TY - JOUR
T1 - Single Proteoliposome High-Content Analysis Reveals Differences in the Homo-Oligomerization of GPCRs
AU - Walsh, Samuel M.
AU - Mathiasen, Signe
AU - Christensen, Sune M.
AU - Fay, Jonathan F.
AU - King, Christopher
AU - Provasi, Davide
AU - Borrero, Ernesto
AU - Rasmussen, Søren G.F.
AU - Fung, Juan Jose
AU - Filizola, Marta
AU - Hristova, Kalina
AU - Kobilka, Brian
AU - Farrens, David L.
AU - Stamou, Dimitrios
N1 - Funding Information:
This work was supported by the Danish Council for Strategic Research (grant number 1311-00002B ) and Innovation Fund Denmark (grant number 5184-00048B ). Partial funds were provided by National Institutes of Health grants DA038882 and DA026434 (to M.F.). E.B. was supported in part by National Institute on Drug Abuse grant T32 DA007135 .
Publisher Copyright:
© 2018 Biophysical Society
PY - 2018/7/17
Y1 - 2018/7/17
N2 - 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.
AB - 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.
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U2 - 10.1016/j.bpj.2018.05.036
DO - 10.1016/j.bpj.2018.05.036
M3 - Article
C2 - 30021106
AN - SCOPUS:85048851627
SN - 0006-3495
VL - 115
SP - 300
EP - 312
JO - Biophysical journal
JF - Biophysical journal
IS - 2
ER -