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  • The concept of GPCRs and


    The concept of GPCRs and EGFRs MAPK Inhibitor Library existing as heteromers is consistent with previous studies. Co-immunoprecipitation studies have shown that agonist stimulation (both AngII and EGF) promotes a multi-protein complex containing the AT1R and EGFR, however these endpoint assays were not in live cells or in real-time [78]. The β1- and β2-adrenergic receptors associate with the EGFR [66], [79], and while isoprenaline enhances β1-adrenergic receptor association with EGFR, this is disrupted by EGF-stimulation. Others have also used BRET to provide evidence for the bile MAPK Inhibitor Library receptor, TGR5, forming preassembled heterodimers with the EGFR in lipid rafts, however agonist stimulation showed no further enhancement in receptor association [80]. Complementary to our studies, Bhattacharya and colleagues showed G protein-coupled receptor 54 interacts with EGFR at the plasma membrane in the absence of agonist. However, only stimulation with its endogenous ligand, kisspeptin, significantly increased their association, while EGF-treatment did not affect the receptor complex [81]. Although it appears that many GPCRs can transactivate EGFRs, heteromerization has only been indicated for a subset. It is still unknown how this interaction affects signaling of either receptor, with the mechanism and/or binding domains that facilitate heteromer formation largely unknown. One possible mechanism that has not been tested directly is the reported contribution of reactive oxygen species [82], [83] and their potential oxidation of the EGFR on dimers formation [84]. Whether the complex subserves actual transactivation is difficult to reconcile because specific inhibitors that prevent complex formation are not available. Remarkably, we observed that the modulation of BRET signals associated with AT1R-EGFR heteromerization differed depending on whether the complex was modulated by activation of the AT1R or the EGFR. The AngII-induced complex modulation is apparently independent of both Gq/11 and EGFR kinase activity. G protein-independent transactivation has been reported for β1-adrenergic receptor and EGFR continued association [66]. In contrast, in our hands, EGF-mediated modulation of the AT1R-EGFR complex was absolutely dependent on the EGFR kinase domain. Moreover, antagonism of the AT1R or inhibition of Gq/11 did not inhibit complex modulation, but rather enhanced the EGF-mediated increase in BRET signal between AT1R-Rluc8 and EGFR-Venus. Whether this enhancement plays any role in receptor activation is unclear, but it is interesting to note that traditional models of monomeric EGFR dimer formation as the active component have been superseded by models that suggest that up to 40% of EGFRs are present as inactive dimers that form higher order active receptor signaling complexes [85], [86], [87], [88]. This negative modulation of EGFR may be mirrored by the AT1R, whereby the AT1R and its associated G protein negatively modulate monomeric/dimeric/oligomeric EGFR. In summary, the combination of different BRET approaches emphasizes the importance of monitoring the most proximal events of EGFR activation, in live cells and in real-time. Using this approach, we propose a Gq/11-independent mechanism of EGFR recruitment, instead providing evidence of ligand-modulated AT1R-EGFR complexes. This work provides a platform to better interrogate the molecular mechanisms underpinning AT1R-EGFR transactivation, and provides powerful new insights into this underappreciated process.
    Significance The heterogeneity of tyrosine kinase inhibitor (TKI) resistance remains a challenge in treating TKI-intrinsic or acquired resistant EGFR-mutant non-small cell lung cancer (NSCLC). In the current study, we report EGFR heterodimer-mediated nuclear PKCδ as a common mediator shared by multiple TKI-resistance mechanisms, and it is upregulated in a significant proportion of patients (∼41.5%) with TKI-resistant NSCLC. Inhibition of PKCδ sensitized NSCLC tumors with mutant EGFR to first- and third-generation TKI. These results provide mechanistic insights into the role of the EGFR-PKCδ axis in TKI resistance and suggest an EGFR addiction in TKI-resistant EGFR-mutant NSCLC.