A system for quantifying dynamic protein interactions defines a role for Herceptin in modulating ErbB2 interactions

Abstract

The orphan receptor tyrosine kinase ErbB2 is activated by each of the EGFR family members upon ligand binding. However, difficulties monitoring the dynamic interactions of the membrane receptors have hindered the elucidation of the mechanism of ErbB2 activation. We have engineered a system to monitor proteinprotein interactions in intact mammalian cells such that different sets of protein interactions can be quantitatively compared. Application of this system to the interactions of the EGFR family showed that ErbB2 interacts stably with the EGFR and ErbB3, but fails to spontaneously homooligomerize. The widely used anticancer antibody Herceptin was found to effectively inhibit the interaction of the EGFR and ErbB2 but not to interfere with the interaction of ErbB2-ErbB3. Treatment of cells expressing EGFR and ErbB2 with Herceptin results in increased EGFR homooligomerization in the presence of EGF and a subsequent rapid internalization and down-regulation of the EGFR. In summary, the protein interaction system described here enabled the characterization of ErbB2 interactions within the biological context of the plasma membrane and provides insight into the mechanism of Herceptin action on cells overexpressing ErbB2.anti-cancer ͉ EGF receptor T he EGF family of receptor tyrosine kinases consists of four members, EGFR, ErbB2, ErbB3, and ErbB4, that become activated in response to ligand-induced dimerization. ErbB2 (HER2͞Neu) does not itself bind any known ligand, and activation of this receptor is believed to be mediated through heterodimerization with any of the other EGF family members. Physical characterization of this process has proven difficult using conventional biochemical methods, but it is of considerable interest because of the role of ErbB2 in breast cancer pathogenesis.ErbB2 is overexpressed in 30% of breast cancers and most clearly associated with a malignant phenotype and poor prognosis, especially if coexpressed with the EGF receptor (EGFR) (1-3). For a subset of breast cancer patients whose tumors overexpress ErbB2, the monoclonal antibody Herceptin has revolutionized treatment by extending lifespan and decreasing recurrence rate in an unprecedented manner (4-6). Although there is evidence that Herceptin targets tumor cells for destruction by the immune system (7), the antibody was originally selected as an inhibitor of tumor cell growth in vitro independent of an immune response (8). Herceptin is not known to block the formation of heterodimers of ErbB2, yet its inhibitory effects on cell proliferation suggest that it interferes with signal transduction by the ErbB family of tyrosine kinases. One reason that the mechanism of action of Herceptin has remained elusive is the difficulty in monitoring the interactions of the ErbB receptors in a quantitative manner using available biochemical methods, including purified or coimmunoprecipitated receptors (9-11).We postulated that the ␤-gal system we recently developed for assays of protein translocation (12) could enable a comparative analysis of the co...

The ERBB3 receptor in cancer and cancer gene therapy

“…116 However, herceptin, a monoclonal antibody to ERBB2 which targets ERBB2/EGFR heterodimers, had no effect on ERBB2/ERBB3; ERBB3/EGFR heterodimers were unstable in this engineered cell expression system. 117 ERBB3/ERBB4 complexes have also been reported 78 and can stimulate cell division. 92 ERBB3 phosphorylation by other kinases-Other kinases may also phosphorylate ERBB3 under some circumstances.…”

Targeting the function of the HER2 oncogene in human cancer therapeutics

“…In pulldown assays, trastuzumab does not inhibit HER2-HER3 interaction (Agus et al, 2002), and in fluorescence resonance energy transfer (FRET)-based assays trastuzumab also does not inhibit HER2 interaction with EGFR or HER3 (Diermeier et al, 2005). In a different model using truncated HER proteins fusing them to b-galactosidase fragments in an enzyme complementation assay, trastuzumab was reported to inhibit EGFR-HER2 interaction but not HER2-HER3 interactions (Wehrman et al, 2006). The artificial nature of the truncated receptors used in the latter study makes it less reliable, specially in light of FRET evidence to the contrary.…”

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

“…In addition to homodimerization, some combinations of heterodimerization between members of the ErbB family have been described (1,2). Several forms of unliganded and liganded homoand heterodimers of HRG receptors have been reported (8)(9)(10)(11)(12)(13)(14)(15). However, discrepancies among these studies have not yet been resolved, and the full model of ErbB heterodimerization remains unclear.…”