Detection of Ras nanoclustering-dependent homo-FRET using fluorescence anisotropy measurements

Ganesh babu Manoharan, Camilo Guzmán, Arafath Kaja Najumudeen, Daniel Abankwa*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

1 Citation (Scopus)

Abstract

The small GTPase Ras is frequently mutated in cancer and a driver of tumorigenesis. The recent years have shown great progress in drug-targeting Ras and understanding how it operates on the plasma membrane. We now know that Ras is non-randomly organized into proteo-lipid complexes on the membrane, called nanoclusters. Nanoclusters contain only a few Ras proteins and are necessary for the recruitment of downstream effectors, such as Raf. If tagged with fluorescent proteins, the dense packing of Ras in nanoclusters can be analyzed by Förster/ fluorescence resonance energy transfer (FRET). Loss of FRET can therefore report on decreased nanoclustering and any process upstream of it, such as Ras lipid modifications and correct trafficking. Thus, cellular FRET screens employing Ras-derived fluorescence biosensors are potentially powerful tools to discover chemical or genetic modulators of functional Ras membrane organization. Here we implement fluorescence anisotropy-based homo-FRET measurements of Ras-derived constructs labelled with only one fluorescent protein on a confocal microscope and a fluorescence plate reader. We show that homo-FRET of both H-Ras- and K-Ras-derived constructs can sensitively report on Ras-lipidation and -trafficking inhibitors, as well as on genetic perturbations of proteins regulating membrane anchorage. By exploiting the switch I/II-binding Ras-dimerizing compound BI-2852, this assay is also suitable to report on the engagement of the K-Ras switch II pocket by small molecules such as AMG 510. Given that homo-FRET only requires one fluorescent protein tagged Ras construct, this approach has significant advantages to create Ras-nanoclustering FRET-biosensor reporter cell lines, as compared to the more common hetero-FRET approaches.

Original languageEnglish
Article number151314
JournalEuropean Journal of Cell Biology
Volume102
Issue number2
DOIs
Publication statusPublished - Jun 2023
Externally publishedYes

Keywords

  • Anisotropy
  • Drug discovery
  • Homo-FRET
  • Nanoclustering
  • Ras

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