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

Ivana Gaziova, Robert A. Davey, Lisa A. Elferink

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMembrane Trafficking: Second Edition
PublisherSpringer New York
Pages381-394
Number of pages14
ISBN (Print)9781493923090, 9781493923083
DOIs
StatePublished - Feb 21 2015
Externally publishedYes

Keywords

  • HeLa cells
  • Hepatocyte growth factor (HGF)
  • MET
  • Receptor endocytosis
  • RNA interference (siRNA)
  • Ube3C
  • Ubiquitination

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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    Gaziova, I., Davey, R. A., & Elferink, L. A. (2015). Identification of factors regulating MET receptor endocytosis by high-throughput siRNA screening. In Membrane Trafficking: Second Edition (pp. 381-394). Springer New York. https://doi.org/10.1007/978-1-4939-2187-4_26