Atomic force microscopy-single-molecule force spectroscopy unveils GPCR cell surface architecture

Etienne Dague,Céline Galés,Alexandre Roland,Samuel Velmont,Emmanuelle Trevisiol,Véronique Pons,Silvia Arcucci,Du N’Guyen,Véronique Lachaize,Jean-Michel Sénard,Jean-Marc Azaïs

doi:10.1038/s42003-022-03162-w

Abstract

G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors. Despite considerable insights into their pharmacology, the GPCR architecture at the cell surface still remains largely unexplored. Herein, we present the specific unfolding of different GPCRs at the surface of living mammalian cells by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). Mathematical analysis of the GPCR unfolding distances at resting state revealed the presence of different receptor populations relying on distinct oligomeric states which are receptor-specific and receptor expression-dependent. Moreover, we show that the oligomer size dictates the receptor spatial organization with nanoclusters of high-order oligomers while lower-order complexes spread over the whole cell surface. Finally, the receptor activity reshapes both the oligomeric populations and their spatial arrangement. These results add an additional level of complexity to the GPCR pharmacology until now considered to arise from a single receptor population at the cell surface.

Highlights

G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors
We present for the first time the use of AFM-SMFS in the force-volume mode to pull out GPCRs from the surface of living cells in their native cellular environment, allowing us to extract information about their architectural arrangement and to map their spatial organization at steadystate and their reshaping upon receptor activation
The assumption that major unfolding events depicted by AFM-SMFS reflected the oligomerization states of GPCRs relies on the theoretical length of each HA-tagged receptor amino acid sequence that we compared to the Gaussian distribution mean of the unfolding distances estimated from the FD curves

Summary

Introduction

G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors. We present the specific unfolding of different GPCRs at the surface of living mammalian cells by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). The receptor activity reshapes both the oligomeric populations and their spatial arrangement These results add an additional level of complexity to the GPCR pharmacology until now considered to arise from a single receptor population at the cell surface. Recent fluorescent single-molecule imaging studies based on total internal reflection fluorescence (TIRF) and superresolution structured illumination microscopy (SIM) indicated that different populations of a given GPCR, relying on different oligomerization states, coexisted at the surface of living cells[2–4]. We report for the first time the use of AFM-SMFS to quantify and spatially map GPCRs through their unfolding at the surface of living mammalian cells. An optimized mathematical analysis of the results revealed the unfolding of mainly GPCR oligomers and a tight connection between GPCR activity and its oligomeric architecture at the cell surface

Methods

Results

Conclusion