Systematic Identification of Interaction Partners of RET Receptor Isoforms
The REarranged during Transfection (RET) receptor tyrosine kinase is pivotal for normal development of human tissues, but is also an oncogene driver involved in several human cancers. Alternative splicing at the 3’ end of the RET gene produces two conserved protein isoforms, RET9 and RET51, that differ in their subcellular localization and protein trafficking, and their functional roles in tumorigenesis and metastatic processes, suggesting that they may also differ in their interactomes. We used a combination of cell-based screening and in silico approaches to identify novel potential interaction partners of RET isoforms. We used a Mammalian Membrane Two-Hybrid (MaMTH) assay using a library of SH2 domain-containing adaptor and signaling proteins, to screen for interactions with each RET isoform. Through different steps of analysis of MaMTH screen data, we narrowed down RET potential interactors. We complemented these studies by ranking our SH2 prey library using sequence profiles of SH2 domains of known RET interaction partners. Predicted interaction candidates were validated using co-immunoprecipitation assays. A subset of these showed differential interactions with RET isoforms that were mediated through RET isoform-specific docking sites. Our results suggest that combinations of distinct interaction partners may contribute to RET isoform-specific functions. In this research we developed a systematic approach to map and characterize RET isoform interactions. Our data suggest that no single method identified all known and novel RET interactions, and that a combination of multiple approaches improves characterization of growth factor receptor interactomes.