ANGIOTENSIN-(1-7) INDUCES MAS RECEPTOR INTERNALIZATION

M. GIRONACCI, H. ADAMO, G. CORRADI, R. SANTOS, P. ORTIZ, O. CARRETERO

MIlan

Congreso; 21st European Meeting on Hypertension and Cardiovascular Prevention; 2011

European Society of Hypertension

Objective: Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor (R) coupled to cardiac, renal and cerebral protective responses. Physiological evidence suggests that Mas R undergoes agonist-dependent desensitization, but underlying molecular mechanism regulating R activity is unknown. We investigated the hypothesis that Mas R desensitizes and internalizes upon stimulation with Ang-(1-7).Design and Method: We generated a chimera between the Mas R and the fluorescent protein YFP (Mas-YFP R). Mas-YFP transfected HEK 293T cells were incubated with Ang-(1-7) and the relative cellular distribution of Mas-YFP R was observed by confocal microscopy.Results: In resting cells, Mas-YFP R was mostly localized on the cell membrane. Ang-(1-7) induced a redistribution in fluorescence after 5 min stimulation changing the localization of Mas-YFP R to intracellular vesicles of various sizes. Following the time course of [125I]Ang-(1-7) endocytosis we observed that up to 65.1+8.7 % of Mas-YFP R underwent endocytosis after 20 min from the plasma membrane which was blocked by the Mas receptor antagonist. Mas-YFP R colocalizes with Rab5, the early endosome antigen 1 and AP-50, indicating that Mas-YFP R is internalized through clathrin-coated pits and targeted to early endosomes after Ang-(1-7) stimulation. Mas-YFP R also colocalized with caveolin-1 suggesting that at some point of R trafficking Mas-YFP R traverses caveolin-1 positive compartments.Conclusion: Mas R undergoes endocytosis upon stimulation with Ang-(1-7) and this event may explain the desensitization of Mas R responsiveness. In this way, Mas R activity and density may be tightly controlled by the cell.