Identification of factors regulating MET receptor endocytosis by high-throughput siRNA screening.

The tyrosine kinase MET, a receptor for hepatocyte growth factor, is a key regulator for normal development and organ renewal via stem cell maintenance. Dysregulated MET signaling contributes to tumor progression and metastasis and is considered a potent therapeutic target for a growing number of malignancies. Toward that goal it is critical to develop high-throughput assays to identify candidate regulators for the termination of MET signaling. We describe here a rapid and efficient method for identifying cellular factors required for MET ubiquitination, which utilizes high-throughput RNA interference screening (HT-siRNA) with a receptor internalization assay and an In-Cell ELISA in a 96-well format. The assay is amenable to a large array of cell surface proteins as well as genome-wide siRNA libraries, with high signal-to-background ratio and low well-to-well variability.