Abstract
Human RON (Recepteur d’Origine Nantais) receptor tyrosine kinase is a cell surface receptor for Macrophage Stimulating Protein (MSP). RON mediates signal transduction pathways that regulate cell adhesion, invasion, motility and apoptosis processes. Elevated levels of RON and its alternatively spliced variants are implicated in the progression and metastasis of tumor cells. The binding of MSP α/β heterodimer to the extracellular region of RON receptor induces receptor dimerization and activation by autophosphorylation of the intracellular kinase domains. The ectodomain of RON, containing the ligand recognition and dimerization domains, is composed of a semaphorin (Sema), Plexins-Semaphorins-Integrins domain (PSI), and four Immunoglobulins-Plexins-Transcription factor (IPT) domains. High affinity association between MSP and RON is mediated by the interaction between MSP β-chain and RON Sema, although RON activation requires intact RON and MSP proteins. Here, we report the structure of RON Sema-PSI domains at 1.85 Å resolution. RON Sema domain adopts a seven-bladed β-propeller fold, followed by disulfide bond rich, cysteine-knot PSI motif. Comparison with the homologous Met receptor tyrosine kinase reveals that RON Sema-PSI contains distinguishing secondary structural features. These define the receptors’ exclusive selectivity towards their respective ligands, RON for MSP and Met for HGF. The RON Sema-PSI crystal packing generates a homodimer with interface formed by the Sema domain. Mapping of the dimer interface using the RON homology to Met, MSP homology to Hepatocyte Growth Factor (HGF), and the structure of the Met/HGF complex shows the dimer interface overlapping with the putative MSPβ binding site. The crystallographically determined RON Sema-PSI homodimer may represent the dimer assembly that occurs during ligand-independent receptor activation and/or the inhibition of the constitutive activity of RONΔ160 splice variant by the soluble RON splice variant, RONΔ85.
Highlights
Human RON (Recepteur d’Origine Nantais) receptor tyrosine kinase is the specific cell-surface receptor for Macrophage Stimulating Protein (MSP), a serum growth factor known as the Hepatocyte Growth Factor-like protein (HGFL)
The purified RON Sema-Plexins-Semaphorins-Integrins domain (PSI)-IPT1 has a molecular mass of 77,114691 Da, consistent with the five predicted glycosylation sites
The Drosophila Schneider 2 (S2) cells produce at least four acid active cathepsins [60], and trace amounts of contaminating proteases in the RON Sema-PSI-IPT1 preparation could cleave the protein at the low pH (4.6) of the crystal growth solution
Summary
Human RON (Recepteur d’Origine Nantais) receptor tyrosine kinase is the specific cell-surface receptor for Macrophage Stimulating Protein (MSP), a serum growth factor known as the Hepatocyte Growth Factor-like protein (HGFL). Both the single chain and thrombin-cleaved RON Sema-PSI-IPT1 formed crystals under identical condition. As the diffraction resolution of the single chain RON crystal was superior to that of the thrombin-cleaved RON Sema-PSI (1.85 Aand 2.5 Aresolution, respectively), the refinement results are provided for the intact RON structure (Table 1).
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