Endocrine Abstracts

It is generally believed that the signal transduction of AT1 angiotensin receptor (AT1R), the major receptor of angiotensin II, requires ligand binding and subsequent adoption of its active conformation. Activated AT1R induces a plethora of signaling pathways, in contrast to inactive AT1R, which was thought to be silent in terms of signaling. We hypothesized that unliganded, but phosphorylated AT1R may recruit β-arrestins, the key proteins of receptor desensitization, internalization, and signaling. Since phosphorylation of AT1R is induced by a variety of other receptors, the β-arrestin activation by heterologously-phosphorylated AT1R may represent a novel mechanism of signal transduction and receptor cross-talk.In this study, we demonstrated that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to AT1R even in the absence of AngII. We also provided evidence that endogenous purinergic receptors can exert the same effect, proving that the interaction can be triggered at physiological levels of PKC activation. We found that heterologous mechanisms of β-arrestin recruitment to AT1R does not demand the active state of the receptor and was dependent on the stability lock. This interaction required the association of phosphorylated serine-threonine clusters in the receptor’s C-terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain, resulting in sustained binding between these proteins. Using improved FlAsH-based β-arrestin2 conformational biosensors in BRET (bioluminescence energy transfer) measurements, we showed that β-arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results reveal that AT1R may also function as a scaffold protein, demonstrating its novel role in signaling and receptor cross-talk.This work was supported by the Hungarian National Research, Development and Innovation Fund (NKFI K116954 and NVKP_16-1-2016-0039).