title: EphA2 receptor activation by monomeric Ephrin-A1 on supported membranes.
abstract: The receptor tyrosine kinase EphA2 interacts with its glycosylphosphatidylinositol (GPI)-linked ephrin-A1 ligand in a juxtacrine configuration. The soluble ephrin-A1 protein, without its GPI membrane linker, fails to activate EphA2. However, preclustered ephrin-A1 protein is active in solution and has been frequently used to trigger the EphA2 receptor. Although this approach has yielded insights into EphA2 signaling, preclustered ligands bypass natural receptor clustering processes and thus mask any role of clustering as a signal regulatory mechanism. Here, we present EphA2-expressing cells with a fusion protein of monomeric ephrin-A1 (mEA1) and enhanced monomeric yellow fluorescent protein that is linked to a supported lipid bilayer via a nickel-decahistidine anchor. The mEA1 is homogeneously dispersed, laterally mobile, and monomeric as measured by fluorescence imaging, correlation spectroscopy, and photon counting histogram analysis, respectively. Ephrin-A1 presented in this manner activates EphA2 on the surface of MDA-MB-231 human breast cancer cells, as measured by EphA2 phosphorylation and degradation. Spatial mutation experiments in which nanopatterns on the underlying substrate restrict mEA1 movement in the supported lipid bilayer reveal spatio-mechanical regulation of this signaling pathway, consistent with recently reported observations using a synthetically cross-linked ephrin-A1 dimer.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Xu
authors.forename: Wan-Chen
authors.initials: WC
authors.lastname: Lin
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Petit
authors.forename: Jay T
authors.initials: JT
authors.lastname: Groves
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002457:Cell Extracts; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D036382:Ephrin-A1 / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D011485:Protein Binding; D036104:Receptor, EphA2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013499:Surface Properties
chemical_list: D001426:Bacterial Proteins; D002457:Cell Extracts; D036382:Ephrin-A1; D008051:Lipid Bilayers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D036104:Receptor, EphA2