Details

Autor(en) / Beteiligte

• Ilyaskina, Olga S.
• Lemoine, Horst
• Bünemann, Moritz

Titel

Lifetime of muscarinic receptor–G-protein complexes determines coupling efficiency and G-protein subtype selectivity

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2018-05, Vol.115 (19), p.5016-5021

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2018

Link zum Volltext

Quelle

EZB Free E-Journals

Beschreibungen/Notizen

• G-protein–coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR–G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M₁, M₂, and M₃ receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein–M₃–R interactions but rather the affinities of Gq and Gₒ proteins to M₃–Rs, their GPCR–G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.