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  • The development of novel approaches to study


    The development of novel approaches to study GPCR-mediated transactivation in live AMD-070 is important, and this study describes a unique BRET-based quantification of Grb2 recruitment to the EGFR as a direct readout of GPCR-mediated transactivation. We have previously used the recruitment of Grb2 to RTKs (EGFR and HER3) as a measure of EGF receptor family activation in HEK293 cells following stimulation with EGF and Heregulin [47]. Moreover, others have also identified such BRET approaches as bona fide readouts of EGFR activation [63], [64]. In this study, we initially aimed to determine whether stimulation of the AT1R could promote Grb2 translocation and interaction with EGFR in a ligand-dependent manner in live cells. AT1R-mediated EGFR transactivation was readily demonstrable, but was consistently lower in magnitude compared to EGF-stimulation. The selective activation of EGFR by the AT1R is consistent with many observations that the degree of signaling elicited by GPCRs appears to be only a subset of that obtained following full activation by EGF ligands [27], [35], [65], [66]. EGFR transactivation was readily detected in HEK293 cells, which is interesting because a previous study reported that ERK1/2 activation was independent of the EGFR in these cells [65]. EGFR-Grb2 recruitment was also recapitulated in other standard cell lines (CHO-K1 and NIH-3T3) and in primary isolated VSMCs, indicating the versatility of this approach to measure EGFR transactivation. We observed that the AT1R and V1bR showed consistent recruitment of Grb2 to the EGFR, but we were surprised that other GPCRs (previously reported to cause EGFR transactivation [67], [68], [69], [70]) did not. It is important to note that a negative response in our assay does not necessarily mean that the GPCR is not transactivating nor should it call into question the veracity of our assay. There are a number of plausible explanations: firstly, a trivial argument might be that these ‘non-transactivating’ GPCRs were non-functional – however, we confirmed their activation by using a readout of ligand-dependent β-arrestin trafficking. Secondly, these GPCRs may not be recruiting Grb2 as part of their activation process or, alternatively, there is an association but it is different in nature to that generated by the AT1R and V1bR. Indeed, for a number of the GPCRs that did not show enhanced EGFR-Grb2 association, we actually observed decreased BRET ratios following ligand stimulation. It could be reasonably argued that this reflects dynamic changes in an equilibrium between association, dissociation and reconfiguration of putative EGFR-Grb2 complexes – where the balance may favor dissociation or reconfiguration that leads to an increased distance between the donor and acceptor moieties. Thirdly, as our assay only reports on the Grb2 recruitment to the EGFR, the possibility exists that receptors other than the EGFR (HER2, HER3 or HER4) and or combinations of dimers between those receptors, are selectively used by these other GPCRs for transactivation. In this regard, it is important to acknowledge that most studies of EGFR transactivation typically use end-point assays, where specificity is inferred via inhibition by the small molecule inhibitor of the EGFR kinase, AG1478. However, high micromolar concentrations of AG1478 (as commonly used) inhibit receptors other than the EGFR [71], [72]. Importantly, we report the recruitment of Grb2 to HER2, but not HER3, in response to AngII stimulation. Whether a specific GPCR is able to transactivate EGFR, HER2, HER3 or HER4 will require further investigation, using the approaches we have developed. It will be important to consider that HER2 is the preferred dimerization partner for other EGFR family members, and therefore potentially acting in cis to modulate other HER receptors. Indeed, HER2 has been shown to be transactivated and/or modulate transactivation [73], [74]. Another major observation of our study was the apparent disconnect between pharmacological inhibitors and their relative effect on the readout of transactivation vis-à-vis ERK1/2 versus BRET-based Grb2 recruitment assays. We clearly showed that candesartan completely inhibited AngII-induced ERK1/2 phosphorylation and recruitment of Grb2 to the EGFR, indicating that EGFR transactivation is due to ligand activation of the AT1R in both systems. Interestingly, cells treated with AG1478 showed a complete inhibition of ERK1/2 activation in response to AngII, however only a partial reduction of AngII-mediated transactivation as measured by BRET-based Grb2 recruitment assay, indicating a possible disconnect between proximal recruitment of Grb2 and the eventual activation of ERK1/2. We would argue that Grb2 recruitment is necessary, but not in itself sufficient, to drive ERK1/2 activation following AT1R activation. Importantly, EGF-mediated recruitment of Grb2 to the EGFR was completely inhibited with pretreatment of AG1478, confirming the efficacy of AG1478.