Editors' ChoiceReceptors

Enhancing Activity from the Inside

Science Signaling  19 Oct 2010:
Vol. 3, Issue 144, pp. ec322
DOI: 10.1126/scisignal.3144ec322

Activation of growth factor receptors of the epidermal growth factor receptor (EGFR) family is more complex than just ligand binding, which stimulates the receptor dimerization and autophosphorylation that are required for receptor activity. Only a fraction of the ligand-bound receptors signal because alleviation of intracellular inhibition is also necessary (see Shilo). Moreover, drugs, such as MIG6, that inhibit the conformational change required for activation reduce ligand-bound receptor signaling. Bill et al. showed that the conformationally active state of EGFRs, the homodimeric ErbB1 and the ErbB2-ErbB3 heterodimer, was promoted by interactions with cytoplasmic cytohesins. Cytohesins [cytohesin-1, cytohesin-2 (known as ARNO), cytohesin-3 (known as Grp1), and cytohesin-4] are a family of guanine nucleotide exchange factors (GEFs) for the ARF family of guanosine triphosphatases. SecinH3 is a chemical inhibitor of cytohesin activity, and exposure of cells with endogenous EGFRs (either ErbB1 or ErbB2-ErbB3) to EGF in the presence of SecinH3 reduced EGFR phosphorylation and downstream signaling, as did knockdown of cytohesins. Overexpression of wild-type ARNO, a GEF-inactivated mutant form, or the Sec7 domain that is the target of SecinH3 stimulated ligand-induced EGFR phosphorylation. Superresolution light microscopy indicated that SecinH3 did not change the size of EGFR clusters, suggesting that SecinH3 did not impair receptor dimerization, and quantification of EGFR abundance at the plasma membrane indicated that SecinH3 did not alter receptor trafficking or internalization. Overexpression of ARNO also did not change the amount of receptor dimers, detected in a cross-linking assay, but did promote receptor phosphorylation, whereas in this cross-linking assay SecinH3 reduced receptor phosphorylation without altering the amount of receptor dimers. SecinH3 also inhibited the phosphorylation of a constitutively, ligand-independently dimerized EGFR fusion protein (lz-EGFR) expressed in cells, and overexpression of ARNO increased the phosphorylation of this fusion protein. Steady-state fluorescence anisotropy of cells expressing fluorescently tagged lz-EGFR showed that overexpression of ARNO reduced anisotropy, indicating a conformational change in the receptor. In vitro assays with purified ARNO, or the ARNO Sec7 domain and the cytoplasmic domain of EGFR, or a deletion construct lacking the C-terminal 188 amino acids showed that ARNO and EGFR interacted directly and that this interaction promoted the autophosphorylation of the receptor peptide. Given the importance of aberrant EGFR signaling in cancers, the authors analyzed primary lung adenocarcinomas and found a correlation between strong cytohesin staining and EGFR phosphorylation. Growth of a lung cancer cell line in culture and in tumor xenografts was inhibited by SecinH3, suggesting that targeting this intracellular activator of EGFR signaling may be therapeutically advantageous.

A. Bill, A. Schmitz, B. Albertoni, J.-N. Song, L. C. Heukamp, D. Walrafen, F. Thorwirth, P. J. Verveer, S. Zimmer, L. Meffert, A. Schreiber, S. Chatterjee, R. K. Thomas, R. T. Ullrich, T. Lang, M. Famulok, Cytohesins are cytoplasmic ErbB receptor activators. Cell 143, 201–211 (2010). [PubMed]

B.-Z. Shilo, Insider influence on ErbB activity. Cell 143, 181–182 (2010). [PubMed]