ABSTRACT
The mammalian RON and the avian sea genes encode tyrosine kinase receptors of poorly characterized biological functions. We recently identified macrophage-stimulating protein as the ligand for Ron; no ligand has yet been found for Sea. In this work we investigated the biological response to macrophage-stimulating protein in mouse liver progenitor cells expressing Ron. These cells were also transfected with a chimeric cDNA encoding the cytoplasmic domain of Sea, fused to the extracellular domain of Trk (nerve growth factor receptor). In the presence of nanomolar concentrations of the respective ligands, both receptors induced cell "scattering", extracellular matrix invasion, and DNA synthesis. When liver progenitor cells were grown in a tri-dimensional type-I collagen matrix, ligand-induced stimulation of either Ron or Sea induced sprouting of branched cell cords, evolving into ductular-like tubules. The motogenic, mitogenic, and morphogenic responses were also elicited by triggering the structurally related hepatocyte growth factor receptor (Met) but not epidermal growth factor or platelet-derived growth factor receptors. These data show that Ron, Sea, and Met belong to a receptor subfamily that elicits a distinctive biological response in epithelial cells.
Subject(s)
Avian Proteins , Liver/cytology , Protein-Tyrosine Kinases/physiology , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Cell Surface/physiology , Stem Cells/cytology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cell Differentiation/physiology , Cells, Cultured , Cricetinae , DNA Replication , In Vitro Techniques , Mice , Microscopy, Electron , Molecular Sequence Data , Phenotype , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptor, trkA , Receptors, Cell Surface/genetics , Receptors, Nerve Growth Factor/genetics , Recombinant Fusion Proteins/genetics , TransfectionABSTRACT
SH3 domains constitute a family of protein-protein interaction modules that bind to peptides displaying an X-proline-X-X-proline (XPXXP) consensus. We report that the SH3 domain of Eps8, a substrate of receptor and non-receptor tyrosine kinases, displays a novel and unique binding preference. By a combination of approaches including (i) screening of phage-displayed random peptide libraries, (ii) mapping of the binding regions on three physiological interactors of Eps8, (iii) alanine scanning of binding peptides and (iv) in vitro cross-linking, we demonstrate that a proline-X-X-aspartate-tyrosine (PXXDY) consensus is indispensable for binding to the SH3 domain of Eps8. Screening of the Expressed Sequence Tags database allowed the identification of three Eps8-related genes, whose SH3s also display unusual binding preferences and constitute a phylogenetically distinct subfamily within the SH3 family. Thus, Eps8 identifies a novel family of SH3-containing proteins that do not bind to canonical XPXXP-containing peptides, and that establish distinct interactions in the signaling network.