Abstract P318: Signaling Pathways Triggered by the Mas Receptor

Abstract

Mas receptor (MasR) is a class A Orphan G-protein-coupled receptor (GPCR). Although angiotensin-(1-7) [Ang-(1-7)] has been reported as its putative ligand, the intracellular signaling pathways activated by the MasR remain only partially characterized. In this study we examined MasR-dependent activation of G protein-mediated and ERK mediated signaling pathways. With that aim in human lung carcinoma A549 cells we evaluated Gq-coupled activity by monitoring changes in intracellular Ca 2+ levels after Ang(1-7) stimulation. Incubation with 100nM Ang-(1-7) failed to increase Ca 2+ levels either in endogenously expressing or MasR overexpressing A549 cells. On the other hand, transfection of HEK293T cells with a wild-type MasR (wt-MasR) construct resulted in a significant decrease in basal cAMP levels (p<0.05) that depended on the amount of wt-MasR protein expressed but was not observed when increasing amounts of mutant MasR lacking the PDZ binding motif were expressed. Pretreatment of wt-MasR expressing cells with pertussis toxin restored basal cAMP levels (p<0.05) whereas no effect was observed in mock-transfected cells. Also, cAMP production after forskolin stimulation was lower in cells expressing wt-MasR than in control cells. These results indicate a high level of constitutive receptor activity towards cAMP modulation involving Gαi-protein. Treatment with Ang-(1-7) increased p-ERK levels in a concentration-dependent manner in both wt-MasR and in mock-transfected HEK293T cells. In view of these results we are analyzing whether ERK activation is mediated through Ang-(1-7) binding to endogenously expressed receptors different from MasR. As MasR has been suggested to participate in cardiovascular and renal functions, comprehensive pharmacological characterization of MasR signaling is essential for developing clinical therapeutics targeting MasR function.