Insights into the Basal Activity and Activation Mechanism of the β1 Adrenergic Receptor Using Native Mass Spectrometry
In the absence of orthosteric ligands, most G protein-coupled receptors (GPCRs) exist in an equilibrium of different conformational states. This equilibrium is shifted by an agonist towards the active state or by an inverse agonist towards the inactive state. The basal activity of the receptor, and its ability to activate intracellular signaling pathways, is defined by the probability that a fraction of the receptor adopts the active state in the absence of ligand. Despite breakthroughs in native MS of membrane proteins, GPCR-transducing complexes have not been studied by this approach until very recently. Here, we investigated different conformational states of the turkey β1 adrenergic receptor (tβ1AR) in complex with two transducing partners: a G protein mimicking nanobody, Nb80, and an engineered truncated Gs protein (miniGs), in the presence of the full agonist isoprenaline by native MS. Interestingly, complex formation with both transducing partners was also observed in the absence of agonist, and allowed us to quantify basal activity of tβ1AR. We followed the stepwise disassembly of the transducing complexes by increasing the concentration of the inverse agonist S32212 in the presence of a constant concentration of isoprenaline. This allowed us to determine the relative binding affinity of S32212 in comparison to isoprenaline by native MS. Our approach provides a fast and sensitive way to detect complexes, study their stability in the presence of different ligands, and determine relative ligand affinities. Native mass spectrometry thus has the potential to become a useful tool to screen for orthosteric and allosteric GPCR drugs.
KeywordsNative electrospray ionization mass spectrometry G-coupled protein receptors
We thank David Sykes for critical reading of the manuscript, Chris Tate for sharing the sequences for the miniG proteins, and Jan Stayer and Brian Kobilka for sharing the plasmid for the Nb80. We thank the Swiss National Science Foundation (grants no. 200020_159929 and 200020_178765 to RZ and 31003A_159748 and CRSII3_141898 to DBV) for financial support of this research.
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