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Regulation of Cell Surface Ligand Dynamics

Steve Wiley, Principal Investigator

Autocrine versus juxtacrine signaling modes, graphic
Autocrine versus juxtacrine signaling modes. In the simplest model, autocrine signaling is regulated by the removal of the prepro-extension from the membrane-anchored ligand (step 1) followed by its controlled release from the membrane (step 2). Orientation restrictions are responsible for the release requirement. In the case of juxtacrine signaling, prepro-extension release is required (step 1), followed by binding to an "auxiliary" molecule on a neighboring cell (step 2). Note that autocrine ligands bind to the cell that produced them, and juxtacrine ligands bind to a neighboring cell. Click for a larger version.

Cells respond to stress by shedding a variety of biologically active proteins, such as proteases and growth factors. For the Regulation of Cell Surface Ligand Dynamics project, researchers at Pacific Northwest National Laboratory (PNNL) are using human mammary epithelial cells (HMEC) as a model to examine the regulation of autocrine and paracrine (soluble) versus juxtacrine (membrane-anchored) ligands. We are determining whether juxtacrine ligands selectively activate a distinctive set of signaling pathways and are investigating the mechanisms by which juxtacrine complexes are inactivated.

In HMEC, at least four distinct ligands for the epidermal growth factor receptor (EGFR) are shed in response to cell stress. We hypothesize that shedding is part of a mechanism by which cells actively interrogate their environment before choosing a particular response pathway. Using engineered cells that express a variety of artificial and natural ligands, we are building quantitative models of this process. We found that different EGFR ligands have distinct biological activities that are conferred by the domains flanking the core receptor-binding domain. Also, we hypothesize that some ligands function predominantly as soluble factors, whereas others operate mostly as membrane-anchored factors. Because the biological effects of juxtacrine and soluble ligands have been reported to be distinct, changes in the pattern of ligand expression and release could specify the net outcome of EGFR activation.

For these ligand regulation studies, we are developing a set of new imaging technologies that can directly visualize ligand oligomerization as well as interaction with other cell surface molecules. In addition, we are developing approaches to rapidly change ligand domains and evaluate multiple nodes in cellular signaling networks. We will use information derived from our studies in conjunction with computational modeling to gain insights concerning the complexities of ligand regulation and to extrapolate our results to the physiologically relevant context of tissues. This work will improve our basic understanding of EGFR system regulation in normal epithelium and will help to identify mechanisms whose dysregulation may be associated with cancer and other diseases. Moreover, our findings will provide general insights into how other membrane-associated growth factor systems can regulate cell-cell interactions.

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