Microscopy Research and Technique. 2019. doi: 10.1002/jemt.2342

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells.

Nadir Planes, Patrick P.M.L. Vanderheyden, Enrico Gratton, Catherina Caballero-George.

Abstract:

The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G-protein-coupled receptors namely Angiotensin II type 1 (AT1) and Endothelin 1 type A (ETA) receptors and their corresponding receptor–ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT1 and ETA receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local diffusivity and the confinement zone of AT1 receptors were reduced by the binding of its antagonist losartan, and the long-range diffusion with the local diffusivity coefficient of ETA receptors was reduced upon exposure to its antagonist BQ123. To the best of our knowledge, this is the first study addressing the protein diffusion laws of these two receptors on living cells using total internal reflection fluorescence microscopy and iMSD.

Biochim Biophys Acta Gen Subj. 2019 May;1863(5):917-924.

Number and brightness analysis to study spatio-temporal distribution of the

angiotensin II AT1 and the endothelin-1 ETA receptors: Influence of ligand binding.

Nadir Planes, Michelle A. Digman, Patrick P.M.L. Vanderheyden, Enrico Gratton, Catherina Caballero-George.

Abstract:

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.