Transmodulation of Cell Responses
Cells are confronted with a variety of extracellular signals, including peptide ligands, amino acids, nucleotides, small organic molecules, and even specific ions. For cells to integrate information from multiple, distinct signals, there must be some mechanism for the signal from one receptor pathway to modify cell responsiveness to a second receptor type. For Pacific Northwest National Laboratory's (PNNL's) Transmodulation of Cell Responses project we are using transmodulation of the epidermal growth factor receptor (EGFR) by the insulin-like growth factor-1 receptor (IGF-1R) in human mammary epithelial cells (HMEC) as our primary model system to develop methods to dissect, isolate, and quantify the molecular pathways involved in EGFR transmodulation.
We are using quantitative kinetic measurements of downstream intermediates in the EGFR pathway to build a mathematical model of transmodulation between IGF-1R and EGFR. The end result will be a dynamic model of the flow of information among distinct receptor types and of how this crosstalk between receptors modifies biological endpoints related to proliferation and survival.
HMEC were treated with a variety of mitogens with or without pretreatment with the tyrosine kinase inhibitor, AG1517 (AG). The cell number from each treatment was measured after some period of time and calculated as fold increase over control. Interestingly, the data shows that HMEC proliferation is dependent on EGFR kinase activity even in response to other mitogens.Click for a larger version.
Cells respond to growth factors and stressors by nuclear translocation of the signaling molecule, mitogen-activated protein kinase (MAPK). In experiments testing whether these responses occur through common mechanisms, HMEC expressing fluorescently tagged MAPK were treated with uridine triphosphate (UTP), a purinergic receptor agonist released from cells in response to cell injury. In live-cell confocal microscopy experiments, MAPK accumulated in nuclei shortly after UTP treatment (arrows), a response comparable to EGF stimulation (view movie). Pre-treating cells with mAb225, an antibody preventing EGF-like ligands from binding the EGFR, blocked MAPK nuclear translocation, suggesting that UTP transactivates the EGFR to facilitate MAPK translocation. Click for a larger version and a short movie [SWF 125KB].