Quantitative analysis of the EGF receptor autocrine system reveals cryptic regulation of cell response by ligand...

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.