Quantitative analysis and modulation of the spatial operation of the EGF receptor autocrine cell signaling system

Abstract

The spatial localization of EGFR autocrine systems is a critical factor for tissue development and homeostasis. It is difficult to analyze these systems, however, because they are both self-contained and recursive. To understand how cellular parameters influence autocrine activity, we investigated a human epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) loop engineered into mouse B82 fibroblasts. We modulated three parameters: ligand secretion, receptor expression and ligand/receptor binding affinity using various molecular and cell biology techniques. We measured autocrine system localization via autocrine ligand release using two different assays: the first indirectly measured the fraction of autocrine ligand captured by measuring ligand escaping the cells and the second measured receptor activation using a microphysiometer-based assay. We analyzed our data using a quantitative model of autocrine cell receptor/ligand 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 (VLT) and the rate of receptor production (VR). At a VLTNR ratio of < 0.3, essentially no ligand was found in the extracellular medium, but a significant number cell receptors (30-40%) were occupied. As the VLTNR ratio increased from 0.3 towards unity, receptor occupancy increased, and significant amounts of ligand appeared in the medium.

(cont.) Above a VLT/VNR ratio of 1.0, receptor occupancy approached saturation and most of the released ligand was lost into the medium. The low affinity autocrine system was less efficient at capturing ligand and had fewer occupied receptors for a given production ratio. The effect of changing the receptor/ligand binding affinity on the fraction ligand capture as a function of the production ratio could be predicted using microphysiometer data and our autocrine cell model. Importantly, our results suggest that cellular processes that control system parameters such as the ratio of ligand production to receptor production and receptor/ligand binding affinity also regulate the overall activity and spatial operation of autocrine systems, thus governing cell behavior.