Single Proteoliposome High: Content Analysis Reveals Differences in the Homo-Oligomerization of GPCRs

Samuel McEwen Walsh, Signe Mathiasen, Sune M. Christensen, Jonathan F. Fay, Christopher King, Davide Provasi, Ernersto Borreor, Søren G. F. Rasmussen, Juan Jose Fung, Marta Filizola, Kalina Hristova, Brian Kobilka, David L. Farrens, Dimitrios Stamou

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Abstract

G-protein-coupled receptors (GPCRs) control vital cellular signaling pathways. GPCR oligomerization is proposed to increase signaling diversity. However, many reports have arrived at disparate conclusions regarding the existence, stability, and stoichiometry of GPCR oligomers, partly because of cellular complexity and ensemble averaging of intrareconstitution heterogeneities that complicate the interpretation of oligomerization data. To overcome these limitations, we exploited fluorescence-microscopy-based high-content analysis of single proteoliposomes. This allowed multidimensional quantification of intrinsic monomer-monomer interactions of three class A GPCRs (β2-adrenergic receptor, cannabinoid receptor type 1, and opsin). Using a billion-fold less protein than conventional assays, we quantified oligomer stoichiometries, association constants, and the influence of two ligands and membrane curvature on oligomerization, revealing key similarities and differences for three GPCRs with decidedly different physiological functions. The assays introduced here will assist with the quantitative experimental observation of oligomerization for transmembrane proteins in general.
Original languageEnglish
JournalBiophysical Journal
Volume115
Issue number2
Pages (from-to)300-3012
ISSN0006-3495
Publication statusPublished - 17 Jul 2018

Cite this

Walsh, S. M., Mathiasen, S., Christensen, S. M., Fay, J. F., King, C., Provasi, D., Borreor, E., Rasmussen, S. G. F., Fung, J. J., Filizola, M., Hristova, K., Kobilka, B., Farrens, D. L., & Stamou, D. (2018). Single Proteoliposome High: Content Analysis Reveals Differences in the Homo-Oligomerization of GPCRs. Biophysical Journal, 115(2), 300-3012. https://www.sciencedirect.com/science/article/pii/S0006349518306775