Gaining New Insight on Receptor Tyrosine Kinase Signaling and Pharmacology

Domain Therapeutics describes a biosensor platform that allows for real-time monitoring of signal transduction pathways engaged upon activation of unmodified receptors

Receptor tyrosine kinases (RTKs) regulate many critical biological processes such as cell growth, differentiation, and survival through the recruitment of intracellular signaling molecules. The binding of growth factors to RTKs promotes their dimerization and subsequent phosphorylation of tyrosine residues in their cytosolic domain. In turn, this leads to the recruitment of intracellular signaling proteins containing Src homology-2 (SH2) and/or phosphotyrosine-binding (PTB) domains, which then engage downstream effectors involved in initiating multiple cellular signaling events.1

Dysregulated RTK activity aberrantly affects many cellular functions, often culminating in cancer. Consequently, RTKs serve as prime targets for new anticancer agents. However, the efficacy of several current drugs is limited by the development of drug-induced adverse events. Furthermore, the emergence of somatic and acquired RTK mutations allow tumors to develop resistance to certain drugs. RTK mutations may alter receptor activity and/or prevent drug binding through various mechanisms (that is, alteration of receptor subcellular localization, dimerization, signaling/trafficking, and kinetics signaling bias).

Currently, it is well recognized that different ligands binding the same receptor can promote different biological outcomes, a concept known as functional selectivity (or biased signaling). Functional selectivity has been extensively described for GPCRs2; yet this novel pharmacological paradigm remains underexplored for RTKs in part due to an emphasis on the discovery of ATP-competitive tyrosine kinase domain inhibitors.

Biased ligands hold therapeutic promise due to their ability to selectively modulate beneficial signaling cascades while lacking activity on those that may produce adverse side effects. Additional efforts and technologies are therefore required to fully understand the proximal and distal mechanisms that underlie the functional selectivity of RTK ligands.

Our understanding of the complexities underlying the activity and pharmacology of RTKs remains incomplete, thus hindering the development of new therapeutics targeting specific downstream pathways. Many of the current methods used to study RTK activation and screen for RTK inhibitors revolve around their kinase activity. However, kinase activity–based assays are limited in throughput and overlook additional key determinants of ligand therapeutic efficacy, including kinetics, localization, and functional selectivity, leading to an incomplete view about a ligand’s signaling signature. Such limitations require the development of novel tools to improve drug discovery in the field of RTKs.

Overview of the bioSensAll RTK platform

The RTK bioSensAll™ platform is a quantitative, livecell, enhanced bystander bioluminescence resonance energy transfer (ebBRET)-based biosensor platform that allows for real-time mapping and monitoring of the signal transduction pathways engaged upon activation of unmodified receptors. Unlike conventional BRET assays, ebBRET exploits theability of luciferase and green fluorescent protein from Renilla reniformis (rGFP) to self-associate with mild affinity and to optimally transfer energy, resulting in enhanced assay windows and sensitivity.3

These naturally interacting chromophores were exploited to develop new, highly dynamic BRET-based trafficking sensors designed to monitor the trafficking of various SH2 domain–containing proteins, specifically interacting with phosphotyrosine residues in the cytosolic tails of active RTKs. Events can be detected from both plasma membrane and early endosome compartments (Figure 1).

Figure 1. A panel of 12 SH2 domain–containing biosensors to study distinct proximal events and trafficking downstream of RTK activation. These sensors can also be used to assess receptor and effector trafficking to endosomal compartments.

To date, we have developed biosensors that monitor the activation of 12 distinct SH2 domain–containing proteins (Grb2, SHC1, PLCG1, PI3KR1-d1, PI3KR1-d2, PI3KR2-d1, PI3KR2-d2, Grb14, SHIP1, SHIP2, SHP-1, and SHP-2). The proximal biosensors forming the basis of the RTK biosensor platform contain a specific SH2 domain of the previously named proteins fused to Renilla luciferase (RlucII, R in Figure 2). The recruitment of biosensors to the plasma membrane upon RTK activation translates into an increased BRET efficiency with a plasma membrane–anchored Renilla reniformis rGFP (rGFP, G in Figure 2). The same translocation principle is used to measure the internalization of RTKs with an early endosome–anchored rGFP (Figure 2).

Figure 2. The RTK bioSensAll™ assay principles for monitoring SH2 domain protein engagement. Upon receptor activation, RlucII-tagged SH2 domain proteins (SH2-R) translocate toward and interact with the phosphorylated RTK following ligand binding. This event brings the ebBRET energy donor (RlucII) in close physical proximity to the plasma membrane–anchored rGFP (G; ebBRET energy acceptor), leading to increased ebBRET. The internalization into early endosomes assay is built on the same translocation principle, bringing SH2-R in close physical proximity to the early endosome–anchored rGFP.

Case study: Spatiotemporal assessment of EGFR and TKIs characterization on EGFR mutants

Epidermal growth factor receptor (EGFR) was used as a model receptor to demonstrate the spectrum of applications of the ebBRETbased biosensor platform in studying RTK biology and pharmacology. EGFR is part of the ErbB receptor family that also includes HER2, HER3, and HER4. The ErbB family of peptide growth factors comprises seven members: EGF, transforming growth factor-α, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, epiregulin, epigen, and the neuregulins.

