title: Identification of factors regulating MET receptor endocytosis by high-throughput siRNA screening.
abstract: 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.
authors.affiliation: Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-0620, USA, ivgaziov@UTMB.EDU.
authors.forename: Ivana
authors.initials: I
authors.lastname: Gaziova
authors.forename: Robert A
authors.initials: RA
authors.lastname: Davey
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Elferink
mesh_terms: D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects*; D015870:Gene Expression; D015723:Gene Library; D006367:HeLa Cells; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D034741:RNA, Small Interfering; D014452:Ubiquitins; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met