Rapid Assays of Oncogenic Aberrant ErbB Receptor Activation Using Fluorescence Microscopy
Abstract
Signaling by the four members of the erbB family of receptor tyrosine kinases involves their ligand-induced homo- and/or hetero-oligomerization. Ligand-induced receptor homodimerization appears to be driven straightforwardly by receptor extracellular domains, and can be recapitulated in vitro. By contrast, hetero-oligomerization cannot be detected in studies of isolated extracellular domains, and has only been observed for receptors in cellular membranes. We were therefore interested in determining which domains of an erbB receptor drive hetero-oligomerization. By analyzing a series of breast cancer cell-lines we found that EGF does not induce robust phosphatidylinositol-3-kinase (PI-3-K) activation in MCF-7 cells, since these cells express little to no EGF receptor. We visualize PI-3-K activation in vivo by observing the cytoplasm-to-plasma membrane translocation of a pleckstrin homology domain (fused to green fluorescent protein) that specifically recognized PtdIns(3,4,5)P3 (Grp1). We found that overexpression of wild-type EGFR in MCF-7 cells restored' the ability of EGF to induce robust PI-3-K activation in these cells. Surprisingly, both a kinase-deficient EGFR mutant and a form of EGFR lacking all cytoplasmic sequences were equally effective in mediating EGE-induced PI-3-K activation. The EGFR extracellular domain tethered' to the plasma membrane by a GPI anchor, or just added in excess (together with EGF) were also capable of inducing PI-3-K activation.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2002
- Accession Number
- ADA408226
Entities
People
- Mark A. Lemmon
- Mitchel B. Berger
Organizations
- University of Pennsylvania