Complex Interactions Between The Angiotensin Ii Type 1 Receptor, The Epidermal Growth Factor Receptor And Trio-Dependent Signaling Partners

Transactivation of the epidermal growth factor receptor (EGFR) by the angiotensin II (AngII) type 1 (AT(1)) receptor is involved in AT(1) receptor-dependent growth effects and cardiovascular pathologies, however the mechanisms underpinning this transactivation are yet to be fully elucidated. Recently, a potential intermediate of this process was identified following the discovery that a kinase called TRIO was involved in AngII/AT(1) receptor-mediated transactivation of EGFR. To investigate the mechanisms by which TRIO acts as an intermediate in AngII/AT(1) receptor-mediated EGFR transactivation we used bioluminescence resonance energy transfer (BRET) assays to investigate proximity between the AT(1) receptor, EGFR, TRIO and other proteins of interest. We found that AngII/AT(1) receptor activation caused a G alpha(q)-dependent increase in proximity of TRIO with G gamma(2) and the AT(1)-EGFR heteromer, as well as trafficking of TRIO towards the Kras plasma membrane marker and into early, late and recycling endosomes. In contrast, we found that AngII/AT(1) receptor activation caused a G alpha(q)-independent increase in proximity of TRIO with Grb2, GRK2 and PKC zeta, as well as trafficking of TRIO up to the plasma membrane from the Golgi. Furthermore, we confirmed the proximity between the AT(1) receptor and the EGFR using the Receptor-Heteromer Investigation Technology, which showed AngII-induced recruitment of Grb2, GRK2, PKC zeta, G gamma(2) and TRIO to the EGFR upon AT(1) coexpression. In summary, our results provide further evidence for the existence of the AT(1)-EGFR heteromer and reveal potential mechanisms by which TRIO contributes to the transactivation process.