Number and brightness analysis to study spatio-temporal distribution of the angiotensin II AT1 and the endothelin-1 ETA receptors: Influence of ligand binding

• Published in: Biochimica et biophysica acta. General subjects Vol. 1863; no. 5; pp. 917 - 924
• Main Authors: Planes, Nadir, Digman, Michelle A., Vanderheyden, Patrick P.M.L., Gratton, Enrico, Caballero-George, Catherina
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.05.2019
• Subjects: agonists; angiotensin II; Angiotensin II AT1; antagonists; antihypertensive agents; dyes; Endothelin 1 ETA; endothelins; energy transfer; fluorescence; Fluorescent microscopy; G-protein coupled receptors; heart failure; hypertension; hypertrophy; ligands; Number and brightness; Oligomerization; pathogenesis; plasma membrane; protein aggregates; spatial distribution; stroke; TIRF; BRET; B; Oligomerization; Endothelin 1 ETA; N&B; EMCCD; TIRF; Ang II; Fluorescent microscopy; ETA; ET-1; GPCRs; GABA; Angiotensin II AT1; FRET; Number and brightness; AT1
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2019.03.004

The angiotensin II AT1 and the endothelin 1 ETA receptors play a crucial role in the pathogenesis of cardiovascular diseases like hypertension, heart failure, stroke, pulmonary hypertension, and cardiac hypertrophy. Both receptors are members of the rhodopsion-like superfamily of G protein-coupled receptors which can exist as monomers, dimers, and higher order aggregates. Recently, oligomerization of these two receptors have been described by several biophysical methods based mainly on luminescence and fluorescence energy transfer. Since this oligomerization can occur either spontaneously or it can be induced by ligand-binding, the aim of this work was to address whether the oligomerization of these receptors occurs upon ligand-binding. For this purpose the Number and Brightness analysis, a method that allows the identification, localization, and quantification of protein aggregates in the plasma membrane of a single cell, was used. An advantage of this method is that it is not limited to certain dyes specially required for Fluorescence Resonance Energy Transfer measurements. Our results showed that stably transfected angiotensin II AT1 receptors and transiently transfected endothelin 1 ETA receptors, were found as monomeric, dimeric, and tetrameric receptor aggregates. Interestingly, the binding of antihypertensive agents like losartan and BQ123, earlier suggested to be inverse agonists, significantly increased the proportion of monomers and reduced the occurrence of dimers on the cell membrane; while the kown endothelin 1 ETA antagonist sitaxentan did not influence the aggregation state of these receptors. •Both the AT1 and the ETA receptors are mainly organized as monomers near and on the plasma membrane.•Different antagonists have different effects on the organization of these two receptors.•N&B method can detect and quantify receptor aggregates near or on the plasma membrane in living cells.