Interaction between the angiotensin-(1-7) Mas receptor and the alamandine Mas-related G protein-coupled receptor member D receptor

GIRONACCI, M.M.; RUKAVINA MIKUSIC, NATALIA LUCÍA; MAZZITELLI LUCIANA; GÓMEZ KARINA; GRECCO HERNAN; SILVA MAURO

Conferencia; Gordon Conferences 2020; 2020

The renin-angiotensin system is composed of the pressor axis, consisting of angiotensin (Ang) II and the receptor (R) AT1 that mediates the pressor and trophic effects of Ang II, and the depressor axis represented by Ang-(1-7) and MasR as the mediator of the vasodilatory, anti-inflammatory and antifibrotic effects of Ang-(1-7). Alamandine, a new component of this system, exerts effects similar to those of Ang-(1-7), but through the stimulation of another R, the Mas-related G protein-coupled receptor member D receptor (MrgDR). It has been recently shown that Ang-(1-7) and alamandine induces anti-inflammatory effects in mice. Since both MasR and MrgDR belong to the G protein coupled receptors (GPCR) family, and that GPCRs heteromerize with other Rs affecting their functionality, we hypothesized that the anti-inflammatory action of both peptides may result from MasR-MrgDR heteromerization. Our aim was to investigate MasR-MrgDR heteromerization and its functional consequences. Human monocyte (THP-1) cells differentiated to macrophages and then exposed to lipopolysaccharide (LPS) and human embryonic kidney (HEK) 293T cells were used. Alamandine and Ang-(1-7) induced a decrease in pro-inflammatory interleukin (IL) 6 levels in human macrophages exposed to LPS. The decrease in IL-6 induced by alamandine was blocked by the MasR and the MrgDR antagonists and when MasR expression was decreased by a MasR siRNA, suggesting MasR-MrgDR interaction. Co-immuno precipitation assay in THP-1 cells confirmed MasR-MrgDR interaction supporting the interaction between both Rs. To avoid the influence from others Rs, MasR-MrgDR interaction and its functional consequences were characterized in transfected HEK293T cells. MasR fused to the yellow fluorescent protein and MrgDR fused to the m-cherry fluorescent protein were transiently co-expressed in HEK293T cells. Fluorescence resonance energy transfer analysis showed that MasR and MrgDR formed a constitutive heteromer, which was not modified by their agonists. Heteromerization among GPCRs has been proposed to alter receptor signaling. Thus, ERK1/2 and Akt phosphorylation after agonist stimulation were examined. Ang-(1-7) and alamandine did not change Akt phosphorylation in cells expressing each receptor alone. In contrast, both Ang-(1-7) and alamandine promoted Akt activation in cells expressing MasR-MrgDR heteromers, and the effect of alamandine was prevented by MasR blockade. Concerning ERK1/2 activation, neither Ang-(1-7) nor alamandine modified ERK1/2 phosphorylation in cells expressing each receptor alone; however, both Ang-(1-7) and alamandine promoted ERK1/2 phosphorylation in MasR-MrgDR-expressing cells and the effect of alamandine was prevented by MasR blockade, supporting MasR-MrgDR heteromerization . Ang-(1-7) but not alamandine elicited an anti-proliferative effect when MasR or MrgDR were expressedalone. Despite the fact that alamandine was without effect in MrgDR-expressing cells, it induced a decrease in cellular proliferation in cells expressing MasR-MrgDR heteromers. Our results showed MasR-MrgDR heteromerization, which may provide an explanation for the anti-inflammatory and anti-proliferative effects of alamandine.