Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells

Abstract

The targeted spatial organization (sorting) of Gprotein-coupled receptors (GPCRs) is essential for their biological function and often takes place in highly curved membrane compartments such as filopodia, endocytic pits, trafficking vesicles or endosome tubules. However, the influence of geometrical membrane curvature on GPCR sorting remains unknown. Here we used fluorescence imaging to establish a quantitative correlation between membrane curvature and sorting of three prototypic class A GPCRs (the neuropeptide Y receptor Y2, the β1 adrenergic receptor and the β2 adrenergic receptor) in living cells. Fitting of a thermodynamic model to the data enabled us to quantify how sorting is mediated by an energetic drive to match receptor shape and membrane curvature. Curvature-dependent sorting was regulated by ligands in a specific manner. We anticipate that this curvature-dependent biomechanical coupling mechanism contributes to the sorting, trafficking and function of transmembrane proteins in general.

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Figure 1: Quantitative analysis of membrane curvature–dependent sorting of TMPs using live-cell imaging of filopodia.
Figure 2: Combined filopodia measurements from multiple cells verify curvature-dependent sorting of the Y2R.
Figure 3: Membrane curvature–dependent sorting of the Y2R is recurrent in artificially pulled cell membrane tethers.
Figure 4: Curvature-dependent sorting of class A GPCRs is governed by structural properties of the proteins and is regulated by agonist activation.

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Acknowledgements

This work was supported by the Lundbeck Foundation (Center of Excellence Biomembranes in Nanomedicine), the Danish Council for Strategic Research (1311-00002B), and the Innovation Fund Denmark (5184-00048B) to D.S.'s research group, by the Danish National Research Foundation (DNRF116) to L.B.O.'s research group and by the Villum Kann Rasmussen Foundation (VKR022593 to P.M.B.).

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D.S. designed and supervised the project. K.R.R. conducted all experiments and data analysis, A.M. assisted with experiments and data analysis, V.T. helped with cell culturing, and N.S.H. helped design experiments and discuss results. A.C.-J. developed the theoretical sorting model and performed the numerical fits. L.B.O. provided the optical tweezers setup and supervised the optical trapping experiments. N.L. and K.R.R. conducted the pulling tether experiments. N.L. provided the expertise in using the laser tweezers setup, and P.M.B. discussed results and data treatment. K.L.M. and V.F.W. provided the plasmid and know-how for expressing the SNAP-tagged Y2R, β1AR and β2AR. K.J.J. and S.L.P. synthesized the PYY3-36 peptide. All authors discussed the results and commented on the manuscript, which was written by K.R.R. and D.S.

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Correspondence to Dimitrios Stamou.

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The authors declare no competing financial interests.

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Rosholm, K., Leijnse, N., Mantsiou, A. et al. Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells. Nat Chem Biol 13, 724–729 (2017). https://doi.org/10.1038/nchembio.2372

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