Altered Exon Usage in the Juxtamembrane Domain of Mouse and Human RON Regulates Receptor Activity and Signaling Specificity

Abstract

Alternative splicing of signaling proteins can contribute to the complexity of signaling networks. We find that expression of mouse RON, but not human RON, results in constitutive receptor autophosphorylation, ligand-independent activation of the mitogen-activated protein kinase pathway, and association of the receptor with c-Src. Using chimeric receptors, we mapped the region for this difference in signaling capacity of mouse and human RON to the juxtamembrane domain. Expression of these receptors in primary erythroid progenitor cells also demonstrated a functional difference in the ability of mouse and human RON to support erythropoietin-independent colony formation that mapped to the juxtamembrane domain. Splicing of the mouse RON receptor tyrosine kinase transcript results in the constitutive deletion of an exon used by all other known RON orthologs that encodes part of the juxtamembrane domain of the receptor. Mutational analysis indicated that the two tyrosines present in this region in human RON, one of which has been previously shown to be a c-Cbl binding site, are not responsible for this difference. However, deletion of this region in the context of human RON enhanced receptor phosphorylation, activation of mitogen-activated protein kinase, and association of c-Src at levels comparable with those observed with mouse RON. These data provide direct evidence that the divergence of exon usage among different species can generate a protein with novel activity and subsequently add to the complexity of cellular signaling regulation.The modular nature of signaling proteins greatly facilitates the rapid evolution of a large number of molecules with various combinations of functional domains, increasing the possibility for combinatorial regulation. Furthermore, individual exons frequently correspond to basic folding and functional modules of a given protein (1). Expectedly, alternative splicing constitutes an important mechanism for protein evolution and diversification. By simply including or excluding certain exons, genes could reorganize their functional motifs to achieve novel activities without taking the risk of deleteriously mutating the coding sequence. Receptor tyrosine kinases (RTKs) 2 play a fundamental role in the regulation of animal development and pathogenesis. Intramolecular interactions, receptor dephosphorylation, and degradation form multiple layers of defense against uncontrolled kinase activity. The binding of growth factors enhances the enzymatic activity of the kinase domain by relieving structural constraints (2), resulting in tyrosine phosphorylation of the receptor that serves as an assembly platform for downstream signaling molecules (3). Alternative splicing, resulting in similar relief of these constraints, is one mechanism by which RTKs could become constitutively activated in tumor cells. Recent studies have identified several transforming transcripts of the human RON receptor tyrosine kinase, generated by alternative splicing of exons 5, 6, and 11 in the extracellular domain in pa...

Hypermethylation of Ron proximal promoter associates with lack of full-length Ron and transcription of oncogenic short-Ron from an internal promoter

“…Previously, we noticed that Stk lacks what corresponds to human exon 13. Recently it was shown (Wei et al, 2005) that this difference represents part of the diversification between human Ron and murine Stk signaling activity.…”

The Receptor Tyrosine Kinase RON Represses HIV-1 Transcription by Targeting RNA Polymerase II Processivity

“…RON mutations were introduced using a QuikChange Mutagenesis kit (Stratagene) and then were subcloned into murine stem cell virus-derived retroviral transfer vector MSCV2.1 as previously described (25,26). Receptor expression was confirmed by immunoblotting (22) using a rabbit anti-RON polyclonal Ab (Santa Cruz Biotechnology) and mouse anti-GAPDH (Fitzgerald) as a loading control.…”

“…Therefore, to gain further insight into mechanisms by which RON repress HIV-1 provirus transcription we used site-directed mutagenesis to generate several mutations in the RON cytoplasmic domain (25,26). In particular, we mutated Y1353 and Y1360, which serve as a multifunctional docking site for several signaling molecules including Gab-1, Grb2, PI3K, phospholipase C, and Src homology protein-2 (32).…”

Met‐Related Receptor Tyrosine Kinase Ron in Tumor Growth and Metastasis

“…The human Ron transcript consists of 20 exons while murine Ron codes for 19 exons. Altered splicing of the murine Ron gene creates a deletion of a small juxtamembrane region that is present in the human Ron gene (Wei et al, 2005). An analysis of the mouse Ron gene promoter region showed the presence of a number of putative transcription factor binding sites important in tumor progression, including binding sites for NF-kβ, Ets-1, and the estrogen receptor (Waltz et al, 1998).…”