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J. Biol. Chem., Vol. 283, Issue 46, 31477-31487, November 14, 2008

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Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells^*

Karin D. Rodland¹, Nikki Bollinger, Danielle Ippolito, Lee K. Opresko, Robert J. Coffey^¶, Richard Zangar, and H. Steven Wiley^||

From the Systems Biology Program, Biological Sciences Division, and ^||Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354 and ^¶Vanderbilt University Medical Center, Nashville, Tennessee 37232

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.

Received for publication, January 17, 2008 , and in revised form, August 22, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health, NCI, Grant CA 46413 (to R. J. C.). This work was also supported by the Biomolecular Systems Initiative Laboratory Directed Research and Development Program at the Pacific Northwest National Laboratory, a multiprogram national laboratory operated by Battelle for the United States Department of Energy under Contract DE-AC05-76RL01830 and by Gastrointestinal Special Program of Research Excellence Grant P50 95103 (to R. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: P7-56, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352. Tel.: 509-376-7605; Fax: 509-376-6767; E-mail: karin.rodland{at}pnl.gov.

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