Label-free cell signaling pathway deconvolution of angiotensin type 1 receptor reveals time-resolved G-protein activity and distinct AngII and AngIIIIV responses

Abstract Angiotensin II (AngII) type 1 receptor (AT 1 R) is a G protein-coupled receptor known for its role in numerous physiological processes and its implication in many vascular diseases. Its functions are mediated through G protein dependent and independent signaling pathways. AT 1 R has several endogenous peptidic agonists, all derived from angiotensinogen, as well as several synthetic ligands known to elicit biased signaling responses. Here, surface plasmon resonance (SPR) was used as a cell-based and label-free technique to quantify, in real time, the response of HEK293 cells stably expressing the human AT 1 R. The goal was to take advantage of the integrative nature of this assay to identify specific signaling pathways in the features of the response profiles generated by numerous endogenous and synthetic ligands of AT 1 R. First, we assessed the contributions of Gq, G12/13, Gi, Gβγ, ERK1/2 and β-arrestins pathways in the cellular responses measured by SPR where Gq, G12/Rho/ROCK together with β-arrestins and ERK1/2 were found to play significant roles. More specifically, we established a major role for G12 in the early events of the AT 1 R-dependent response, which was followed by a robust ERK1/2 component associated to the later phase of the signal. Interestingly, endogenous AT 1 R ligands (AngII, AngIII and AngIV) exhibited distinct responses signatures with a significant increase of the ERK1/2-like components for both AngIII and AngIV, which points toward possibly distinct physiological roles for the later. We also tested AT 1 R biased ligands, all of which affected both the early and later events. Our results support SPR-based integrative cellular assays as a powerful approach to delineate the contribution of specific signaling pathways for a given cell response and reveal response differences associated with ligands with distinct pharmacological properties.