appears in the extracellular medium.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: DeWitt
authors.forename: J Y
authors.initials: JY
authors.lastname: Dong
authors.forename: H S
authors.initials: HS
authors.lastname: Wiley
authors.forename: D A
authors.initials: DA
authors.lastname: Lauffenburger
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D019898:Autocrine Communication / Q000187:drug effects; D001940:Breast / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008954:Models, Biological; D014162:Transfection
chemical_list: D000906:Antibodies; D008024:Ligands; D004815:Epidermal Growth Factor; D066246:ErbB Receptors