The EGFR belongs to the ErbB family of receptor tyrosine
The EGFR belongs to the ErbB family of receptor tyrosine kinases (RTKs), comprised of four members: EGFR (ErbB1, HER1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) . HER receptors are activated by a group of epidermal growth factor (EGF) ligands and undergo homo- or hetero-dimerization during their activation, with HER2 being the preferred dimer partner . Activated and tyrosine-phosphorylated HER receptors recruit growth factor receptor-bound protein 2 (Grb2) as an initial step in a complex cascade of growth signaling activation. HER receptors can also be ‘transactivated’ following prior activation of other receptors. Most notably, Ullrich and colleagues first described GPCR-mediated EGFR transactivation, and subsequently proposed a model, whereby activated GPCRs stimulated matrix metalloproteases (MMP), including the ADAMs (A Disintegrin and Metalloprotease), to cleave inactive membrane-bound EGF ligands to activate EGFR , . This model has been broadly accepted, although others have suggested a more complex mechanism (perhaps cell-specific) involving non-receptor tyrosine kinases (e.g., src and Pyk2) , , , ,  and the activation of additional second messengers including calcium , , , protein kinase C , , , , ROS ,  and β-arrestin , . Definitive resolution of the precise mechanism of GPCR-EGFR transactivation requires approaches to monitor this process in living cells, in real-time, as well as the capacity to identify key proteins and interactions involved. While some progress has been made on the latter problem – a siRNA screen was recently used to identify novel mediators of AT1R-EGFR transactivation  – to date most readouts of EGFR transactivation have been biochemical, end-point assays that do not capture live cell dynamics or kinetics.
Commonly, GPCR-mediated EGFR transactivation has been defined utilizing the activation of ERK1/2 that is inhibited by the small molecule antagonist of EGFR (i.e., AG1478). However, there are several limitations in using ERK1/2 as a surrogate readout of transactivation. EGFR signal transduction is complex with ERK1/2 AG 1879 representing a signal quite distal from the initial step of EGFR activation. Arguably, a more direct readout would be to examine upstream events, including activation and autophosphorylation of the EGFR. Although a direct readout of phospho-EGFR may mitigate the limitations of the ERK1/2 assay, both phospho-EGFR and phospho-ERK1/2 assays are endpoint approaches and only provide a snapshot of cellular events. More recently, proximity-based assays, such as resonance energy transfer (RET) assays and fluorescein arsenical hairpin (FlAsH) biosensors have evolved to enable dynamic analysis of protein-protein and intra-molecular interactions in real-time , . The development and validation of proximity-based assays has enabled their wide use to characterize receptor interactions. They have become extremely useful tools for studying GPCR biology, and specifically the interaction of GPCRs with G proteins and arrestins , , , .
In this study, we employed a Bioluminescence-RET (BRET)-based assay to monitor the most proximal event in EGFR activation, namely Grb2 interaction with the activated EGFR. We demonstrate, in live cells and in real-time, that both AngII- and EGF-stimulation can promote Grb2 translocation to the activated EGFR, indicating AT1R transactivation of the EGFR. Having established this approach, we extend it to investigate the molecular processes involved and report evidence for the formation of AT1R and EGFR complexes.
Materials and methods
Discussion GPCR-mediated EGFR transactivation is commonly defined in terms of the activation of ERK1/2, which although informative, is an indirect and distal readout. Herein, we describe a BRET assay, based on the recruitment of Grb2 to the EGFR to quantitatively monitor, in living cells and in real-time, the proximal activation of EGFR. Importantly, we identified that the molecular requirements for EGFR transactivation differed depending on the readout used. For the Grb2-EGFR assay, we showed that EGFR transactivation is independent of Gq/11 coupling and does not apparently involve the AT1R carboxy-terminal tail or interaction with arrestin. Using a series of additional BRET-based assays, we provide evidence of preformed complexes between the AT1R and EGFR that show different attributes, depending on which receptor is activated. In summary, the capacity to interrogate proximal EGFR transactivation provides a platform to better define the complex molecular processes involved.