Linda J. Pike, Ph.D.
Associate Professor
Biochemistry and Molecular Biophysics
Molecular Cell Biology Program
Biochemistry Program
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Office Phone: 314-362-9502
Lab Phone: 314-362-9501
Other Phone:
FAX: 314-362-7183
Box: 8231
Lab Address: 1905A South Bldg
Email: pike@wustl.edu
Website: http://biochem.wustl.edu/~pike
Keywords: signal transduction; cell signaling; cancer; EGF receptor; lipid rafts
Short Research Description: EGF receptor-mediated cell signaling and the control of cell growth |
Research Abstract:
The EGF receptor is a ubiquitously expressed cell surface receptor tyrosine kinase. Stimulation of cells with EGF leads to increases in metabolic activity and ultimately in cell proliferation. The EGF receptor and is homologous ErbB family members are over-expressed in many human tumors and several clinically useful drugs are directed against these proteins. My laboratory is interested in the the regulation of cell signaling via the EGF receptor and ErbB family members. We are particularly interested in the early steps that follow the binding of ligand and in understanding how the structure of the membrane controls receptor signaling.
In the membrane, the EGF receptor is thought to exist as a dimer, but upon binding ligand, the receptor dimerizes and its tyrosine kinase is activated. Using ligand binding analyses, we have shown that the EGF receptor exhibits negative cooperativity between binding sites in the active dimer and we are currently doing a mutational analysis to define the structural aspects of the EGF receptor that underlie this allosteric phenomenon. We have recently developed a split luciferase enzyme complementation system that allows us to image EGF receptor dimerization in real time in live cells. The method allowed us to identify sequential, ligand-induced conformational changes in the receptor and provided insight into the regulation of EGF receptor function by MAP kinase. We will be using this system to further analyze the earliest steps in EGF receptor-mediated signal transduction and to define the mechanism by which MAP kinase desensitizes EGF receptor function.
The EGF receptor partitions into low-density, cholesterol-enriched membrane domains known as lipid rafts. We have recently used fluorescence cluster analysis to determine the number of EGF receptor molecules that cluster in a single lipid raft. The data suggest that at rest, EGF receptors exist as both monomers and dimers. Upon depletion of cholesterol, which disrupts lipid rafts, the receptors become more clustered, occurring in groups of four receptors. Addition of excess cholesterol shifts all receptors to monomeric form. This suggests that cholesterol regulates the ability of the EGF receptor to oligomerize. Since the EGF receptor must dimerize to signal, and increased clustering would favor dimerization, these findings explain the observation that cholesterol depletion enhances EGF-stimulated receptor autophosphorylation. |
Selected Publications:
Macdonald JM. and Pike LJ. Heterogeneity in EGF-binding Affinities Arises from Negative Cooperativity in an Aggregating System. Proc. Natl. Acad. Sci. U.S.A. 2008 105:112-117.
Saffarian S, Li Y, Elson EL, and Pike LJ. Oligomerization of the EGF Receptor Investigated by Live Cell Fluorescence Intensity Distribution Analysis. Biophys J 2007 93:1021-1031.
Macdonald J, Li Z, Su W and Pike LJ. The Membrane Proximal Disulfides of the EGF Receptor Extracellular Domain are Required for High Affinity Binding and Signal Transduction but do not Play a Role in the Localization of the Receptor to Lipid Rafts, Biochim. Biophys. Acta Mol Cell Res 2006 1763: 870-878.
Macdonald JL, Pike LJ. A simplified method for the preparation of detergent-free lipid rafts. J Lipid Res 2005 46:1061-1067.
Pike, LJ., Han, X. , and Gross, RW. EGF Receptors Are Localized to Lipid Rafts That Contain a Balance of Inner and Outer Leaflet Lipids: A Shotgun Lipidomics Study, J. Biol. Chem. 2005 280: 26796-26804. |