Abstract 022: Evidence for AT2-receptor-MAS Dimerisation from Fluorescence Resonance Energy Transfer (FRET) Imaging and Fluorescence Cross Correlation Spectroscopy (FCCS)

Abstract

The angiotensin AT2-receptor (AT2R) and the receptor MAS share a strinkingly similar spectrum of signaling mechanisms and protective, physiological actions. Furthermore, cross-inhibition by the respective receptor antagonists has been observed. Therefore we hypothesised that a physical interaction between these two receptors may exist. HEK-293 cells were transfected with vectors encoding MAS or AT2R fused in the C-terminus with the fluorophores CFP or YFP for FRET and GFP or mCherry for FCCS. FRET with photobleaching was used to detect, whether MAS and AT2R are localised in very close proximity (1-10nm) in cell membranes thus indicating dimerisation. FCCS was used to follow simultaneously occurring fluctuations in fluorescence intensity of both labeled molecules. Several controls were applied such as co-transfection of equal amounts of fused and non-fused MAS/AT2R expression vectors for competition, co-tranfection of coding and uncoding pcDNA vectors or co-transfection with an unrelated transmembrane receptor. Experiments were conducted under baseline conditions and in cells treated with AT2R/MAS agonists and antagonists Significant FRET efficiency of 10.8±0.8% was measured for AT2-YFP/MAS-CFP strongly indicating heterodimerisation. FRET efficiency was not altered by AT2R or MAS agonists or antagonists. Non-fluorescent MAS and AT2R competed with fluorescent receptors as indicated by a 50% reduction in FRET efficiency (6.0±0.6%), while empty vectors did not compete (9.6±0.6%). No FRET efficiency was observed with an unrelated transmembrane receptor (0.44±1.44%) indicating specificity of receptor interactions. Both, MAS and AT2R also formed homodimers (7.4±0.8% for MAS, 9.2±0.8% for AT2R). Hetero- and homodimerisations were absent if amino acid C35 of the AT2R was mutated (3,9 ± 1,2%). FCCS corroborated the FRET results and revealed a significantly enhanced cross correlation in cells tranfected with fluorophore-tagged MAS/AT2R when compared to vectors only expressing the fluorophores (8.5±3% vs 11.1±4%; p<0.0001). Our data strongly suggest that MAS and the AT2R form homo- and heterodimers. Studies to investigate the physiological relevance of MAS/AT2R dimerisation are currently being conducted.