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

by Selleckchem | Sep 12 2023

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.

Comments:

The passage describes the use of fluorescence resonance energy transfer (FRET) to analyze the organization of the small GTPase Ras on the plasma membrane. Ras is frequently mutated in cancer and plays a key role in tumorigenesis. It forms nanoclusters, proteo-lipid complexes on the membrane, which are crucial for recruiting downstream effectors like Raf. By tagging Ras with fluorescent proteins and analyzing FRET, the dense packing of Ras in nanoclusters can be studied. Loss of FRET indicates decreased nanoclustering and can reflect alterations in Ras lipid modifications, trafficking, and other upstream processes.

The passage discusses the implementation of homo-FRET measurements using Ras-derived constructs labeled with a single fluorescent protein. This approach allows for sensitive detection of Ras-lipidation and -trafficking inhibitors, as well as genetic perturbations affecting membrane anchorage proteins. Additionally, the engagement of the K-Ras switch II pocket by small molecules, such as AMG 510, can be assessed using the Ras-dimerizing compound BI-2852.

Compared to hetero-FRET approaches, which require multiple fluorescent protein tags, homo-FRET only necessitates one fluorescent protein-tagged Ras construct. This advantage facilitates the creation of Ras-nanoclustering FRET-biosensor reporter cell lines. These cellular FRET screens using Ras-derived fluorescence biosensors can be powerful tools for identifying chemical or genetic modulators that affect functional Ras membrane organization.