INTRODUCTION Autocrine signaling was first described two decades ago as a partner to tumorigenic phenomena (Sporn and Todaro, 1980). In this mode of signaling, cells make both a receptor and the ligand that activates the receptor. Although originally envisioned as a pathological phenomenon, autocrine regulation is now recognized as a mechanism that can regulate cell responses under many different physiological conditions. These include prostate epithelial development (Kim et al., 1999), mammary epithelial development (Li et al., 1992; Wang et al., 1994; Wojcik et al., 1999), immune cell proliferation (Boussiotis et al., 1994; Duprez et al., 1985), tissue response to injury (Campochiaro et al., 1994; Piepkorn et al., 1998; Sporn and Roberts, 1992) and liver regeneration (Bissell et al., 1995; Hioki et al., 1996; Russell et al., 1993). More recently, autocrine signaling has been identified as a major mechanism of receptor cross-talk or ‘transactivation’. In these cases, induction of one receptor system induces the release of an autocrine factor that activates a second type of receptor. Although the physiological significance of transactivation is unclear, it is thought to act as a mechanism to integrate information from multiple receptor systems (Hackel et al., 1999). A prominent example of autocrine signaling is the EGF receptor (EGFR) system. Several different ligands bind to the EGFR including EGF (Carpenter and Wahl, 1990), transforming growth factor α (TGF-α) (Derynck, 1992), amphiregulin (Shoyab et al., 1989), heparin-binding EGF-like growth factor (HB-EGF) (Higashiyama et al., 1991), betacellulin (Shing et al., 1993), and epiregulin (Toyoda et al., 1995). Most cells that depend on the EGFR for proliferation and differentiation also make one or more of the cognate ligands. The EGFR system can be involved in both disease and normal tissue homeostasis, depending on the expression levels of both ligand and receptor. For example, whereas mice knockouts of EGFR show epithelial defects and multi-organ failure (Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgill et al., 1995), over-expression of EGFR ligands can induce liver and mammary tumor formation (Jhappan et al., 1990; Sandgren et al., 1990). Although autocrine signaling is an important regulator of cell 2301 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 <5×10 -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. Key words: Autocrine, EGF, Computational model SUMMARY Quantitative analysis of the EGF receptor autocrine system reveals cryptic regulation of cell response by ligand capture Ann E. DeWitt 1 , Jian Ying Dong 2 , H. Steven Wiley 3 and Douglas A. Lauffenburger 1,4, * 1 Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 2 Newborn Medicine, Children’s Hospital, Boston, MA 02115, USA 3 Fundamental Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA 4 Division of Bioengineering and Environmental Health and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA *Author for correspondence (e-mail: lauffen@mit.edu) Accepted 29 March 2001 Journal of Cell Science 114, 2301-2313 © The Company of Biologists Ltd RESEARCH ARTICLE