All EGFR ligands are synthesized as transmembrane precursors that undergo extracellular domain cleavage to release soluble ligands, which then bind to and activate EGFR through favoring receptor homo- and/or heterodimerization. Interestingly, binding of different EGF family members to the same receptor has been reported to stimulate different biological responses (that is, biased signaling).4

EGFR is involved in key biological processes, and its deregulation is associated with the development of many cancers. Moreover, mutations of EGFR have been described to affect receptor signaling and to be responsible for the appearance of drug resistance during treatment in clinical settings.

Herein, we demonstrate how ebBRET biosensors were used to characterize and differentiate the signaling signatures of two EGFR ligands: EGF and epiregulin. Real-time studies of plasma membrane and early endosome recruitment of the SH2(PLC-γ1) biosensor—that is, the biosensor based on the SH2 domains of phospholipase C-γ1—highlighted how the RTK biosensors permit agonist signaling kinetics to be discriminated on timescales ranging from milliseconds to hours.

Our investigation further revealed that epiregulin was less potent than EGF in promoting the recruitment of SH2(PLC-γ1) effector to the plasma membrane. In addition, we observed that EGF was more efficient at recruiting SH2(PLC-γ1) effector to the plasma membrane compared to epiregulin, whereas epiregulin-induced SH2(PLC-γ1) effector recruitment to the plasma membrane displayed faster kinetics relative to that observed with EGF. In contrast, we demonstrated that EGF, but not epiregulin, stimulated SH2(PLC-γ1) effector engagement at the early endosome compartment (Figure 3a).

Figure 3. Evaluation of EGFR signaling at the plasma membrane and early endosomes and characterization of tyrosine kinase inhibitors using ebBRET-based technology. (A) HEK293 cells were co-transfected with plasmids coding for the human EGFR, the SH2(PLC-γ1) effector, and the plasma membrane– or early endosome–anchored proteins, as indicated. On the day of BRET, cells were stimulated for 10 minutes (plasma membrane) or 1 hour (early endosomes) with increasing amounts of EGF or epiregulin, and subsequently BRET was measured (top and bottom left). To assess the kinetics of recruitment of PLC-γ1 effector to the plasma membrane (top right) or trafficking to the early endosomes (bottom left), cells were stimulated with an EC80 of EGF or epiregulin, and BRET was immediately recorded every 30 seconds over a period of 1 hour.

Currently, EGFR is specifically targeted in different types of cancer using tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. Therefore, the inhibitory effects of different TKIs were assessed with wildtype-EGFR (EGFR-wt) and two EGFR mutants (EGFR-T790M and EGFR-C797S), prevalent in non-small cell lung carcinoma (NSCLC). These acquired mutations are known to arise during treatment with first- and third-generation EGFR TKIs.5

Initially, we monitored the real-time recruitment of the SH2(PLC-γ1) biosensor to the plasma membrane following stimulation of EGFR-wt and mutated receptors with EGF. Reversal of effector engagement upon addition of the first-generation TKI, gefitinib (Iressa®), or the third-generation TKI, osimertinib (Tagrisso®), was then investigated. Interestingly, different kinetics of inhibition between the two TKIs were observed on EGFR-wt with gefitinib displaying faster inhibitory activity (maximal inhibition within 60 s) compared to osimertinib (maximal inhibition at 5 min). Furthermore, Gefitinib reversed the activity of EGFR-wt and the EGFR-C797S mutant but was ineffectiveon the EGFR-T790M mutant (Figure 3b).

(B) HEK293 cells were co-transfected with plasmids encoding human wt-EGFR or the indicated EGFR mutants, along with SH2(PLC-γ1) effector and the plasma membrane-anchored protein. Cells were first stimulated with an EC80 of EGF, and the BRET signal was immediately measured every 30 seconds over a period of 30 minutes. Thereafter, a 10-µM solution of the indicated TKIs (gefitinib or osimertinib) was added to the wells, and BRET was recorded for an additional 30 minutes.


The case study presented above demonstrates the applicability of the RTK bioSensAll platform to perform in-depth characterization of RTK biology and pharmacology. This technology presents qualitative and quantitative functional readouts using the SH2 domain–containing biosensors that allow real-time spatiotemporal monitoring of ligand activity across various effector proteins/pathways. Furthermore, these biosensors are applicable for the identification of biased ligands and biased signaling in response to different ligands.

Such technology enabled us to identify unique signaling signatures triggered by different RTK ligands using unmodified receptors. Moreover, the RTK biosensor platform allowed us to differentiate the impact of different TKIs on various RTK mutations. This work highlighted the platform’s capacity to be exploited as a tool to develop next-generation TKIs effective against RTK variants.

The bioSensAll platform offers a unique combination of capabilities: it can profile RTK signal transduction; it is compatible with high-throughput screening; and it allows real-time kinetic measurements across multiple effector pathways to better understand RTK complex biology. This deeper level of understanding will enable the identification of safer and more effective RTK-targeting drugs against various mutations identified in different cancers.


Bio: Florence Gross is research associate and Laurent Sabbagh, PhD, is associate director at Domain Therapeutics North America. 


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