PMC:3405059 / 2133-9360 JSONTXT

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RON (Recepteur d’Origine Nantais) receptor tyrosine kinase is the specific cell-surface receptor for Macrophage Stimulating Protein (MSP), a serum growth factor also known as the Hepatocyte Growth Factor-like protein (HGFL). RON, encoded by the MST1R gene, is a member of the Class VI receptor tyrosine kinase family (EC:2.7.10.1) along with the proto-oncogene Met receptor tyrosine kinase (Met). The extracellular regions and the cytoplasmic kinase domains of RON and Met share 33% and 64% amino acid sequence identities, respectively [1]. RON is widely expressed in macrophages, epithelial tissues, adenocarcinoma cells, bronchial epithelial cells, granulocytes, and monocytes [2], [3], [4]. The interaction of RON with MSP transduces multiple signaling pathways that regulate cellular morphogenesis, adhesion, invasion and motility [5]. RON is also associated with the MSP-mediated inflammatory activities upon cellular stresses and with innate immune responses to bacterial infections [6], [7], [8]. High levels of RON are detected in patients with ulcerative colitis and deep endometriosis and also in several types of epithelial cancers, implicating RON in tumor progressions and cancer pathogenesis [5], [9], [10], [11]. In addition, alternatively spliced variants of RON promote the metastasises of lung, breast, colon, ovarian, prostate, pancreatic, thyroid and gastric cancers [12], [13], [14], [15], [16], [17], [18], [19], [20]. Thus, RON has become an important target for cancer therapy using anti-RON monoclonal antibodies, small molecule kinase inhibitors, and small interfering RNAs [21], [22], [23].\nRON comprises an extracellular ligand binding domain (ectodomain), a single pass trans-membrane segment and a cytoplasmic tyrosine kinase domain. The ectodomain can be subdivided into the N-terminal semaphorin (Sema) domain, a small cysteine-rich Plexins-Semaphorins-Integrins (PSI) motif, and four Immunoglobulins-Plexins-Transcription factor (IPT) domains. Cellular RON is produced as a glycosylated, single chain precursor (Pro-RON), which undergoes a furin protease cleavage at Arg309–Gly310 in the Sema domain prior to its transport from the Golgi to the apical surface of the cell [4], [23]. This disulfide-linked heterodimer is the mature form of RON. RON α-chain contains the N-terminal half of the Sema domain (∼40 kDa) and the β-chain (145 kDa) consists of the second half of the Sema domain, the PSI motif, the four IPT units, the transmembrane region and the cytoplasmic kinase domain. The current model for the MSP-mediated activation of RON begins with the binding of MSP to the receptor, leading to the formation of signaling-competent 2∶2 MSP:RON complex on the cell surface. RON dimerization then promotes the autophosphorylation of the functional tyrosine residues in trans, and the up regulation of the intrinsic kinase activity [7], [24], [25]. The phosphorylated kinase domains provide docking sites for cytoplasmic adaptor and signal tranducer proteins to initiate downstream signaling cascades [7], [24]. Some of these signal transduction pathways involve the participations of ras/mitogen activated protein kinase (MAPK), phosphatidyl inositol-3 kinase (PI-3K)/Akt, focal adhesion kinase (FAK), and β-catenin proteins [5], [26].\nThe RON specific ligand, MSP, is a serum protein that stimulates the chemotaxis of mouse peritoneal resident macrophages when exposed to the endotoxin-activated serum [7], [27]. Liver hepatocytes produce a single chain precursor MSP (Pro-MSP), which circulates in blood as biologically inactive scatter factor [28]. Under cellular stress, pro-MSP is cleaved by a type II transmembrane serine proteases, matriptase and hepsin, at Arg483-Val484 to produce the biologically active, disulfide-linked MSP α/β heterodimer [29], [30]. MSP belongs to the plasminogen-like growth factor family along with Hepatocyte Growth Factor (HGF), the specific ligand of Met. The two ligands share 43% amino acid sequence identity [31]. The 50 kDa MSP α-chain (MSPα) contains a N-terminal domain and four Kringle domains. The 30 kDa β-chain (MSPβ) adopts a chymotrypsin-like serine-protease fold [7], [31], [32] but lacks the catalytic triad of serine proteases, and accordingly, is devoid of proteolytic activity (the catalytic triad counterparts in MSP are Gln522, Gln568, and Tyr661) [32]. Pro-MSP exhibits no binding affinity to RON, whereas the mature MSP α/β binds to RON and stimulates the autophosphorylation of tyrosine residues located on the intracellular kinase domain [28]. Both α and β chains of MSP heterodimer are required for RON activation since MSPα or MSPβ alone are incapable of receptor induction [28], [33], [34]. Cell-based binding studies showed similar binding affinities of RON to MSP α/β heterodimer and to MSPβ alone (EC50 ∼0.20 nM), indicating that MSPβ contains the high affinity binding site for RON [33]. Surface Plasmon Resonance studies determined a dissociation constant of ∼13 nM between RON full-length extracellular domain and MSPβ [35]. The ability of RON Sema domain to compete with the membrane-bound, full length RON in binding to MSPβ suggested that the MSPβ binding site is localized on the Sema domain [36]. The cell based binding and competition assays also showed that MSPα binds to RON with ∼100-fold lower affinity than that of MSPβ (EC50 = 17 nM) [33], [34], [36]. Without MSPβ, MSPα has weak or no affinity for full-length RON, RON Sema, and RONΔ85 splice variant containing only the Sema and PSI domains [16], [34]. However, it remains unknown whether MSPα interacts with any of the RON IPT domains.\nIn addition to MSP, co-immunoprecipitation and co-localization experiments suggest that RON has other partner proteins. It forms heterodimers with Met, plexin B1–B3, β1 integrin, and epidermal growth factor receptor (EGFR). These interactions control cellular adhesion, migration and invasion processes [37], [38], [39], [40], [41], [42], [43]. For example, RON/EFGR complex can migrate from the cell surface to the nucleus to act as a transcriptional regulator in human bladder cancer cells [44]. RON also associates with several hyaluronan binding proteins. RON/hyaluronidase 2 complex on the cell surface prevents RON’s participation in retroviral transformation of human bronchial epithelial cells [45], [46]. The v6 splice variant of the hyaluronan receptor CD44 associates with RON/MSP during the migration of human colon adenocarcinoma cells, and another hyaluronan receptor, RHAMM (Receptor for Hyaluronic Acid-Mediated Motility) was co-localized with RON at the apical surface of ciliated cells in response to oxidative stress [47], [48].\nDespite a wide range of cellular responses regulated by RON, the basic mechanisms by which MSP and other protein partners mediate RON’s activity are unknown at atomic detail. Here, we report the crystal structure of RON Sema-PSI domains at 1.85 Å. The structure reveals unique MSP specificity determinants. It also suggests possible mechanisms for the ligand-independent RON dimerization, which occurs at high RON expression levels and with RONΔ160 splice variant, present in a wide range of human tumors and tumor-derived epithelial cell lines [23], [49]."}