Mapping and modeling receptor topography during signal transduction

Bridget S. Wilson (University of New Mexico)

Abstract

The ErbB family of growth factor receptors are widely expressed by cells of epithelial and mesenchymal lineages. EGFR and ErbB2 overexpression can lead to ligand-independent signaling and is linked to carcinogenesis. We use a combination of microscopy approaches and mathematical modeling to explore the early steps in receptor signaling. We have mapped distributions of ErbB receptors on membranes of SKBR3 breast cancer cells by immunoelectron microscopy. The most abundant receptor, ErbB2, is phosphorylated, clustered and active. Kinase inhibitors ablate ErbB2 phosphorylation without dispersing clusters. Modest coclustering of ErbB2 and EGFR, even after EGF treatment, suggests that both are predominantly involved in homointeractions. Heregulin leads to dramatic clusters of ErbB3 that contain some ErbB2 and EGFR and abundant PI 3-kinase. Other docking proteins, Shc and STAT5, respond differently to receptor activation. Levels of Shc at the membrane increase 2-5 fold with EGF while preassociated STAT5 becomes strongly phosphorylated. These data suggest that the distinct topography of receptors and their docking partners modulates signaling activities. The EM approach is complemented by live cell imaging approaches, such as single particle tracking of quantum dot probes to monitor diffusional properties of monomers and dimers. These data provide the foundation for our modeling efforts.

To explore spatial aspects of signal initiation, we have developed an agent-based computational model, SPS (Signaling Pathways Simulator). The stochastic modeling framework incorporates important spatio-temporal aspects of signaling, including protein clustering, protein motion and biochemical reactions within an idealized cellular geometry. We investigate mechanisms of receptor dimerization and activation as functions of time and receptor conformation, density and spatial distribution. For example, we have tested the hypothesis that the primary factor driving ligand-independent signaling is the fluctuation of EGFR conformation. Our model predicts that in A431 cells, uniformly distributed receptors would need to spend only 0.14% of the time in the open conformation to reach an average of 14% of EGFR in dimers at steady state. Results indicate that receptor density is a principal factor in the ability to form a measurable amount of active homodimers in the absence of ligands. Our results further predict that receptor clustering exacerbates the density-dependent homodimerization of unoccupied receptors and slightly lowers the estimate (to 0.12%) of receptors in the open state. We propose that membrane spatial organization is a contributor to the carcinogenesis process, particularly at moderate expression levels.

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